transforming-growth-factor-beta and Breast-Neoplasms

Breast cancer is a complex and diverse condition that disrupts multiple signaling pathways essential for cell proliferation, survival, and differentiation. Recently, the significant involvement of long-chain non-coding RNAs (lncRNAs) in controlling key signaling pathways associated with breast cancer development has been discovered. This review aims to explore the interaction between lncRNAs and various pathways, including the AKT/PI3K/mTOR, Wnt/β-catenin, Notch, DNA damage response, TGF-β, Hedgehog, and NF-κB signaling pathways, to gain a comprehensive understanding of their roles in breast cancer. The AKT/PI3K/mTOR pathway regulates cell growth, survival, and metabolic function. Recent data suggests that specific lncRNAs can influence the functioning of this pathway, acting as either oncogenes or tumor suppressors. Dysregulation of this pathway is commonly observed in breast cancer cases. Moreover, breast cancer development has been associated with other pathways such as Wnt/β-catenin, Notch, TGF-β, Hedgehog, and NF-κB. Emerging studies have identified lncRNAs that modulate breast cancer's growth, progression, and metastasis by interacting with these pathways. To advance the development of innovative diagnostic tools and targeted treatment options, it is crucial to comprehend the intricate relationship between lncRNAs and vital signaling pathways in breast cancer. By fully harnessing the therapeutic potential of lncRNAs, there is a possibility of developing more effective and personalized therapy choices for breast cancer patients. Further investigation is necessary to comprehensively understand the role of lncRNAs within breast cancer signaling pathways and fully exploit their therapeutic potential.

Topics: Animals; beta Catenin; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Hedgehogs; Humans; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Breast cancer (BC) as one of the most common causes of human deaths among women, is always considered one of the global health challenges. Despite various advances in diagnostic and therapeutic methods, a significant percentage of BC patients have a poor prognosis due to the lack of therapeutic response. Therefore, investigating the molecular mechanisms involved in BC progression can improve the therapeutic and diagnostic strategies in these patients. Cytokine and growth factor-dependent signaling pathways play a key role during BC progression. In addition to cytokines and growth factors, long non-coding RNAs (lncRNAs) have also important roles in regulation of such signaling pathways. Therefore, in the present review we discussed the role of lncRNAs in regulation of PI3K/AKT, MAPK, and TGF-β signaling pathways in breast tumor cells. It has been shown that lncRNAs mainly have an oncogenic role through the promotion of these signaling pathways in BC. This review can be an effective step in introducing the lncRNAs inhibition as a probable therapeutic strategy to reduce tumor growth by suppression of PI3K/AKT, MAPK, and TGF-β signaling pathways in BC patients. In addition, considering the oncogenic role and increased levels of lncRNAs expressions in majority of the breast tumors, lncRNAs can be also considered as the reliable diagnostic markers in BC patients.

Topics: Animals; Breast Neoplasms; Cytokines; Female; Humans; Mammary Neoplasms, Animal; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Transforming Growth Factor beta

Transforming growth factor β (TGF-β), a pluripotent cytokine and a multifunctional growth factor has a crucial role in varied biological mechanisms like invasion, migration, epithelial-mesenchymal transition, apoptosis, wound healing, and immunosuppression. Moreover, it also has an imperative role both in normal mammary gland development as well as breast carcinogenesis. TGF-β has shown to have a paradoxical role in breast carcinogenesis, by transitioning from a growth inhibitor to a growth promoter with the disease advancement. The inter-communication and crosstalk of TGF-β with different signaling pathways has strengthened the likelihood to explore it as a comprehensive biomarker. In the last two decades, TGF-β has been studied extensively and has been found to be a promising biomarker for early detection, disease monitoring, treatment selection, and tumor progression making it beneficial for disease management. In this review, we focus on the signaling pathways and biological activities of the TGF-β family in breast cancer pathogenesis and its role as a circulatory and independent biomarker for breast cancer progression and metastasis. Moreover, this review highlights TGF-β as a drug target, and the underlying mechanisms through which it is involved in tumorigenesis that will aid in the development of varied therapies targeting the different stages of breast cancer.

Topics: Breast; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Female; Humans; Transforming Growth Factor beta

The most effective way to control severity and mortality rate of the novel coronavirus disease (COVID-19) is through sensitive diagnostic approaches and an appropriate treatment protocol. We aimed to identify the effect of adding corticosteroid and Tocilizumab to a standard treatment protocol in treating COVID-19 patients with chronic disease through hematological and lab biomarkers.. This study was performed retrospectively on 68 COVID-19 patients with chronic disease who were treated by different therapeutic protocols. The patients were categorized into four groups: control group represented the patients' lab results at admission before treatment protocols were applied; group 1 included patients treated with anticoagulants, Hydroxychloroquine, and antibiotics; group 2 comprised patients treated with Dexamethasone; and group 3 included patients treated with Dexamethasone and Tocilizumab.. The study paves the way into the effectiveness of combining Dexamethasone with Tocilizumab in treatment COVID-19 patients with chronic diseases.. La forma más eficaz de controlar la gravedad y la tasa de mortalidad de la enfermedad del nuevo coronavirus (COVID-19) es mediante enfoques de diagnóstico sensibles y un protocolo de tratamiento adecuado. Nuestro objetivo fue identificar el efecto de agregar corticosteroides y tocilizumab a un protocolo de tratamiento estándar en el tratamiento de pacientes con COVID-19 con enfermedad crónica a través de biomarcadores hematológicos y de laboratorio.. Este estudio se realizó de forma retrospectiva en 68 pacientes COVID-19 con enfermedad crónica que fueron tratados por diferentes protocolos terapéuticos. Los pacientes se clasificaron en cuatro grupos: el grupo de control representaba los resultados de laboratorio de los pacientes en el momento de la admisión antes de que se aplicaran los protocolos de tratamiento; el grupo 1 incluyó a pacientes tratados con anticoagulantes, hidroxicloroquina y antibióticos; el grupo 2 estaba compuesto por pacientes tratados con dexametasona; y el grupo 3 incluyó a pacientes tratados con dexametasona y tocilizumab.. El estudio allana el camino hacia la eficacia de la combinación de dexametasona con tocilizumab en el tratamiento de pacientes con COVID-19 con enfermedades crónicas.. The Child-Mother Index constitutes a potential useful risk factor indicator for statistical analyses on data after birth. The value of the Child-Mother Index based on the estimated fetal weight before birth deserves evaluation.. Six ceria supports synthesized by various synthesis methodologies were used to deposit cobalt oxide. The catalysts were thoroughly characterized, and their catalytic activity for complete methane oxidation was studied. The supports synthesized by direct calcination and precipitation with ammonia exhibited the best textural and structural properties as well as the highest degree of oxidation. The remaining supports presented poorer textural properties to be employed as catalytic supports. The cobalt deposited over the first two supports presented a good dispersion at the external surface, which induced a significant redox effect that increased the number of Co. Some studies show that children with obesity are more likely to receive a diagnosis of depression, anxiety, or attention-deficit hyperactivity disorder (ADHD). But this does not necessarily mean obesity causes these conditions. Depression, anxiety, or ADHD could cause obesity. A child's environment, including family income or their parents' mental health, could also affect a child's weight and mental health. Understanding the nature of these relationships could help scientists develop better interventions for both obesity and mental health conditions. Genetic studies may help scientists better understand the role of the environment in these conditions, but it's important to consider both the child's and their parents’ genetics in these analyses. This is because parents and children share not only genes, but also environmental conditions. For example, families that carry genetic variants associated with higher body weight might also have lower incomes, if parents have been affected by biases against heavier people in society and the workplace. Children in these families could have worse mental health because of effects of their parent’s weight, rather than their own weight. Looking at both child and adult genetics can help disentangle these processes. Hughes et al. show that a child's own body mass index, a ratio of weight and height, is not strongly associated with the child’s mental health symptoms. They analysed genetic, weight, and health survey data from about 41,000 8-year-old children and their parents. The results suggest that a child's own BMI does not have a large effect on their anxiety symptoms. There was also no clear evidence that a child's BMI affected their symptoms of depression or ADHD. These results contradict previous studies, which did not account for parental genetics. Hughes et al. suggest that, at least for eight-year-olds, factors linked with adult weight and which differ between families may be more critical to a child's mental health than a child’s own weight. For older children and adolescents, this may not be the case, and the individual’s own weight may be more important. As a result, policies designed to reduce obesity in mid-childhood are unlikely to greatly improve the mental health of children. On the other hand, policies targeting the environmental or societal factors contributing to higher body weights, bias against people with higher weights, and poor child mental health directly may be more beneficial.. The development of an efficient photocatalyst for C2 product formation from CO. Оценка антиастенического эффекта последовательной терапии левокарнитином (ЛК) и ацетилкарнитином (АЛК) пациентов с артериальной гипертензией и/или ишемической болезнью сердца (ИБС) с астеническим синдромом (АС).. В открытое сравнительное исследование были включены 120 пациентов в возрасте 54—67 лет с артериальной гипертензией и/или ИБС с АС. Пациенты 1-й группы (. У больных 1-й группы отмечено статистически значимое уменьшение различных проявлений АС. Отличия носили достоверный характер по сравнению как с исходным уровнем, так и со 2-й группой. Установлено эндотелийпротективное действие ЛК и АЛК.. Полученные результаты свидетельствуют, что у таких коморбидных пациентов использование ЛК и АЛК уменьшает выраженность проявлений АС, а установленные эндотелиотропные свойства препаратов позволяют рекомендовать их в составе комплексной персонифицированной терапии пациентов с сердечно-сосудистыми заболеваниями.. Naproxen sodium 440 mg/diphenhydramine 50 mg combination demonstrated improvement in sleep maintenance (WASO) vs. naproxen sodium 550 mg and higher efficiency in average daily pain reduction compared with the comparison groups. The treatment was well tolerated There were no serious or unexpected adverse events reported in the study.. Сравнительный анализ эффективности и безопасности новой комбинации напроксена натрия и дифенгидрамина у пациентов с неспецифическим болевым синдромом в пояснично-крестцовом отделе спины (M54.5 «Боль внизу спины») и нарушением сна (G47.0 «Нарушения засыпания и поддержания сна [бессонница]»).. Проведено проспективное многоцентровое рандомизированное открытое сравнительное в параллельных группах клиническое исследование. Пациенты были рандомизированы в 3 группы. Больные 1-й группы получали напроксен натрия (440 мг) и дифенгидрамин (50 мг), 2-й — напроксен натрия (550 мг), 3-й — парацетамол (1000 мг) и дифенгидрамин (50 мг). Исследуемые препараты пациенты принимали однократно перед сном в течение 3 дней. Все пациенты также принимали 275 мг (1 таблетка) напроксена натрия в качестве препарата фоновой терапии. Первичным критерием эффективности было общее время бодрствования после наступления сна (WASO), измеряемое методом актиграфии. Также использовались критерии оценки продолжительности и качества сна и выраженности боли.. Анализ эффективности проведен для ITT популяции (. Применение комбинации напроксена натрия (440 мг) и дифенгидрамина (50 мг) характеризовалось более выраженным поддержанием сна по сравнению с напроксеном натрия 550 мг и более высокой эффективностью в отношении снижения интенсивности боли по сравнению со 2-й и 3-й группами. Отмечена хорошая переносимость препарата, серьезных нежелательных явлений зарегистрировано не было.

Topics: Acetaminophen; Acetylcarnitine; Acetylcholinesterase; Acids; Acinetobacter baumannii; Acinetobacter Infections; Adaptation, Psychological; Adolescent; Adsorption; Adult; Aged; Alcohol Drinking; Alzheimer Disease; Amikacin; Ammonia; Anaerobiosis; Animals; Anorexia; Anti-Bacterial Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Anxiety; Aptamers, Nucleotide; Asthenia; Attention Deficit Disorder with Hyperactivity; Bacterial Proteins; Beryllium; beta-Lactamases; Biofuels; Biomass; Biosensing Techniques; Bismuth; Blister; Body Mass Index; Body Surface Area; Boronic Acids; Brain; Breast Neoplasms; Butyrylcholinesterase; Cannabis; Carbapenems; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carboxylic Acids; Carcinoma, Hepatocellular; Cardiovascular Diseases; Carnitine; Case-Control Studies; Catalysis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Child; China; Cholinesterase Inhibitors; Clarithromycin; Clostridioides; Clostridioides difficile; Clostridium Infections; Cohort Studies; Colistin; Colitis; Colon; Coloring Agents; Coronary Artery Bypass; Creatinine; Crystalloid Solutions; Cytokines; Depression; Dextran Sulfate; Dextrans; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Diarrhea; Dietary Supplements; Diphenhydramine; Disease Models, Animal; Disease Outbreaks; Double-Blind Method; Doxorubicin; Drosophila; Drug Tapering; Dysbiosis; Electrons; Escherichia coli; Extracellular Vesicles; Fatigue; Female; Fermentation; gamma-Cyclodextrins; Gastrointestinal Microbiome; Glucose; Graft Survival; Graft vs Host Disease; Head and Neck Neoplasms; Heart Arrest, Induced; Hematopoietic Stem Cell Transplantation; High-Intensity Interval Training; Hippocampus; Humans; Hydrogen-Ion Concentration; Hypertension; Incidence; Interferon-gamma; Italy; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Lab-On-A-Chip Devices; Lactoferrin; Larva; Length of Stay; Lignin; Liver; Liver Neoplasms; Liver Transplantation; Living Donors; Low Back Pain; Lung; Lung Volume Measurements; Macrophages; Male; Melphalan; Men; Mendelian Randomization Analysis; Meropenem; Methane; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Mitochondrial Proteins; Molecular Docking Simulation; Molecular Structure; Mothers; Motivation; Mycoplasma; Mycoplasma hominis; Mycoplasma Infections; NAD; Nanocomposites; Nanoparticles; Nanotubes, Carbon; Naproxen; Neovascularization, Pathologic; Neurons; Nitrates; Nucleolin; Opuntia; Paratyphoid Fever; Phenotype; Phosphatidylinositol 3-Kinases; Phytochemicals; Plant Extracts; Pregnancy; Prevalence; Prospective Studies; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Wistar; Resveratrol; Retrospective Studies; Rifampin; Risk Factors; RNA, Messenger; Selenium; Sleep; Social Behavior; Soil; Soil Pollutants; Squamous Cell Carcinoma of Head and Neck; Staphylococcus aureus; Structure-Activity Relationship; Suicidal Ideation; Suicide; Superoxide Dismutase-1; Surveys and Questionnaires; Swimming; Syndrome; Tannins; Temperature; Transforming Growth Factor beta; Transplantation Conditioning; Treatment Outcome; Triple Negative Breast Neoplasms; Troponin T; Tumor Microenvironment; United Kingdom; Ureaplasma; Ureaplasma urealyticum; Urinary Tract Infections; Viscum; Waste Disposal Facilities; Wastewater; Water; Water Pollutants, Chemical; Wolfiporia; Young Adult

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Estrogen Antagonists; Female; Humans; Signal Transduction; Tamoxifen; Transforming Growth Factor beta

In recent years, it has been shown that breast cancer consists not only of neoplastic cells, but also of significant alterations in the surrounding stroma or tumor microenvironment. These alterations are now recognized as a critical element for breast cancer development and progression, as well as potential therapeutic targets. Furthermore, there is no doubt that ion channels are deregulated in breast cancer and some of which are prognostic markers of clinical outcome. Their dysregulation is also associated with aberrant signaling pathways. The number of published data on ion channels modifications by the microenvironment has significantly increased last years. Here, we summarize the state of the art on the cross talk between the tumor microenvironment and ion channels, in particular collagen 1, EGF, TGF-β, ATP, hypoxia, and pH, on the development and progression of breast cancer.

Topics: Breast Neoplasms; Female; Humans; Ion Channels; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Cancer is a complex disease, with various pre-existing health ailments enhancing its pathology. In cancer, the extracellular environment contains various intrinsic physiological factors whose levels are altered with aging and pre-existing conditions. In obesity, the tumor microenvironment and metastases are enriched with factors that are both derived locally, and from other physiological compartments. Similarly, in obesity, the cancer cell environment both at the site of origin and at the secondary site i.e., metastatic niche, contains significantly more phenotypically-altered adipocytes than that of un-obese cancer patients. Indeed, obesity has been linked with cancer progression, metastasis, and therapy resistance. Adipocytes not only interact with tumor cells, but also with adjacent stromal cells at primary and metastatic sites. This review emphasizes the importance of bidirectional interactions between adipocytes and breast tumor cells in breast cancer progression and its bone metastases. This paper not only chronicles the role of various adipocyte-derived factors in tumor growth, but also describes the significance of adipocyte-derived bone metastatic factors in the development of bone metastasis of breast cancer. It provides a molecular view of the interplay between the adipocytes and tumor cells involved in breast cancer bone metastasis. However, more research is needed to determine if targeting cancer-associated adipocytes holds promise as a potential therapeutic approach for breast cancer bone metastasis treatment. Interplay between adipocytes and breast cancer cells at primary cancer site and metastatic bone microenvironment. AMSC Adipose-derived mesenchymal stem cell, CAA Cancer associated adipocytes, CAF Cancer associated fibroblast, BMSC Bone marrow derived mesenchymal stem cell, BMA Bone marrow adipocyte.

Topics: Adiponectin; Adiposity; Bone Neoplasms; Breast Neoplasms; Estrogens; Female; Humans; Lipolysis; Mesenchymal Stem Cells; RANK Ligand; Transforming Growth Factor beta; Tumor Microenvironment

Investigating dietary polyphenolic compounds as antitumor agents are rising due to the growing evidence of the close association between immunity and cancer. Cancer cells elude immune surveillance for enhancing their progression and metastasis utilizing various mechanisms. These mechanisms include the upregulation of programmed death-ligand 1 (PD-L1) expression and Epithelial-to-Mesenchymal Transition (EMT) cell phenotype activation. In addition to its role in stimulating normal embryonic development, EMT has been identified as a critical driver in various aspects of cancer pathology, including carcinogenesis, metastasis, and drug resistance. Furthermore, EMT conversion to another phenotype, Mesenchymal-to-Epithelial Transition (MET), is crucial in developing cancer metastasis. A central mechanism in the upregulation of PD-L1 expression in various cancer types is EMT signaling activation. In breast cancer (BC) cells, the upregulated level of PD-L1 has become a critical target in cancer therapy. Various signal transduction pathways are involved in EMT-mediated PD-L1 checkpoint overexpression. Three main groups are considered potential targets in EMT development; the effectors (E-cadherin and Vimentin), the regulators (Zeb, Twist, and Snail), and the inducers that include members of the transforming growth factor-beta (TGF-β). Meanwhile, the correlation between consuming flavonoid-rich food and the lower risk of cancers has been demonstrated. In BC, polyphenols were found to downregulate PD-L1 expression. This review highlights the effects of polyphenols on the EMT process by inhibiting mesenchymal proteins and upregulating the epithelial phenotype. This multifunctional mechanism could hold promises in the prevention and treating breast cancer.

Topics: Antigens, CD; B7-H1 Antigen; Breast Neoplasms; Cadherins; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Polyphenols; Programmed Cell Death 1 Receptor; Signal Transduction; Transforming Growth Factor beta; Vimentin

The mammary gland (MG) is a unique organ responsible for milk synthesis, secretion, and involution to prepare the gland for subsequent lactation. The mammary epithelial cells (MECs), which are the milk synthesizing units of the MG, proliferate, differentiate, undergo apoptosis and regenerate following a cyclic pathway of lactation - involution - lactation, fine-tuning these molecular events through hormones, growth factors and other regulatory molecules. The developmental stages of the MG are embryonic, prepubertal, pubertal, pregnancy, lactation and involution, with major developmental processes occurring after puberty. The involution stage includes interesting physiological processes such as MEC apoptosis, matrix remodeling, and the generation of cells regaining the shape of a virgin MG. Signal transducer and activator of transcription 3 (STAT3) is the established master regulator of this process and aberrant expression of STAT3 leads to subnormal involution and may induce neoplasia. Several studies have reported on the molecular mechanism of MG involution with substantial knowledge being gained about this process; however, a deep understanding of this phenomenon has yet to be attained. This review focuses deeply on the molecular details of post-lactational regression, the signaling pathways involved in the lactation-involution cycle, and the latest developments in STAT3-associated MG neoplasia. Deep insight into the involution process will pave the way towards understanding the biology, apoptosis, and oncogenesis of the MG.

Topics: Animals; Apoptosis; Breast Neoplasms; Cytokines; Disease Progression; Epithelial Cells; Extracellular Matrix; Female; Glycolipids; Glycoproteins; Humans; Lactation; Leukemia Inhibitory Factor; Lipid Droplets; Mammary Glands, Animal; Mice; MicroRNAs; Models, Biological; Pregnancy; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

Toll-like receptors (TLRs) may play dual roles in human cancers. TLR4 is a key molecule which may participate in both friend and foe roles against breast cancer. This review article collected recent data regarding the mechanisms used by TLR4 in the eradication of breast cancer cells and induction of the tumor cells, and discussed the mechanisms involved in the various functions of TLR4. The literature searches revealed that TLR4 is a key molecule that participates in breast cancer cell eradication or induction of breast cancer development and also transformation of the normal cells. TLR4 eradicates breast cancer cells via recognition of their DAMPs and then induces immune responses. Over-expression of TLR4 and also alterations in its signaling, including association of some intrinsic pathways such as TGF-β signaling and TP53, are the crucial factors to alter TLR4 functions against breast cancer.

Topics: Animals; Breast Neoplasms; Female; Humans; Neoplasm Metastasis; Risk Factors; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The TGF-β signaling pathway plays an important role in cancer cell proliferation, growth, inflammation, angiogenesis, and metastasis. The role of TGF-β signaling in the pathogenesis of breast cancer is complex. TGF-β acts as a tumor suppressor in the early stages of disease, and as a tumor promoter in its later stages. Over-activation of the TGF-β signaling pathway and over-expression of the TGF-β receptors are frequently found in breast tumors. Suppression of TGF-β pathway using biological or pharmacological inhibitors is a potentially novel therapeutic approach for breast cancer treatment. This review summarizes the regulatory role of TGF-β signaling in the pathogenesis of breast cancer for a better understanding and hence a better management of this disease.

Topics: Breast Neoplasms; Clinical Trials as Topic; Female; Humans; Imidazoles; Quinoxalines; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Epithelial to mesencyhmal transition (EMT) has a central role in tumor metastasis and progression. EMT is regulated by several growth factors and pro-inflammatory cytokines. The most important role in this regulation could be attributed to transforming growth factor-β (TGF-β). In breast cancer, TGF-β effect on EMT could be potentiated by Fos-related antigen, oncogene HER2, epidermal growth factor, or mitogen-activated protein kinase kinase 5 - extracellular-regulated kinase signaling. Several microRNAs in breast cancer have a considerable role either in potentiation or in suppression of EMT thus acting as oncogenic or tumor suppressive modulators. At present, possibilities to target EMT are discussed but the results of clinical translation are still limited. In prostate cancer, many cellular events are regulated by androgenic hormones. Different experimental results on androgenic stimulation or inhibition of EMT have been reported in the literature. Thus, a possibility that androgen ablation therapy leads to EMT thus facilitating tumor progression has to be discussed. Novel therapy agents, such as the anti-diabetic drug metformin or selective estrogen receptor modulator ormeloxifene were used in pre-clinical studies to inhibit EMT in prostate cancer. Taken together, the results of pre-clinical and clinical studies in breast cancer may be helpful in the process of drug development and identify potential risk during the early stage of that process.

Topics: Benzopyrans; Breast Neoplasms; Cadherins; Cell Plasticity; Cytokines; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Male; MAP Kinase Kinase 5; Metformin; MicroRNAs; Prostatic Neoplasms; Proto-Oncogene Proteins c-fos; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta

Matrix metalloproteinase 9 (MMP-9) has been frequently noticed in the breast cancers. In this study, we aim to investigate the associations of MMP-9 with the activation of transforming growth factor beta (TGF-β)/SMAD signalling and the malignancy of breast malignant tumour cells.. The distributions of MMP-9 and TGF-β in the tissues of canine breast cancers were screened by immunohistochemical assays. A recombinant plasmid expressing mouse MMP-9 was generated and transiently transfected into three different breast cancer cell lines. Cell Counting Kit-8 and colony formation assay were used to study cell viability. Migration and invasion ability were analysed by wound assay and transwell filters. Western blot and quantitative real-time PCR were used to determine the protein and mRNA expression.. Remarkable strong MMP-9 and TGF-β signals were observed in the malignant tissues of canine breast cancers. In the cultured three cell lines receiving recombinant plasmid expressing mouse MMP-9, the cell malignancy was markedly increased, including the cell colony formation, migration and epithelial-mesenchymal transition. The levels of activated TGF-β, as well as SMAD4, SMAD2/3 and phosphorylation of SMAD2, were increased, reflecting an activation of TGF-β/SMAD signalling. We also demonstrated that the inhibitors specific for MMP-9 and TGF-β sufficiently blocked the overexpressing MMP-9 induced the activation of SMAD signalling and enhancement on invasion in the tested breast cancer cell lines.. Overexpression of MMP-9 increases the malignancy of breast cancer cell lines, largely via activation of the TGF-β/SMAD signalling.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Matrix Metalloproteinase 9; Phosphorylation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Lack of early diagnosis methods and the development of drug resistance are among the main reasons for increased mortality rates within breast cancer patients. These two aspects are governed by specific pro-carcinogenic modifications, where TGBβ-induced EMT is one of the leading actors. Endowment of the epithelial cells with mesenchymal characteristics allows them to migrate and invade secondary tissues in order to form malignant sites and also confers chemoresistance. TGFβ which role switches from the tumor suppressor cytokine to the oncogenic one favoring the tumor microenvironment regulates this process.. This review aims to comprehensively present the updated TGFβ-induced EMT in breast cancer, including the regulatory role of the non-coding RNAs with focus on the miR-200 family and newly discovered lncRNAs such as HOTAIRM1. Additionally, a new phenotype, P-EMT, also modulated by miR-200 and miR-34 families that form complex feedback loops with TGFβ, SNAI1 and ZEB1/2 is presented under an updated form.. The hallmarks of EMT are becoming increasingly associated with aggressive forms of breast cancer and low survival rates among patients. Considering that this phenotypical switch can trigger drug resistance, invasion and metastasis, inhibition of EMT could represent an important milestone in mammary cancer treatment.. The present review assembles the most recent data regarding TGFβ induced EMT, including the input of non-coding RNAs, contributing to the possible development of new targeted treatment strategies for cancer patients.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; MicroRNAs; RNA, Untranslated; Signal Transduction; Transforming Growth Factor beta

This article focusses on the role of TGF-β and its signaling crosstalk with the RHO family GTPases RAC1 and RAC1b in the progression of breast and pancreatic carcinoma. The aggressive nature of these tumor types is mainly due to metastatic dissemination. Metastasis is facilitated by desmoplasia, a peculiar tumor microenvironment and the ability of the tumor cells to undergo epithelial-mesenchymal transition (EMT) and to adopt a motile and invasive phenotype. These processes are controlled entirely or in part by TGF-β and the small RHO GTPase RAC1 with both proteins acting as tumor promoters in late-stage cancers. Data from our and other studies point to signaling crosstalk between TGF-β and RAC1 and the related isoform, RAC1b, in pancreatic and mammary carcinoma cells. Based on the exciting observation that RAC1b functions as an endogenous inhibitor of RAC1, we propose a model on how the relative abundance or activity of RAC1 and RAC1b in the tumor cells may determine their responses to TGF-β and, ultimately, the metastatic capacity of the tumor.

Topics: Animals; Breast Neoplasms; Humans; Pancreatic Neoplasms; rac1 GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta

Fanconi anemia (FA), the most common form of inherited bone marrow failure, predisposes to leukemia and solid tumors. FA is caused by the genetic disruption of a cellular pathway that repairs DNA interstrand crosslinks. The impaired function of this pathway, and the genetic instability that results, is considered the main pathogenic mechanism behind this disease. The identification of breast cancer susceptibility genes (for example, BRCA1/FANCS and BRCA2/FANCD1) as being major players in the FA pathway has led to a surge in molecular studies, resulting in the concept of the FA-BRCA pathway. In this review, we discuss recent advances in the molecular pathogenesis of FA from three viewpoints: (a) new FA genes, (b) modifier pathways that influence the cellular and clinical phenotypes of FA and (c) non-canonical functions of FA genes that may drive disease progression independently of deficient DNA repair. Potential therapeutic approaches for FA that are relevant to each will also be proposed.

Topics: Aldehydes; Autophagy; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Disease Progression; DNA Repair; Fanconi Anemia; Female; Genetic Predisposition to Disease; Humans; Male; Molecular Targeted Therapy; Transforming Growth Factor beta

Breast cancer is the most prevalent female malignancy throughout the world. Conventional treatment strategies for breast cancer consist of chemotherapy, radiation, surgery, chemoradiation, hormone therapy, and targeted therapies. Among them, targeted therapies show advantages to reduce cost and toxicity for being possible for individualized treatments based on the intrinsic subtypes of breast cancer. With deeper understanding of key signaling pathways concerning tumor growth and survival, growth factor-controlled signaling pathways are frequently dysregulated in the development and progression of breast cancer. Thus, targeted therapies against growth factor-mediated signaling pathways have been shown to have promising efficacy in both preclinical animal models and human clinical trials. In this chapter, we will briefly introduce inhibitors and monoclonal antibodies that target the main growth factor-modulated scenarios including epidermal growth factor receptor (EGFR), transforming growth factor beta (TGF-β), insulin-like growth factor 1 receptor (IGF1R), and fibroblast growth factor receptor (FGFR) signaling pathways in breast cancer therapy.

Topics: Antibodies, Monoclonal; Breast Neoplasms; ErbB Receptors; Female; Humans; Molecular Targeted Therapy; Receptor, Fibroblast Growth Factor, Type 1; Receptor, IGF Type 1; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of various cancer biologies, including cancer cell migration, invasion, angiogenesis, proliferation, as well as apoptosis, and it is one of indispensable signaling pathways during cancer metastasis. TGF-β signaling pathway can regulate and be regulated by a series of molecular and signaling pathways where microRNAs (miRNAs) seem to play important roles. miRNAs are small non-coding RNAs that can regulate expressions of their target genes. Emerging evidence suggest that miRNAs participate in various biological and pathologic processes such as cancer cells apoptosis, proliferation, invasion, migration, and metastasis by influencing multiple signaling pathways. In this article, we focus on the interaction between miRNAs and TGF-β in breast cancer (BC) metastasis through modulating invasion-metastasis-related factors, including epithelial-to-mesenchymal transition (EMT), cancer stem cells (CSCs), matrix metalloproteinase (MMP), tissue inhibitors of MMPs (TIMPs), cell adhesion molecules (CAMs), and tumor microenvironment (TME). Through a clear understanding of the complicated links between TGF-β pathway and miRNAs, it may provide a novel and safer therapeutic target to prevent BC metastasis.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinases; MicroRNAs; Models, Biological; Neoplasm Metastasis; Neoplasm Proteins; Signal Transduction; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Microenvironment

Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor β (TGFβ), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFβ signaling is considered to be abnormal in the context of cancer development; however, TGFβ acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFβ, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.

Topics: Animals; Breast Neoplasms; Cellular Reprogramming; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Neoplastic Stem Cells; RNA, Untranslated; Signal Transduction; Transforming Growth Factor beta

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. MicroRNAs (miRNAs) are naturally-occurring, non-coding small RNA molecules that can modulate protein coding-genes, which makes it contributing to nearly all the physiological and pathological processes. Progression of breast cancer and resistance to endocrine therapies have been attributed to the possibility of hormone-responsive miRNAs involved in the regulation of certain signaling pathways.. This review introduces better understanding of miRNAs to provide promising advances for treatment. miRNAs have multiple targets, and they were found to regulate different signaling pathways; consequently it is important to characterize their mechanisms of action and their cellular targets in order to introduce miRNAs as novel and promising therapies.. This review summarizes the molecular mechanisms of miRNAs in TGF-beta signaling, apoptosis, metastasis, cell cycle, ER-signaling, and drug resistance.. Finally, miRNAs will be introduced as promising molecules to be used in the fight against breast cancer and its developed drug resistance.

Topics: Animals; Apoptosis; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Transforming Growth Factor beta

Despite the advancement of treatment modalities, many cancer patients experience tumor recurrence and metastasis at regional or distant sites. Evolving understanding of tumor biology has led to the hypothesis that tumors may possess a stem cell-like subpopulation known as cancer stem cells (CSCs) that may be involved in driving tumor propagation and pathogenesis. Like normal stem cells (NSCs), CSCs can be identified by markers such as CD133, CD44, and ALDH. CSCs have the ability to self-renew and differentiate into different tumor components through stemness pathways, such as Wnt, TGF-β, STAT, and Hippo-YAP/TAZ, among others. In NSCs, stemness pathways are strictly regulated and control many important biologic processes, including embryogenesis and intestinal crypt cellular regulation. In contrast, stemness pathways in CSCs are significantly dysregulated. Combining current drugs with the targeting of these stemness pathways may significantly improve patient prognosis. The aim of this supplement is to update clinicians on the accumulated evidence characterizing the role of CSCs in tumor initiation, heterogeneity, therapy resistance, and recurrence and metastasis, and the potential for effectively treating patients.

Topics: Biomarkers, Tumor; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gastrointestinal Neoplasms; Hematologic Neoplasms; Humans; Hyaluronan Receptors; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Neoplasms; Neoplastic Stem Cells; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

Early pregnancy is associated with a reduction in a woman's lifetime risk for breast cancer. However, different studies have demonstrated an increase in breast cancer risk in the years immediately following pregnancy. Early and long-term risk is even higher if the mother age is above 35 years at the time of first parity. The proinflammatory microenvironment within the mammary gland after pregnancy renders an "ideal niche" for oncogenic events. Signaling pathways involved in programmed cell death and tissue remodeling during involution are also activated in breast cancer. Herein, the major signaling pathways involved in mammary gland involution, signal transducer and activator of transcription (STAT3), nuclear factor-kappa B (NF-κB), transforming growth factor beta (TGFβ), and retinoid acid receptors (RARs)/retinoid X receptors (RXRs), are reviewed as part of the complex network of signaling pathways that crosstalk in a contextual-dependent manner. These factors, also involved in breast cancer development, are important regulatory nodes for signaling amplification after weaning. Indeed, during involution, p65/p300 target genes such as MMP9, Capn1, and Capn2 are upregulated. Elevated expression and activities of these proteases in breast cancer have been extensively documented. The role of these proteases during mammary gland involution is further discussed. MMPs, calpains, and cathepsins exert their effect by modification of the extracellular matrix and intracellular proteins. Calpains, activated in the mammary gland during involution, cleave several proteins located in cell membrane, lysosomes, mitochondria, and nuclei favoring cell death. Besides, during this period, Capn1 is most probably involved in the modulation of preadipocyte differentiation through chromatin remodeling. Calpains can be implicated in cell anchoring loss, providing a proper microenvironment for tumor growth. A better understanding of the role of any of these proteases in tumorigenesis may yield novel therapeutic targets or prognostic markers for breast cancer.

Topics: Breast Neoplasms; Female; Humans; Lactation; Mammary Glands, Human; NF-kappa B; Peptide Hydrolases; Pregnancy; Risk Factors; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) refers to tne transition during which epithelial cells undergo the loss of apical-basal polarity, acquisition of migration capability and transformation into mesenchymal cells. EMT induces breast cancer in situ to developing into metastasis and associates with the drug resistence. The multiple elements including signal pathways, transcriptional factors and downstream genes orchestrate the transition. Among them, the transforming growth factor β (TGF-β) signaling pathway plays a key role in the regulation of EMT in breast cancer. And this paper reviews the development of TGF-β signaling pathway induced EMT in breast cancer.

Topics: Breast Neoplasms; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Pain is a clinical sign of inflammation found in a wide variety of chronic pathologies, including cancer. The occurrence of pain in patients carrying breast tumors is reported and is associated with aspects concerning disease spreading, treatment, and surgical intervention. The persistence of pain in patients submitted to breast surgery is estimated in a range from 21% to 55% and may affect patients before and after surgery. Beyond the physical compression exerted by the metastatic mass expansion and tissue injury found in breast cancer, inflammatory components that are significantly produced by the host-tumor interaction can significantly contribute to the generation of pain. In this context, cytokines have been studied aiming to establish a cause-effect relationship in cancer pain-related syndromes, especially the proinflammatory ones. Few reports have investigated the relationship between pain and cytokines in women carrying advanced breast cancer. In this scenario, the present review analyzes the main cytokines produced in breast cancer and discusses the evidences from literature regarding its role in specific clinical features related with this pathology.

Topics: Bone Neoplasms; Breast Neoplasms; Cytokines; Female; Granulocyte Colony-Stimulating Factor; Humans; Pain, Intractable; Paraneoplastic Polyneuropathy; Transforming Growth Factor beta

Pregnancy at an early age has a strong protective effect against breast cancer in humans and rodents. Postulated mechanisms underlying this phenomenon include alterations in the relative dynamics of hormone and growth factor-initiated cell fate-determining signaling pathways within the hierarchically organized mammary gland epithelium. Recent studies in epithelial cell subpopulations isolated from mouse and human mammary glands have shown that early pregnancy decreases the proportion of hormone receptor-positive cells and causes pronounced changes in gene expression as well as decreased proliferation in stem/progenitor cells. The changes include downregulation of Wnt and transforming growth factor β (TGFβ) signaling. These new findings highlight the importance of cell-cell interactions within the mammary gland epithelium in modulating cancer risk and provide potential targets for breast cancer prevention strategies.

Topics: Age Factors; Animals; Breast Neoplasms; Cell Differentiation; Cell Proliferation; Epithelial Cells; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mice; Parity; Pregnancy; Stem Cells; Transforming Growth Factor beta; Wnt Signaling Pathway

All-trans retinoic acid (ATRA) is the most important active metabolite of vitamin A controlling segmentation in the developing organism and the homeostasis of various tissues in the adult. ATRA as well as natural and synthetic derivatives, collectively known as retinoids, are also promising agents in the treatment and chemoprevention of different types of neoplasia including breast cancer. The major aim of the present article is to review the basic knowledge acquired on the anti-tumor activity of classic retinoids, like ATRA, in mammary tumors, focusing on the underlying cellular and molecular mechanisms and the determinants of retinoid sensitivity/resistance. In the first part, an analysis of the large number of pre-clinical studies available is provided, stressing the point that this has resulted in a limited number of clinical trials. This is followed by an overview of the knowledge acquired on the role played by the retinoid nuclear receptors in the anti-tumor responses triggered by retinoids. The body of the article emphasizes the potential of ATRA and derivatives in modulating and in being influenced by some of the most relevant cellular pathways involved in the growth and progression of breast cancer. We review the studies centering on the cross-talk between retinoids and some of the growth-factor pathways which control the homeostasis of the mammary tumor cell. In addition, we consider the cross-talk with relevant intra-cellular second messenger pathways. The information provided lays the foundation for the development of rational and retinoid-based therapeutic strategies to be used for the management of breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; ErbB Receptors; Female; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PubMed; Receptor Cross-Talk; Receptors, Notch; Retinoids; Transforming Growth Factor beta; Tretinoin; Wnt Proteins

The progression of cancer through stages that guide a benign hyperplastic epithelial tissue towards a fully malignant and metastatic carcinoma, is driven by genetic and microenvironmental factors that remodel the tissue architecture. The concept of epithelial-mesenchymal transition (EMT) has evolved to emphasize the importance of plastic changes in tissue architecture, and the cross-communication of tumor cells with various cells in the stroma and with specific molecules in the extracellular matrix (ECM).. Among the multitude of ECM-embedded cytokines and the regulatory potential of ECM molecules, this article focuses on the cytokine transforming growth factor β (TGFβ) and the glycosaminoglycan hyaluronan, and their roles in cancer biology and EMT. For brevity, we concentrate our effort on breast cancer.. Both normal and abnormal TGFβ signaling can be detected in carcinoma and stromal cells, and TGFβ-induced EMT requires the expression of hyaluronan synthase 2 (HAS2). Correspondingly, hyaluronan is a major constituent of tumor ECM and aberrant levels of both hyaluronan and TGFβ are thought to promote a wounding reaction to the local tissue homeostasis. The link between EMT and metastasis also involves the mesenchymal-epithelial transition (MET). ECM components, signaling networks, regulatory non-coding RNAs and epigenetic mechanisms form the network of regulation during EMT-MET.. Understanding the mechanism that controls epithelial plasticity in the mammary gland promises the development of valuable biomarkers for the prognosis of breast cancer progression and even provides new ideas for a more integrative therapeutic approach against disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Topics: Animals; Breast Neoplasms; Epithelial-Mesenchymal Transition; Extracellular Matrix; Humans; Neoplastic Stem Cells; Smad Proteins; Transforming Growth Factor beta

The purpose of this study is to identify metastasis-associated genes/signaling pathways in basal-like breast tumors. Kaplan-Meier analysis of two public meta-datasets and functional classification was used to identify genes/signaling pathways significantly associated with distant metastasis free survival. Integrated analysis of expression correlation and interaction between mRNAs and miRNAs was used to identify miRNAs that potentially regulate the expression of metastasis-associated genes. The novel metastatic suppressive role of miR-17-5p was examined by in vitro and in vivo experiments. Over 4,000 genes previously linked to breast tumor progression were examined, leading to identification of 61 and 69 genes significantly associated with shorter and longer DMFS intervals of patients with basal-like tumors, respectively. Functional annotation linked most of the pro-metastatic genes to epithelial mesenchymal transition (EMT) process and three intertwining EMT-driving pathways (hypoxia, TGFB and Wnt), whereas most of the anti-metastatic genes to interferon signaling pathway. Members of three miRNA families (i.e., miR-17, miR-200 and miR-96) were identified as potential regulators of the pro-metastatic genes. The novel anti-metastatic function of miR-17-5p was confirmed by in vitro and in vivo experiments. We demonstrated that miR-17-5p inhibition in breast cancer cells enhanced expression of multiple pro-metastatic genes, rendered cells metastatic properties, and accelerated lung metastasis from orthotopic xenografts. In contrast, intratumoral administration of miR-17-5p mimic significantly reduced lung metastasis. These results provide evidence supporting that EMT activation and IFN pathway inactivation are markers of metastatic progression of basal-like tumors, and members of miR-17, miR-200, and miR-96 families play a role in suppressing EMT and metastasis. The metastasis-associated genes identified in this study have potential prognostic values and functional implications, thus, can be exploited as therapeutic targets to prevent metastasis of basal-like breast tumors.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kaplan-Meier Estimate; Lung Neoplasms; MicroRNAs; Neoplasm Metastasis; Neoplasms, Basal Cell; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Metastatic spread of breast cancer cells, facilitated by the epithelial-mesenchymal transition (EMT) process, is responsible for the majority of breast cancer mortality. Increased levels of hyaluronan due to deregulation of hyaluronan-synthesizing enzymes, like HAS2, and expression of CD44, the key receptor for hyaluronan, are correlated to poor outcome of patients with basal-like breast cancer. TGFβ induces HAS2 and CD44, both of which are required in the course of efficient TGFβ-induced EMT processes by mammary epithelial cells. Elucidation of the molecular mechanisms underlying tumor-stroma interactions in breast cancer including the regulation of HAS2 and CD44 expression may contribute to the development of better strategies to treat breast cancer patients.

Topics: Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Disease Progression; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

The tumor-promoting arm of transforming growth factor beta (TGF-β) receptor signaling contributes to advanced cancer progression and is considered a master regulator of breast cancer metastasis. In mammals, there are six distinct members in the tumor-necrosis factor receptor (TNFR)-associated factor (TRAF) family (TRAF1-TRAF6), with the function of TRAF4 not being extensively studied in the past decade. Although numerous studies have suggested that there is elevated TRAF4 expression in human cancer, it is still unknown in which oncogenic pathway TRAF4 is mainly implicated. This review highlights TGF-β-induced SMAD-dependent signaling and non-SMAD signaling as the major pathways regulated by TRAF4 involved in breast cancer metastasis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Transformation, Neoplastic; Female; Humans; Neoplasm Metastasis; Prognosis; Signal Transduction; Smad Proteins; TNF Receptor-Associated Factor 4; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Elucidation of the regulatory controls on epithelial plasticity is pivotal not only to better understand the nature of metastasis but also for the design of targeted therapies to prevent the earliest steps in migration and invasion from the primary tumor. This review will highlight the role of the novel TRIM protein DEAR1 (annotated as TRIM62) in the regulation of apical-basal polarity and acinar morphogenesis as well as its function as a chromosome 1p35 tumor suppressor and negative regulator of TGFβ-driven epithelial-mesenchymal transition (EMT). DEAR1 binds to and promotes the ubiquitination of SMAD3, the major effector of TGFβ-mediated EMT, as well as downregulates SMAD3 targets SNAIL1/2, master transcriptional regulators of EMT. Cumulative results suggest a novel paradigm for DEAR1 in the regulation of the breast tumor microenvironment, polarity, and EMT. Because DEAR1 undergoes loss-of-function mutations, homozygous deletion, as well as copy-number losses in multiple epithelial cancers, including breast cancer, DEAR1 has clinical use as a predictive and prognostic biomarker as well as for stratifying breast cancers and potentially other epithelial tumor types for targeted therapies aimed at the pathways regulated by DEAR1.

Topics: Animals; Breast Neoplasms; Cell Polarity; Epithelial-Mesenchymal Transition; Epithelium; Female; Humans; Neoplasms, Glandular and Epithelial; Signal Transduction; Transforming Growth Factor beta; Tripartite Motif Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination

The human epidermal growth factor receptor 2 (HER2) is a major player in the survival and proliferation of tumour cells and is overexpressed in up to 30 % of breast cancer cases. A considerable amount of work has been undertaken to unravel the activity and function of HER2 to try and develop effective therapies that impede its action in HER2 positive breast tumours. Research has focused on exploring the HER2 activated phosphoinositide-3-kinase (PI3K)/AKT and rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) pathways for therapies. Despite the advances, cases of drug resistance and recurrence of disease still remain a challenge to overcome. An important aspect for drug resistance is the complexity of the HER2 signaling network. This includes the crosstalk between HER2 and hormone receptors; its function as a transcription factor; the regulation of HER2 by protein-tyrosine phosphatases and a complex network of positive and negative feedback-loops. This review summarises the current knowledge of many different HER2 interactions to illustrate the complexity of the HER2 network from the transcription of HER2 to the effect of its downstream targets. Exploring the novel avenues of the HER2 signaling could yield a better understanding of treatment resistance and give rise to developing new and more effective therapies.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Ion Channels; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mucin-1; Phosphatidylinositol 3-Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; ras Proteins; Receptor, ErbB-2; Receptors, Androgen; Receptors, Estrogen; Receptors, Leptin; Signal Transduction; STAT Transcription Factors; Transforming Growth Factor beta

Skeletal metastases are an incurable complication afflicting the majority of patients who die from advanced breast cancer. They are most often osteolytic, characterized by net bone destruction and suppressed new bone formation. Life expectancy from first diagnosis of breast cancer bone metastases is several years, during which time skeletal-related events - including pain, fracture, hypercalcemia, and spinal cord compression - significantly degrade quality of life. The bone marrow niche can also confer hormonal and chemo-resistance. Most treatments for skeletal metastases target bone-destroying osteoclasts and are palliative. Recent results from the Breast cancer trials of Oral Everolimus-2 trial suggest that agents such as the mammalian target of rapamycin inhibitor everolimus may have efficacy against breast cancer bone metastases in part via stimulating osteoblasts as well as by inhibiting tumor growth. Selective estrogen receptor modulators similarly inhibit growth of estrogen receptor-positive breast cancers while having positive effects on the skeleton. This review discusses the future role of bone-anabolic agents for the specific treatment of osteolytic breast cancer metastases. Agents with both anti-tumor and bone-anabolic actions have been tested in the setting of multiple myeloma, a hematological malignancy that causes severe osteolytic bone loss and suppression of osteoblastic new bone formation. Stimulation of osteoblast activity inhibits multiple myeloma growth - a strategy that might decrease breast cancer burden in osteolytic bone metastases. Proteasome inhibitors (bortezomib and carfilzomib) inhibit the growth of myeloma directly and are anabolic for bone. Drugs with limited anti-tumor activity but which are anabolic for bone include intermittent parathyroid hormone and antibodies that neutralize the WNT inhibitors DKK1 and sclerostin, as well as the activin A blocker sotatercept and the osteoporosis drug strontium ranelate. Transforming growth factor-beta inhibitors have little tumor antiproliferative activity but block breast cancer production of osteolytic factors and are also anabolic for bone. Some of these treatments are already in clinical trials. This review provides an overview of agents with bone-anabolic properties, which may have utility in the treatment of breast cancer metastatic to the skeleton.

Topics: Antineoplastic Agents; Bone Density Conservation Agents; Bone Neoplasms; Boronic Acids; Bortezomib; Breast Neoplasms; Everolimus; Female; Humans; Oligopeptides; Osteoblasts; Parathyroid Hormone; Proteasome Inhibitors; Pyrazines; Recombinant Fusion Proteins; Sirolimus; Thiophenes; Transforming Growth Factor beta

There is increasing evidence that cancer stem cells (CSCs) play a critical role in breast cancer initiation, progression, metastasis and drug resistance. It is thought that they are either generated from normal mammary stem/progenitor cells or from mammary epithelial cells by epithelial-mesenchymal transition. Breast CSCs are characterized by the activation of stemness-related pathways, such as the Notch and Wnt pathways, and by the expression of certain stem cell markers, such as CD44, EpCAM and ALDH1. CSCs form a minor population, whose proportion depends on various factors, including environmental conditions. Since CSCs are highly resistant to chemotherapy, additional treatment of breast cancer patients with CSC-specific drugs, such as salinomycin and gamma-secretase inhibitors which target the Wnt or Notch pathway, respectively, will be required. Interestingly, an equilibrium seems to exist between CSCs and non-stem cancer cells, and there are indications that CSCs can be recruited from non-stem cancer cells. As a consequence, it may be necessary to combine a therapy targeting CSCs with common chemotherapy that targets the bulk tumor to avoid the regeneration of CSCs.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Phenotype; Protein Structure, Tertiary; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

Metastatic colonization represents the final step of metastasis, and is the major cause of cancer mortality. Metastasis as an "inefficient" process requires the right population of tumor cells in a suitable microenvironment to form secondary tumors. Cancer stem cells are the only capable population of tumor cells to progress to overt metastasis. On the other hand, the occurrence of appropriate microenvironmental conditions within the target tissue would be critical for metastasis formation. Metastatic niche seems to be the specialized microenvironment to support tumor initiating cells at the distant organ. Master regulators not only determine cancer stem cell state, but also may have regulatory roles in metastatic niche elements. Meanwhile, both cancer stem cell and metastatic niche may function like two sides of the metastatic coin. Hypoxia inducible factors have multiple roles in regulation of both sides of this coin. TGF-β superfamily, also, have been considered as master regulators of epithelial to mesenchymal transition and metastasis and may play crucial roles in regulation of metastatic niche as well. In this regard, we hypothesize the presence of a possible emerging molecular pathway in the biological process of breast cancer metastasis. In this process, non-Smad TGF-β-induced metastasis connects cancer stem cell and metastatic niche formation through a central path, "Metastasis Pathway".

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Humans; Hypoxia-Inducible Factor 1; Neoplasm Metastasis; Neoplastic Stem Cells; Transforming Growth Factor beta; Tumor Microenvironment

The components that comprise the extracellular matrix (ECM) are integral to normal tissue homeostasis as well as the development and progression of breast tumors. The secretion, construction, and remodeling of the ECM are each regulated by a complex interplay between tumor cells, fibroblasts and macrophages. Transforming growth factor-β (TGF-β) is an essential molecule in regulating the cellular production of ECM molecules and the adhesive interactions of cells with the ECM. Additionally, hypoxic cell signals, initiated by oxygen deprivation, additional metabolic factors or receptor activation, are associated with ECM formation and the progression of breast cancer. Both TGF-β and hypoxic cell signals are implicated in the functional and morphological changes of cancer-associated-fibroblasts and tumor-associated-macrophages. Moreover, the enhanced recruitment of tumor and stromal cells in response to hypoxia-induced chemokines leads to increased ECM deposition and remodeling, increased blood vessel formation, and enhanced tumor migration. Thus, elucidation of the collaborative networks between tumor and stromal cells in response to the combined signals of TGF-β and hypoxia may yield insight into treatment parameters that target both tumor and stromal cells.

Topics: Breast Neoplasms; Cell Communication; Extracellular Matrix; Female; Fibroblasts; Humans; Hypoxia; Phosphorylation; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

By contrast with developmental epithelial-mesenchymal transition (EMT), where epithelial characteristics undergo transformation to a mesenchymal-like phenotype in a coordinated fashion, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment. Therefore, a partial form of EMT has been proposed as variably participating in the establishment of invasive phenotype in different types of breast carcinoma, in keeping with their morphological and phenotypical diversity. A complex network of signalling pathways and transcription factors appears responding to various growth factors and cytokines released by stromal and neoplastic elements, endowing the system with abundant regulatory opportunities. The process of EMT is largely elusive in histopathological preparations, prompting doubts regarding its significance in tumour progression. This might be related to the presumed focal occurrence of EMT in the majority of tumours. Detailed topological studies might facilitate understanding of the orchestration of events taking place in vivo. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.

Topics: beta Catenin; Breast Neoplasms; Cadherins; Epithelial-Mesenchymal Transition; Female; Humans; Monomeric GTP-Binding Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Proteins

Transforming growth factor-β (TGF-β) is a ubiquitous cytokine playing an essential role in cell proliferation, differentiation, apoptosis, adhesion and invasion, as well as in cellular microenvironment. In malignant diseases, TGF-β signaling features a growth inhibitory effect at an early stage but aggressive oncogenic activity at the advanced malignant state. Here, we update the current understanding of TGF-β signaling in cancer development and progression with a focus on breast cancer. We also review the current approaches of TGF-β signaling-targeted therapeutics for human malignancies.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Disease Progression; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) ligand is a multifunctional growth factor that regulates various cell behavior, such as cell proliferation, differentiation, migration, and apoptosis. Because TGF-β is a potent growth inhibitor, abnormalities in TGF-β signaling result in carcinogenesis. In addition to tumor suppressor function, TGF-β acts as an oncogenic factor. In particular, TGF-β signaling plays an important role during metastasis of breast cancer. Recently, epithelial-mesenchymal transition (EMT) has been shown to confer malignant properties such as cell motility and invasiveness to cancer cells and plays crucial roles during cancer metastasis. Moreover, breast stem-like cells exhibit EMT properties. Because TGF-β is a potent regulator of EMT as well as cell stemness, TGF-β signaling might play a crucial role in the regulation of breast cancer stem cells.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Breast cancer is the second leading cause of death due to cancer in women. Accumulating evidence shows a correlation between overexpression of miR-155 and breast cancer development. The microRNA (miRNA) encoded by mir-155 is known to be oncogenic in multiple tumors. This review summarizes the signaling pathways that are regulated by miR-155 in breast cancer and discusses therapeutic possibilities related to miR-155.

Topics: Breast Neoplasms; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Janus Kinases; MicroRNAs; Models, Biological; Neoplasm Proteins; Oncogenes; RNA, Neoplasm; Signal Transduction; STAT Transcription Factors; Transforming Growth Factor beta

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.

Topics: Breast Neoplasms; Cell Cycle Checkpoints; Female; Humans; Neoplasm Metastasis; Phosphorylation; Signal Transduction; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta

It is increasingly evident that the microenvironment of bone can influence the cancer phenotype in many ways that favor growth in bone. The ability of cancer cells to adhere to bone matrix and to promote osteoclast formation are key requirements for the establishment and growth of bone metastases. Several cytokine products of breast cancers (e.g. PTHrP, IL-11, IL-8) have been shown to act upon host cells of the bone microenvironment to promote osteoclast formation, allowing for excessive bone resorption. The increased release of matrix-derived growth factors, especially TGF-β, acts back upon the tumor to facilitate further tumor expansion and enhance cytokine production, and also upon osteoblasts to suppress bone formation. This provides a self-perpetuating cycle of bone loss and tumor growth within the skeleton. Other contributing factors favoring tumor metastasis and colonization in bone include the unique structure and stiffness of skeletal tissue, along with the diverse cellular composition of the marrow environment (e.g. bone cells, stromal fibroblasts, immune cells), any of which can contribute to the phenotypic changes that can take place in metastatic deposits that favor their survival. Additionally, it is also apparent that breast cancer cells begin to express different bone specific proteins as well as proteins important for normal breast development and lactation that allow them to grow in bone and stimulate bone destruction. Taken together, these continually emerging areas of study suggest new potential pathways important in the pathogenesis of bone metastasis and potential areas for targeting therapeutics.

Topics: Bone and Bones; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cytokines; Female; Humans; Interleukin-11; Interleukin-8; Neoplasms; Osteoblasts; Osteoclasts; Osteogenesis; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta

Estrogen receptor-α (ERα) and transforming growth factor (TGF)-β signaling pathways are major regulators during mammary gland development, function and tumorigenesis. Predominantly, they have opposing roles in proliferation and apoptosis. While ERα signaling supports growth and differentiation and is antiapoptotic, mammary gland epithelia cells are very sensitive to TGF-β-induced cell cycle arrest and apoptosis. Their regulatory pathways intersect, and ERα blocks TGF-β pathway by multiple means, including direct interactions of its signaling components, Smads. However, relatively little is known of the dysfunction of their interactions in cancer. A better understanding would help to develop new strategies for breast cancer treatment.

Topics: Animals; Breast Neoplasms; Estrogen Receptor alpha; Female; Humans; Signal Transduction; Transforming Growth Factor beta

Wnt5a is a member of the Wingless-related/MMTV-integration family of secreted growth factors, which are involved in a wide range of cellular processes. Wnt signaling can be broadly divided into two categories the canonical, ß-catenin-dependent pathway and the non-canonical ß-catenin-independent pathway. Wnt5a is a non-canonical signaling member of the Wnt family. Loss of Wnt5a is associated with early relapse of invasive breast cancer, increased metastasis, and poor survival in humans. It has been shown that TGF-ß directly regulates expression of Wnt5a in mammary gland and that Wnt5a mediates the effects of TGF-ß on branching during mammary gland development. Here we review the evidence suggesting Wnt5a acts as an effector of TGF-ß actions in breast cancer. It is suggested that the tumor suppressive functions of TGF-ß involve Wnt5a-mediated antagonism of Wnt/ß-catenin signaling and limiting the stem cell population. Interactions between TGF-ß and Wnt5a in metastasis appear to be more complex, and may depend on specific cues from the microenvironment as well as activation of specific intracellular signaling pathways.

Topics: Animals; Breast Neoplasms; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Proto-Oncogene Proteins; Transforming Growth Factor beta; Wnt Proteins; Wnt-5a Protein

Tumors such as breast, lung, and prostate frequently metastasize to bone, where they can cause intractable pain and increase the risk of fracture in patients. When tumor cells metastasize to bone, they interact with the microenvironment to promote bone destruction primarily through the secretion of osteolytic factors by the tumor cells and the subsequent release of growth factors from the bone. Our recent data suggest that the differential rigidity of the mineralized bone microenvironment relative to that of soft tissue regulates the expression of osteolytic factors by the tumor cells. The concept that matrix rigidity regulates tumor growth is well established in solid breast tumors, where increased rigidity stimulates tumor cell invasion and metastasis. Our studies have indicated that a transforming growth factor-β (TGF-β) and Rho-associated kinase (ROCK)-dependent mechanism is involved in the response of metastatic tumor cells to the rigid mineralized bone matrix. In this review, we will discuss the interactions between ROCK and TGF-β signaling, as well as potential new therapies that target these pathways.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Denosumab; Diphosphonates; Female; Humans; Kruppel-Like Transcription Factors; Male; Nuclear Proteins; Parathyroid Hormone-Related Protein; RANK Ligand; rho-Associated Kinases; Transforming Growth Factor beta; Zinc Finger Protein Gli2

Breast cancer is a heterogeneous disease comprised of at least five major tumor subtypes that coalesce as the second leading cause of cancer death in women in the United States. Although metastasis clearly represents the most lethal characteristic of breast cancer, our understanding of the molecular mechanisms that govern this event remains inadequate. Clinically, ~30% of breast cancer patients diagnosed with early-stage disease undergo metastatic progression, an event that (a) severely limits treatment options, (b) typically results in chemoresistance and low response rates, and (c) greatly contributes to aggressive relapses and dismal survival rates. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that regulates all phases of postnatal mammary gland development, including branching morphogenesis, lactation, and involution. TGF-β also plays a prominent role in suppressing mammary tumorigenesis by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Genetic and epigenetic events that transpire during mammary tumorigenesis conspire to circumvent the tumor suppressing activities of TGF-β, thereby permitting late-stage breast cancer cells to acquire invasive and metastatic phenotypes in response to TGF-β. Metastatic progression stimulated by TGF-β also relies on its ability to induce epithelial-mesenchymal transition (EMT) and the expansion of chemoresistant breast cancer stem cells. Precisely how this metamorphosis in TGF-β function comes about remains incompletely understood; however, recent findings indicate that the initiation of oncogenic TGF-β activity is contingent upon imbalances between its canonical and noncanonical signaling systems. Here we review the molecular and cellular contributions of noncanonical TGF-β effectors to mammary tumorigenesis and metastatic progression.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Disease Progression; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Signal Transduction; Transforming Growth Factor beta

TGFβ contributes to mammary gland development and has paradoxical roles in breast cancer because it has both tumor suppressor and tumor promoter activity. Another member of the TGFβ superfamily, activin, also has roles in the developing mammary gland, but these functions, and the role of activin in breast cancer, are not well characterized. TGFβ and activin share the same intracellular signaling pathways, but divergence in their signaling pathways are suggested. The purpose of this review is to compare the spatial and temporal expression of TGFβ and activin during mammary gland development, with consideration given to their functions during each developmental period. We also review the contributions of TGFβ and activin to breast cancer resistance and susceptibility. Finally, we consider the systemic contributions of activin in regulating obesity and diabetes; and the impact this regulation has on breast cancer. Elevated levels of activin in serum during pregnancy and its influence on pregnancy associated breast cancer are also considered. We conclude that evidence demonstrates that activin has tumor suppressing potential, without definitive indication of tumor promoting activity in the mammary gland, making it a good target for development of therapeutics.

Topics: Activins; Animals; Breast Neoplasms; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Signal Transduction; Transforming Growth Factor beta

Contrary to the long believed hypothesis, it is now evident that breast cancer cells can disseminate from the early phases of the oncogenesis; and that such early disseminated cells sometimes survive at the sites of dissemination and may outgrow after a long latency of years and decades. For cancer cells to leave their origin, they must at least transiently loosen their adhesion with adjacent epithelial cells and stroma, and become motile while avoiding anoikis. Such processes resemble epithelial-mesenchymal transdifferentiation (EMT), which normally takes place in situations such as embryogenesis and wound healing. Interestingly, the occurrence of an EMT-like process in breast cancer cells has been implicated in the generation of cancer stem-like cells, in which TGFβ1 signalling often plays core roles. Here, I discuss the current knowledge regarding cancerous EMT and its signalling pathways with the aim to consider the possible mechanisms of early dissemination, and also the generation of cancer stem-like cells in mammary tumour.

Topics: Animals; Breast Neoplasms; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Female; Humans; Signal Transduction; Transforming Growth Factor beta

The contribution of transforming growth factor β (TGF-β) signaling to breast cancer has been studied for more than two decades. In an early phase TGF-β may act as a tumour suppressor, while later, when cells have become resistant to its anti-mitogenic effects, the role of TGF-β switches towards malignant conversion and progression. TGF-β stimulates cell invasion and modifies the microenvironment to the advantage of cancer cells. Studies have shown that TGF-β promotes bone and lung metastasis via different mechanisms. The therapeutic strategies to target the TGF-β pathway in breast cancer are becoming increasingly clear. This review will focus on the role TGF-β in breast cancer invasion and metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Female; Humans; Neoplasm Invasiveness; Transforming Growth Factor beta

Despite its tumor suppressive role in normal mammary epithelial cells, TGFβ has been reported to promote the migration, invasion and survival in breast cancer cells overexpressing the HER2 (ERBB2; neu) oncogene, and to accelerate the metastasis of neu-induced mammary tumors in mice. A clearer understanding of the molecular mechanisms underlying the crosstalk between TGFβ and HER2 has started to emerge. In recent studies reviewed here, the synergistic effect of TGFβ and HER2 on tumor progression has been shown to likely be a combined result of two distinct features: (1) loss of TGFβ's tumor suppressive effect through functional alterations in the anti-mitogenic effect of Smad-mediated transcription, and (2) gain of pro-survival and pro-migratory function through HER2-dependent mechanisms. In HER2-overexpressing breast cancer, this crosstalk results in increased cancer cell proliferation, survival and invasion, accelerated metastasis in animal models, and resistance to chemotherapy and HER2-targeted therapy. Thus, the transformed cellular context imparted by constitutively active HER2 signaling, as a consequence of HER2 gene amplification or overexpression, aborts the tumor suppressive role of TGFβ and facilitated the oncogenic role of this pathway. In turn, TGFβ potentiates oncogenic HER2 signaling by inducing shedding of the ERBB ligands and clustering of HER2 with integrins. Here we discuss recent studies examining Smad-dependent and -independent mechanisms of crosstalk between TGFβ and HER2. Therefore, blockade of TGFβ:HER2 crosstalk may suppress breast cancer progression and metastasis, and enhance the efficiency of conventional therapies in patients with HER2-overexpressing breast cancer.

Topics: Animals; Breast Neoplasms; Female; Genes, erbB-2; Humans; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta

Many advanced tumors produce excess amounts of Transforming Growth Factor-β (TGF-β), which is a potent growth inhibitor of normal epithelial cells. However, in tumors its homeostatic action on cells can be diverted along several alternative pathways. Thus, TGF-β signaling has been reported to elicit a preventative or tumor suppressive effect during the earlier stages of tumorigenesis, but later in tumor development, when carcinoma cells become refractory to TGF-β-mediated growth inhibition, response to TGF-β signaling elicits predominantly tumor progressing effects. This is not a simple switch from suppression to progression, but more like a rheostat, involving multiple complementary and antagonizing activities that slowly tip the balance from one to the other. This review will focus on the multiple activities of TGF-β in regulation of two epithelial tumor types, namely squamous cell carcinoma and breast cancer. Basic findings in current mouse models of cancer are presented, as well as a discussion of the complicating issue of outcome of altered TGFβ signaling depending on genetic variability between mouse strains. This review also discusses the role TGF-β within the tumor microenvironment particularly its ability to polarize the microenvironment towards a pro-tumorigenic milieu.

Topics: Animals; Breast Neoplasms; Carcinoma, Squamous Cell; Disease Models, Animal; Female; Humans; Mutation; Smad Proteins; Transforming Growth Factor beta

Induction of epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSCs) can occur as the result of embryonic pathway signaling. Activation of Hedgehog (Hh), Wnt, Notch, or transforming growth factor-β leads to the upregulation of a group of transcriptional factors that drive EMT. This process leads to the transformation of adhesive, non-mobile, epithelial-like tumor cells into cells with a mobile, invasive phenotype. CSCs and the EMT process are currently being investigated for the role they play in driving metastatic tumor formation in breast cancer. Both are very closely associated with embryonic signaling pathways that stimulate self-renewal properties of CSCs and EMT-inducing transcription factors. Understanding these mechanisms and embryonic signaling pathways may lead to new opportunities for developing therapeutic agents to help prevent metastasis in breast cancer. In this review, we examine embryonic signaling pathways, CSCs, and factors affecting EMT.

Topics: Animals; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Hedgehog Proteins; Humans; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

The Hedgehog (HH) and TGF-β signaling pathways represent essential regulators of cell proliferation and differentiation during embryogenesis. Pathway deregulation is a characteristic of various cancers. Recently, evidence for a convergence of these pathways at the level of the GLI2 transcription factor in the context of tumor initiation and progression to metastasis has emerged. This short review summarizes recent knowledge about GLI2 function and mechanisms of action downstream of TGF-β in cancer.

Topics: Bone Neoplasms; Breast Neoplasms; Disease Progression; Female; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Melanoma; Neoplasm Metastasis; Neoplasms; Nuclear Proteins; Signal Transduction; Skin Neoplasms; Smad Proteins; Transforming Growth Factor beta; Zinc Finger Protein Gli2

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to inhibit mammary tumorigenesis by directly inducing mammary epithelial cells (MECs) to undergo cell cycle arrest or apoptosis, and to secrete a variety of cytokines, growth factors, and extracellular matrix proteins that maintain cell and tissue homeostasis. Genetic and epigenetic events that transpire during mammary tumorigenesis typically inactivate the tumor suppressing activities of TGF-beta and ultimately confer this cytokine with tumor promoting activities, including the ability to stimulate breast cancer invasion, metastasis, angiogenesis, and evasion from the immune system. This dramatic conversion in TGF-beta function is known as the "TGF-beta paradox" and reflects a variety of dynamic alterations that occur not only within the developing mammary carcinoma, but also within the cellular and structural composition of its accompanying tumor microenvironment. Recent studies have begun to elucidate the critical importance of mammary tumor microenvironments in manifesting the TGF-beta paradox and influencing the response of developing mammary carcinomas to TGF-beta. Here we highlight recent findings demonstrating the essential function of tumor microenvironments in regulating the oncogenic activities of TGF-beta and its stimulation of metastatic progression during mammary tumorigenesis.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Endothelial Cells; Epithelial Cells; Female; Fibroblasts; Humans; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Bone, as well as liver and lung, is one of the most preferential metastatic target sites for cancers including breast, prostate, and lung cancers and the consequences are always devastating. Like other metastasis, breast cancer bone metastasis consists of several steps from the escape of primary site to the colonization in target site. This review focuses on several key steps including: 1. Invasion and escape from primary tumor site. 2. Target migration toward bone. 3. Specific adhesion and arrest in bone. 4. Establishment of metastasis in bone. The factors involved in this process will provide good targets for therapy.

Topics: Bone Neoplasms; Breast Neoplasms; Cadherins; Cell Adhesion; Female; Humans; Insulin-Like Growth Factor I; Neoplasm Invasiveness; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is an essential process that drives polarized, immotile mammary epithelial cells (MECs) to acquire apolar, highly migratory fibroblastoid-like features. EMT is an indispensable process that is associated with normal tissue development and organogenesis, as well as with tissue remodeling and wound healing. In stark contrast, inappropriate reactivation of EMT readily contributes to the development of a variety of human pathologies, particularly those associated with tissue fibrosis and cancer cell invasion and metastasis, including that by breast cancer cells. Although metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby EMT mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis, and EMT. In addition, TGF-beta also functions as a powerful tumor suppressor in MECs, whose neoplastic development ultimately converts TGF-beta into an oncogenic cytokine in aggressive late-stage mammary tumors. Recent findings have implicated the process of EMT in mediating the functional conversion of TGF-beta during breast cancer progression, suggesting that the chemotherapeutic targeting of EMT induced by TGF-beta may offer new inroads in ameliorating metastatic disease in breast cancer patients. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of TGF-beta during its regulation of EMT in normal and malignant MECs.

Topics: Animals; Breast Neoplasms; Cell Dedifferentiation; Cell Differentiation; Cell Transdifferentiation; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Mesenchymal Stem Cells; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Transforming Growth Factor beta (TGFbeta) signaling influences most aspects of cellular function in addition to playing a major role in organ development, remodeling, and repair. Given the wide range of effects induced by TGFbeta, it is not surprising that alterations in TGFbeta signaling have been implicated in development and progression of many different cancer types. Within the context of breast cancer itself, TGFbeta is known to have a dual nature, being both tumor-suppressive during early breast cancer development and tumor-promoting during breast cancer metastasis. Targets for breast cancer therapeutics are greatly needed to decrease morbidity and mortality from this devastating disease. Here, we summarize what is known about TGFbeta in breast cancer progression and discuss potential TGFbeta targets for breast cancer therapeutics.

Topics: Animals; Breast Neoplasms; Disease Progression; Female; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Models, Animal; Molecular Targeted Therapy; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Stem Cells; Transforming Growth Factor beta

Bone is the most common metastatic site for breast cancer, and bone metastases can cause pain as well as risk of pathological fractures. Emerging treatments for metastatic bone disease have arisen from advances in our understanding of the unique cellular and molecular mechanisms that contribute to bone metastasis. The interaction between tumor cells and the bone microenvironment results in a 'vicious cycle' that increases both bone destruction and tumor burden. The tumor secretes factors, such as parathyroid hormone-related peptide, that stimulate osteoclastogenesis. Similarly, the bone stroma produces growth factors, such as transforming growth factor β, that promote tumor growth in bone. Therapeutic targeting of these microenvironmental factors is under intensive investigation. Other attractive therapeutic targets include signaling molecules, such as receptor activator of nuclear factor κB ligand, Src kinase, and cathepsin K, all of which regulate osteoclast function, and chemokine receptor 4, which is involved in the homing of tumor cells to bone. In this Review, we describe the progress and future directions of novel bone-targeted therapies that may reduce or prevent destructive bone metastasis from breast cancer. Novel modalities for predicting and monitoring treatment response will also be described.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cathepsin K; Chemoreceptor Cells; Denosumab; Diphosphonates; Female; Humans; Imidazoles; Ligands; NF-kappa B; Parathyroid Hormone; RANK Ligand; src-Family Kinases; Transforming Growth Factor beta; Zoledronic Acid

In most human breast cancers, lowering of TGFbeta receptor- or Smad gene expression combined with increased levels of TGFbetas in the tumor microenvironment is sufficient to abrogate TGFbetas tumor suppressive effects and to induce a mesenchymal, motile and invasive phenotype. In genetic mouse models, TGFbeta signaling suppresses de novo mammary cancer formation but promotes metastasis of tumors that have broken through TGFbeta tumor suppression. In mouse models of "triple-negative" or basal-like breast cancer, treatment with TGFbeta neutralizing antibodies or receptor kinase inhibitors strongly inhibits development of lung- and bone metastases. These TGFbeta antagonists do not significantly affect tumor cell proliferation or apoptosis. Rather, they de-repress anti-tumor immunity, inhibit angiogenesis and reverse the mesenchymal, motile, invasive phenotype characteristic of basal-like and HER2-positive breast cancer cells. Patterns of TGFbeta target genes upregulation in human breast cancers suggest that TGFbeta may drive tumor progression in estrogen-independent cancer, while it mediates a suppressive host cell response in estrogen-dependent luminal cancers. In addition, TGFbeta appears to play a key role in maintaining the mammary epithelial (cancer) stem cell pool, in part by inducing a mesenchymal phenotype, while differentiated, estrogen receptor-positive, luminal cells are unresponsive to TGFbeta because the TGFBR2 receptor gene is transcriptionally silent. These same cells respond to estrogen by downregulating TGFbeta, while antiestrogens act by upregulating TGFbeta. This model predicts that inhibiting TGFbeta signaling should drive the differentiation of mammary stem cells into ductal cells. Consequently, TGFbeta antagonists may convert basal-like or HER2-positive cancers to a more epithelioid, non-proliferating (and, perhaps, non-metastatic) phenotype. Conversely, these agents might antagonize the therapeutic effects of anti-estrogens in estrogen-dependent luminal cancers. These predictions need to be addressed prospectively in clinical trials and should inform the selection of patient populations most likely to benefit from this novel anti-metastatic therapeutic approach.

Topics: Animals; Breast Neoplasms; Humans; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

The TGFbeta signaling pathway is conserved from flies to humans and has been shown to regulate such diverse processes as cell proliferation, differentiation, motility, adhesion, organization, and programmed cell death. Both in vitro and in vivo experiments suggest that TGFbeta can utilize these varied programs to promote cancer metastasis through its effects on the tumor microenvironment, enhanced invasive properties, and inhibition of immune cell function. Recent clinical evidence demonstrating a link between TGFbeta signaling and cancer progression is fostering interest in this signaling pathway as a therapeutic target. Anti-TGFbeta therapies are currently being developed and tested in pre-clinical studies. However, targeting TGFbeta carries a substantial risk as this pathway is implicated in multiple homeostatic processes and is also known to have tumor-suppressor functions. Additionally, clinical and experimental results show that TGFbeta has diverse and often conflicting roles in tumor progression even within the same tumor types. The development of TGFbeta inhibitors for clinical use will require a deeper understanding of TGFbeta signaling, its consequences, and the contexts in which it acts.

Topics: Animals; Breast Neoplasms; Disease Models, Animal; Humans; Mice; Neoplasm Metastasis; Prognosis; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor (TGF)-beta signaling pathway plays a critical role in breast cancer development and progression. Limited data from human studies, however, are currently available to link biomarkers in this pathway directly to the risk and survival of breast cancer. Most of the previous epidemiologic studies have focused on evaluating polymorphisms in the TGFB1 gene (T+29C, rs1982073) and the TGFBR1 gene (9A/6A), and the results have been inconsistent. The present review summarizes epidemiologic evidence regarding the association of genetic polymorphisms in the TGF beta pathway genes with breast cancer risk and survival and provides rationale and new approaches to continuing the research in this area.

Topics: Breast Neoplasms; Female; Humans; Polymorphism, Genetic; Risk Factors; Signal Transduction; Survival Rate; Transforming Growth Factor beta

Endocrine therapy is often given together with postoperative radiotherapy in patients with breast cancer and positive hormone-receptor status. However, few experimental or clinical studies address the combined effects of hormone and radiation therapy. Preclinical models have shown changes in tumour cell kinetics with the addition of tamoxifen, and some show reduced tumour cell death with concurrent anti-oestrogen treatment and radiotherapy. Although data from in-vitro studies support the notion of antagonistic effects of concurrent tamoxifen and radiotherapy on tumour cells, in-vivo research suggests a synergistic effect that could be attributable to micro-environmental changes in tumour responsiveness to ionising radiation and hormone therapy. Retrospective studies suggest that in practical application, concurrent administration of tamoxifen with radiotherapy does not compromise local control but might increase toxicity. Preliminary results from simultaneous treatment with aromatase inhibitors and radiation indicate that this combination of endocrine and radiation therapy could enhance cytotoxicity and improve tumour response. Further studies are needed to clarify the physiological mechanisms activated by oestrogens, which will allow a more thorough understanding of the complex interactions between 17beta-oestradiol and P53/P21(WAF1/CIP1)/Rb pathways and of the interaction between endocrine therapy and radiotherapy.

Topics: Aromatase Inhibitors; Breast Neoplasms; Cell Cycle; Combined Modality Therapy; Estradiol; Female; Humans; Radiation Tolerance; Selective Estrogen Receptor Modulators; Tamoxifen; Transforming Growth Factor beta

The transforming growth factor beta (TGFbeta) is a potent regulator of tumor initiation, progression and metastasis. It has been known for many years that TGFbeta signaling in the carcinoma cell can suppress or promote tumor progression depending on the context of stimulation. While the impact of TGFbeta on the carcinoma cell is significant, it is now generally accepted that primary and metastatic carcinoma progression is regulated by an intricate network of host-tumor cell interactions. Interestingly, recent results have revealed that gain or loss of TGFbeta signaling in carcinoma cells can promote metastasis through carcinoma cell derived TGFbeta dependent host-tumor cell interactions in vivo. Further, gain or complete abrogation of TGFbeta signaling was shown to result in gene expression signatures that correlated with poor patient prognosis in breast cancer. Specifically, the TGFbeta responsive gene expression signature correlated with poor prognosis for estrogen receptor negative (ER(-)) breast cancer while complete abrogation of TGFbeta signaling resulted in a correlation with poor outcome in lymph node positive (LN(+)) and ER(+) breast cancers. Importantly, in both cases the correlation with poor prognosis was linked to carcinoma cell derived interactions with the adjacent microenvironment. Together the current results suggest that, in addition to intrinsic carcinoma cell signaling, TGFbeta dependent host-tumor cell interactions should be considered when designing therapeutic strategies to manage carcinoma progression.

Topics: Breast Neoplasms; Carcinoma; Chemokines; Disease Progression; Female; Humans; Receptors, Estrogen; Signal Transduction; Transforming Growth Factor beta

Breast cancer most frequently causes bone metastases in solid tumors. It has been known that there is a vicious cycle consisting of tumor cells, osteoblasts, osteoclasts and various humoral factors in osteolytic lesions. Although systemic therapy is a main treatment of bone metastases, local therapies, such as radiotherapy and surgical therapy, are also promptly needed when bone-related complications occur. In recent years, anti-osteoclast agents, bisphosphonates significantly contribute to the delay of occurrence of bone-related complications. Postoperative adjuvant therapy significantly reduces the incidence of recurrence in breast cancer patients. Chemotherapy and LH-RH agonists cause ovarian function suppression in premenopausal patients, and aromatase inhibitors cause estrogen deprivation in postmenopausal patients. These effects cause unbalance of bone metabolism, loss of bone density and increase in the incidence of fractures. Improvement of these bone-related adverse effects and careful follow-ups are needed for breast cancer patients.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Chemotherapy, Adjuvant; Diphosphonates; Female; Humans; Medroxyprogesterone Acetate; Osteolysis; Parathyroid Hormone-Related Protein; RANK Ligand; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta is a potent growth inhibitor of various types of cells, whereas it stimulates deposition of extracellular matrix proteins and induction of epithelial-to-mesenchymal transition (EMT). TGF-beta thus plays two distinct and opposing roles in cancer progression. In the present study, we have developed a useful method that enables monitoring of tumor and its TGF-beta signaling within the same animal using in vivo bioluminescent imaging.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Diagnostic Imaging; Female; Humans; Luciferases; Luminescent Proteins; Mice; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that regulates cell growth, differentiation, and extracellular matrix formation. Studies on genetically engineered animal models have demonstrated that TGF-beta-mediated signaling pathway plays a critical role in both normal development and tumorigenesis of the breast. In pathogenesis of breast cancer, the role of TGF-beta appears featured with growth-inhibitory effects at early stages of carcinogenesis, but aggressive oncogenesis with transition to more advanced malignant states. The TGF-beta signaling pathway is also tissue-context and ligand content-dependent. Therein, therapeutic modulation of TGF-beta signaling may be a multifactorial event.

Topics: Breast; Breast Neoplasms; Female; Humans; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Although the role of Wnt signalling in breast cancer is far from being fully understood, in the last years its importance has been reported frequently. Besides stimulation by canonical Wnt signalling, the downstream effectors beta-catenin and the transcriptional modulators of the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family can also be activated by other inputs including the TGF-beta pathway. Wnt and TGF-beta signalling are both major signal transduction pathways, which provide important cues during development and tumor progression. However, particularly TGF-beta has a complicated influence on oncogenesis, which ranges from suppressive to promoting activity. Signalling pathways activated in parallel with TGF-beta might determine the oncogenic influence, and therefore place signals cooperating with TGF-beta into the limelight. During early development Wnt and TGF-beta signalling collaborate extensively. Here we provide an overview of the known interactions of Wnt with TGF-beta signalling in development and metastasis, particularly in breast cancer. We want to focus on the Wnt-activated transcription factor complex beta-catenin/LEF-1, its upstream activators, its downstream targets and consequences on the cellular level in response to beta-catenin/LEF-1 activation.

Topics: Animals; beta Catenin; Breast Neoplasms; Female; Humans; Lymphoid Enhancer-Binding Factor 1; Models, Biological; Signal Transduction; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins

Transforming growth factor-beta (TGF-beta) belongs to a family of multifunctional growth factors that participates in the regulation of a variety of cellular activities. Beside induction of growth inhibition and differentiation of epithelial cells, TGF-beta has been shown to promote epithelial-mesenchymal transition in most epithelial tumors. While inhibition of epithelial cell proliferation in response to TGF-beta is mainly mediated by the well-characterized Smad pathway and subsequent inhibition of gene transcription, the molecular mechanism leading to TGF-beta-induced invasiveness and metastasis of epithelial tumors is less clear. Recent results from several groups suggest that the induction of tumorigenic activity by TGF-beta includes not only signaling by Smads, but also by Rho-GTPases and mitogen-activated protein kinases (MAP kinases). Activation of the MAP kinases extracellular signal-regulated kinases (ERK) 1 and 2 as well as c-jun N-terminal kinase (JNK) has been identified as important steps in TGF-beta-induced, Smad4-independent signal transduction in epithelial cells. Recent results identify a role of activated ERK and JNK and their association with focal complexes in TGF-beta-induced, Smad4-independent cell migration of breast carcinoma cells, and are reviewed here.

Topics: Breast Neoplasms; Cell Line, Tumor; Enzyme Activation; Epithelial Cells; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Metastasis is a final stage of tumor progression. Breast and prostate cancer cells preferentially metastasize to bone, wherein they cause incurable osteolytic and osteoblastic lesions. The bone matrix is rich in factors, such as transforming growth factor-beta and insulin-like growth factors, which are released into the tumor microenvironment by osteolysis. These factors stimulate the growth of tumor cells and alter their phenotype, thus promoting a vicious cycle of metastasis and bone pathology. Physical factors within the bone microenvironment, including low oxygen levels, acidic pH, and high extracellular calcium concentrations, may also enhance tumor growth. These elements of the microenvironment are potential targets for chemotherapeutic intervention to halt tumor growth and suppress bone metastasis.

Topics: Bone Neoplasms; Breast Neoplasms; Female; Humans; Hydrogen-Ion Concentration; Male; Molecular Biology; Osteolysis; Prostatic Neoplasms; Somatomedins; Transforming Growth Factor beta

During its lifetime, the mammary gland undergoes many phases of development and differentiation. Much of this occurs during puberty, when the ductal epithelium expands by branching morphogenesis, invading the surrounding fat pad to form an organised mammary tree. Throughout its existence, the epithelium will go through several cycles of proliferation and cell death during pregnancy, lactation and involution. Many of the signalling mechanisms which control the initial invasion of the fat pad by the epithelium, and regulate its continuing plasticity, can be harnessed or corrupted by tumour cells in order to support their aberrant growth and progression towards invasion. This is true not just for the epithelial cells themselves but also for cells in the surrounding microenvironment, including fibroblasts, macrophages and adipocytes. This review examines the complex web of signalling and adhesion interactions controlling branching morphogenesis, and how their alteration can promote malignancy. Current in vivo and in vitro mammary gland models are also discussed. (Part of a Multi-author Review).

Topics: Animals; Breast Neoplasms; Cell Adhesion; Epidermal Growth Factor; Humans; Mammary Glands, Animal; Mammary Glands, Human; Matrix Metalloproteinases; Models, Biological; Neoplasm Invasiveness; Oncogene Proteins v-erbB; Stem Cells; Transcription Factors; Transforming Growth Factor beta

Skeletal-related complications occur commonly in many solid tumors including breast, prostate and lung cancer as well as multiple myeloma. In addition, malignancies and their associated treatment may result in bone loss or osteoporosis. This review will focus solely on recent data associated with metastatic bone disease with a focus on breast cancer, prostate cancer and multiple myeloma. Bone loss or osteoporosis associated with cancer will be covered in a separate article in this issue.. Recent progress in understanding the pathophysiology of bone metastases has pointed to several novel pathways: transforming growth factor beta; receptor activator of nuclear factor beta ligand and osteoprotegerin; and Wnt signaling pathways and associated factors such as dickkopf-1 and endothelin-1.. The identification of new pathways is important in metastatic bone disease from cancer and has allowed for the development of novel therapeutics aimed at preventing the devastating complications of bone metastases. Bisphosphonates remain the predominant therapy in use for the treatment and prevention of skeletal-related adverse effects from cancer.

Topics: Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Endothelin-1; Female; Humans; Intercellular Signaling Peptides and Proteins; Male; Multiple Myeloma; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Humans; Mice; Models, Biological; Precancerous Conditions; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

Topics: Breast Neoplasms; Carrier Proteins; DNA-Binding Proteins; ErbB Receptors; Female; Humans; Neoplasm Invasiveness; Receptors, Estrogen; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

The antiestrogen tamoxifen is one of the most successful drugs in the endocrine treatment of breast cancer and significantly reduces the risk of recurrence and death. Antiestrogens act by inhibiting the production of growth-stimulatory factors as well as by activating peptides with growth-inhibitory effects like transforming growth factor- beta (TGF-beta). In hormone-responsive breast cancer cells treatment with antiestrogens leads to the conversion of TGF-beta1 into a biologically active form. Expression of TGF-beta2 and TGF-beta receptor (TbetaR) II is induced via a transcriptional mechanism involving p38 MAP kinase. Inhibition of p38 abolishes antiestrogen-dependent growth inhibition. However, the role of TGF-beta in breast cancer progression is ambiguous, as it was shown to display both tumor-suppressing and -enhancing effects. A polymorphism in the promoter of TGF-beta2 that enhances expression of the protein was associated with lymph node metastasis in breast cancer patients, pointing to a role of TGF-beta2 in the process of invasion. An immunohistochemical study on TbetaRI and TbetaRII expression in breast cancer tissues indicates that the estrogen receptor (ER) status of a tumor is an important marker and a potential mediator of the transition of TGF-beta from tumor suppressor to tumor promoter. In ER-negative tumors, expression of TbetaRII was associated with a subset of tumors that appeared to be highly aggressive, leading to strongly reduced overall survival times. Further characterization of the influence of ER expression on TGF-beta signal transduction shows that ER-alpha plays a crucial role in TGF-beta signaling.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Estrogen Receptor Modulators; Female; Humans; Signal Transduction; Transforming Growth Factor beta

Although the stroma in which carcinomas arise has been previously regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of tumor cells, it is now generally acknowledged that stromal changes are required for the establishment of cancer. In the present article, we discuss three recent publications that highlight the complex role the stroma has during the development of cancer and the potential for targeting the stroma by therapeutic approaches.

Topics: Animals; Breast Neoplasms; Cell Communication; Cell Transformation, Neoplastic; Epithelial Cells; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mice; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

It is generally accepted that transforming growth factor beta (TGFbeta) is both a tumor suppressor and tumor promoter. Whereas loss or attenuation of TGFbeta signal transduction is permissive for transformation, introduction of dominant-negative TGFbeta receptors into metastatic breast cancer cells has been shown to inhibit epithelial-to-mesenchymal transition, motility, invasiveness, survival, and metastases. In addition, there is evidence that excess production and/or activation of TGFbeta by cancer cells can contribute to tumor progression by paracrine mechanisms involving neoangiogenesis, production of stroma and proteases, and subversion of immune surveillance mechanisms in tumor hosts. These data provide a rationale in favor of blockade of autocrine/paracrine TGFbeta signaling in human mammary tumors with therapeutic intent. Several treatment approaches are currently in early clinical development and have been the focus of our laboratory. These include (1) ligand antibodies or receptor-containing fusion proteins aimed at blocking ligand binding to cognate receptors and (2) small-molecule inhibitors of the type I TGFbeta receptor serine/threonine kinase. Many questions remain about the viability of anti-TGFbeta treatment strategies, the best molecular approach (or combinations) for inhibition of TGFbeta function in vivo, the biochemical surrogate markers of tumor response, the molecular profiles in tumors for selection into clinical trials, and potential toxicities, among others.

Topics: Animals; Breast Neoplasms; Female; Humans; Transforming Growth Factor beta

The most common skeletal complication of breast cancer is osteolytic bone metastasis. Bone metastases are present in 80% of patients with advanced disease and cause significant morbidity. They are most often osteolytic, but can be osteoblastic or mixed. Tumor cells, osteoblasts, osteoclasts and bone matrix are the four components of a vicious cycle necessary for the initiation and development of bone metastases. Tumor cell gene expression is modified by interaction with bone-derived factors. For example, parathyroid hormone related protein (PTHrP), a tumor cell factor, is upregulated by bone-derived transforming growth factor beta (TGFbeta). Tumor cell factors, in turn, act upon bone cells to cause dysregulated bone destruction and formation. PTHrP increases osteoblast expression of RANK (receptor activator of NFkappaB) ligand which, in turn, activates osteoclasts. PTHrP-independent osteolytic factors, such as interleukin [IL]-11 and IL-8, also contribute to the vicious cycle. Other tumor-bone interactions, such as stimulation of tumor-homing through the CXCR4 chemokine receptor by its bone-derived ligand stromal-derived factor-1 (SDF-1), may be responsible for the site-specific predilection of breast cancer for bone. These factors and their roles in fueling the vicious cycle may identify novel targets for therapies to prevent metastasis.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Female; Humans; Osteolysis; Parathyroid Hormone-Related Protein; Signal Transduction; Trans-Activators; Transforming Growth Factor beta

Regulatory T-cells (Treg) protect the host from autoimmune disease by suppressing self-reactive immune cells. As such, Treg may also block antitumor immune responses. Recent observations by us and others showed that the prevalence of Treg is increased in cancer patients, particularly in the tumor environment. Our studies in a mouse pancreas cancer model suggest that the tumor actively promotes the accrual of Treg through several mechanisms involving activation of naturally occurring Treg as well as conversion of non-Treg into Treg. Our studies focus on further defining these mechanisms with the ultimate goal of designing strategies that block Treg-mediated suppression in cancer patients.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cancer Vaccines; Carcinoma, Ductal, Breast; CD4 Antigens; Dendritic Cells; Female; Forkhead Transcription Factors; Humans; Immune Tolerance; Lymphocyte Depletion; Mice; Mice, Knockout; Neoplasms; Pancreatic Neoplasms; Phenotype; Receptors, Interleukin-2; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Members of the transforming growth factor-beta (TGF-beta) family of ligands exhibit potent growth-suppressive and/or apoptosis-inducing effects on different types of cells. They perform essential roles in the elimination of damaged or abnormal cells from healthy tissues. On the other hand, TGF-betas have also been shown to act as tumor-promoting cytokines in a number of malignancies that are capable of stimulating extracellular matrix production, cell migration, invasion, angiogenesis, and immune suppression. Dissecting the complex, multifaceted roles of different TGF-beta-related peptides especially during the development of pathological conditions and in carcinogenesis is an area of continuous research and development. The characterization of EGF-CFC proteins as essential co-receptors that contribute to the modulation of the physiological activities of some of the TGF-beta ligands will be beneficial for future medical research and the adaptation and possible readjustment of currently applied therapeutic regimes.

Topics: Animals; Breast Neoplasms; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta

Topics: Breast Neoplasms; Female; Humans; Transforming Growth Factor beta

The complexity of mechanisms leading to the appearance and progression of cancer is a challenge being addressed by large-scale studies, such as proteomics. Simultaneous monitoring of thousands of proteins uncovers novel signaling mechanisms, thus revising our knowledge of tumorigenesis. Transforming growth factor (TGF)-beta is a secreted polypeptide that is known to inhibit tumor growth at the early stages of cancer, but promote metastasis at the later stages. Proteomics-based studies have significantly widened our knowledge of TGF-beta-dependent regulation of cell proliferation, apoptosis, DNA damage repair and transcription. This leads to better understanding of the TGF-beta role in human breast tumorigenesis, and opens the way for the development of novel anticancer treatments and drugs, with some of the drugs already entering clinics. This review discusses recent advances in proteomics studies of TGF-beta signaling and its contribution to the understanding and treatment of breast cancer.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Humans; Protein Binding; Proteomics; Signal Transduction; Transforming Growth Factor beta

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Topics: Activins; Animals; Anti-Mullerian Hormone; Antineoplastic Agents; Biomarkers, Tumor; Bone Morphogenetic Proteins; Breast Neoplasms; Female; Glycoproteins; Humans; Inhibins; Nodal Protein; Signal Transduction; Testicular Hormones; Transforming Growth Factor beta

Members of the TGFbeta superfamily and EGF-CFC family, such as Nodal and Cripto, are important mediators of anterior-posterior and left-right axis specification during embryogenesis. In this paper, we review the role of Nodal and Cripto as critical morphogen-like molecules, with an emphasis on Nodal and EGF-CFC signaling during embryonic pattern formation. New evidence from gene expression and transgenic mouse studies have shown that both Nodal and Cripto-1 are expressed within the mammary duct and that modulation of these genes can disrupt normal branching morphogenesis resulting in epithelial disorganization and defective ductal architecture. We describe these new findings and propose that Cripto and Nodal are candidate mammary morphogens. Finally, the data linking overexpression of Cripto and perturbations of Cripto signaling to cell transformation and tumor formation are discussed. The fact that Cripto can modulate multiple pathways suggests it may act to deregulate growth inhibitors/homeostasis factors early in the cell transformation process and then activate prosurvival pathways dependent on MAPK and PI3K/Akt later in fully transformed phenotypes.

Topics: Animals; Body Patterning; Breast; Breast Neoplasms; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Mammary Glands, Animal; Mammary Neoplasms, Animal; Membrane Glycoproteins; Neoplasm Proteins; Nodal Protein; Signal Transduction; Transforming Growth Factor beta

Interactions between bone marrow-derived cytokines, growth factors, and tumors play a critical role in both the homing of tumors to the bone and the development of bone metastasis. Bone is a storehouse of latent growth factors produced by stromal cells and osteoblasts that, when activated during osteoclastic bone resorption, can enhance the growth of tumor cells.. This article reviews the role these factors may play in bone metastasis.. Several studies have shown that breast carcinoma cells, which induce osteoclastic bone resorption, release growth factors that enhance tumor growth. In addition, bone-derived growth factors and chemokines, such as stromal cell-derived factor 1 and monocyte chemoattractant protein 1, can act as chemoattractants to attract tumor cells to bone. Finally, the interaction between tumor cells and bone marrow stromal cells can result in increased production of cytokines and growth factors, such as interleukin 6 or the ligand for the receptor activator of nuclear factor kappaB, that can enhance bone destruction, tumor growth, and angiogenesis.. Stromal cell-derived cytokines and growth factors as well as growth factors that are released during the bone resorption process play a critical role in the development of bone metastasis. Interruption of this symbiotic relation between tumors that induce bone destruction and release of bone-derived growth factors can have beneficial effects on blocking both bone destruction and decreasing tumor burden within bone.

Topics: Bone Marrow; Bone Neoplasms; Breast Neoplasms; Cytokines; Humans; Interleukin-6; Platelet-Derived Growth Factor; Receptors, Vascular Endothelial Growth Factor; Stromal Cells; Transforming Growth Factor beta

Osteolytic and osteoblastic metastases are often the cause of considerable morbidity in patients with advanced prostate and breast carcinoma. Breast carcinoma metastasis to bone occurs because bone provides a favorable site for aggressive behavior of metastatic cancer cells. A vicious cycle arises between cancer cells and the bone microenvironment, which is mediated by the production of growth factors such as transforming growth factor beta and insulin growth factor from bone and parathyroid hormone-related protein (PTHrP) produced by tumor cells. Osteolysis and tumor cell accumulation can be interrupted by inhibiting any of these limbs of the vicious cycle. For example, bisphosphonates (e.g., pamidronate, ibandronate, risedronate, clodronate, and zoledronate) inhibit both bone lesions and tumor cell burden in bone in experimental models of breast carcinomametastasis. Neutralizing antibodies to PTHrP, which inhibit PTHrP effects on osteoclastic bone resorption, also reduce osteolytic bone lesions and tumor burden in bone. Other pharmacologic approaches to inhibit PTHrP produced by breast carcinoma cells in the bone microenvironment also produce similar beneficial effects. Identification of the molecular mechanisms responsible for osteolytic metastases is crucial in designing effective therapy for this devastating complication.

Topics: Bone and Bones; Bone Neoplasms; Breast Neoplasms; Female; Humans; Osteoclasts; Parathyroid Hormone-Related Protein; Peptide Hormones; Transforming Growth Factor beta

The microenvironment in which cancer arises is often regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of the tumour. However, a major function of the microenvironment is to suppress cancer, and its disruption is required for the establishment of cancer. In addition, tumour cells can further distort the microenvironment to promote growth, recruit non-malignant cells that provide physiological resources, and facilitate invasion. In this review, the authors discuss the contribution of the microenvironment, i.e., the stroma and its resident vasculature, inflammatory cells, growth factors and the extracellular matrix (ECM), in the development of cancer, and focus on two components as potential therapeutic targets in breast cancer. First, the ECM, which imparts crucial signalling via integrins and other receptors, is a first-line barrier to invasion, modulates aggressive behaviour and may be manipulated to provide novel impediments to tumour growth. Second, the authors discuss the involvement of TGF-beta1 as an example of one of many growth factors that can regulate ECM composition and degradation and that play complex roles in cancer. Compared to the variable routes taken by cells to become cancers, the response of tissues to cancer is relatively consistent. Therefore, controlling and eliminating cancer may be more readily achieved indirectly via the tissue microenvironment.

Topics: Breast Neoplasms; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Cytokines; Drug Design; Extracellular Matrix; Female; Fibronectins; Growth Substances; Humans; Laminin; Matrix Metalloproteinases; Models, Biological; Neoplasm Proteins; Proteoglycans; Stromal Cells; Tenascin; Transforming Growth Factor beta

Breast cancers frequently metastasize to the skeleton and cause bone destruction. Tumor cells secrete factors that stimulate osteoclasts. The consequent osteolytic resorption releases active factors from the bone matrix, in particular transforming growth factor-beta (TGF-beta). The released factors then stimulate tumor cell signaling, which causes breast cancer cells to make increased amounts of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), interleukin-11 (IL-11), and vascular endothelial growth factor (VEGF). Therefore, tumor cell-bone cell interactions cause a vicious cycle in which tumor cells stimulate bone cells to cause bone destruction. As a consequence, the local microenvironment is enriched with factors that fuel tumor growth in bone. Transforming growth factor-beta is of particular importance because it increases breast cancer production of PTHrP. Parathyroid hormone-related protein then stimulates osteoblasts to express RANK (receptor activator of nuclear factor kappa B) ligand, which in turns enhances osteoclast formation and activity. Breast cancer osteolytic metastasis can be interrupted at four points in the vicious cycle: by neutralizing PTHrP biologic activity, by blocking the TGF-beta signaling pathway in the tumor cells, by inhibiting PTHrP gene transcription, and by inhibiting bone resorption.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Osteolysis; Parathyroid Hormone-Related Protein; Signal Transduction; Transforming Growth Factor beta

The progression of breast cancer depends on the establishment of a neovasculature, by a process called angiogenesis. Angiogenesis is an invasive cellular event that requires the co-ordination of numerous molecules including growth factors and their receptors, extracellular proteins, adhesion molecules, and proteolytic enzymes. TGFbeta has emerged to be a major modulator of angiogenesis by regulating endothelial cell proliferation, migration, extracellular matrix (ECM) metabolism, and the expression of adhesion molecules. It is a potent growth inhibitor of normal mammary epithelial cells and a number of breast cancer cell lines. It seems that TGFbeta exerts pleiotropic effects in the oncogenesis of breast cancers in a contextual manner, i.e., it suppresses tumourigenesis at an early stage by direct inhibition of angiogenesis and tumour cell growth. However, over-production of TGFbeta by an advanced tumour may accelerate disease progression through indirect stimulation of angiogenesis and immune suppression. The cell membrane antigen CD105 (endoglin) binds TGFbeta1 and TGFbeta3 and is preferentially expressed in angiogenic vascular endothelial cells. The reduction of CD105 levels in HUVEC leads to in vitro angiogenesis inhibition and massive cell mortality in the presence of TGFbeta1. CD105 null mice die in utero with impaired vasculature, indicating the pivotal role of CD105 in vascular development. The administration of an immunotoxin-conjugate, mab to CD105, induces long-term and complete regression of breast cancer growth in SCID mice. Therefore, CD105 is a promising vascular target for antiangiogenic therapy.

Topics: Animals; Antigens, CD; Breast Neoplasms; Endoglin; Female; Humans; Mice; Mice, SCID; Neovascularization, Pathologic; Receptors, Cell Surface; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Mammary development depends on branching morphogenesis, namely the bifurcation and extension of ductal growth points (end buds) and secretory lobules into a more or less fatty stroma. Because breast carcinomas are overwhelmingly ductal in origin, this review focuses on stromal influences guiding postnatal ductal development and there is only the briefest account of the role of embryonic stroma (mesenchyme). The stroma as the necessary target for endocrine mammogens and the source of stimulatory growth factors is described and the importance of mammary epithelium-induced modifications of the periductal stroma is emphasized. Evidence is presented that if they are to grow, end buds must condition proximal fatty stroma by recruiting white blood cells as well as inducing stromal cell division and, possibly, estrogen receptors. The induction of a fibrous stromal tunic around the end bud is described and its likely role as a complex ductal morphogen is discussed; a possible role in growth inhibition is also considered. Although the signals governing fibrotic induction, ductal morphogenesis, and growth inhibition are unknown, a role for transforming growth factor-beta is highly likely and is discussed. Finally, a need for new conceptual and experimental approaches to understanding stromal-epithelial signaling is discussed.

Topics: Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Communication; Cell Division; Cell Transformation, Neoplastic; Female; Humans; Leukocytes; Receptors, Estrogen; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

The mammary gland undergoes morphogenesis through the entire reproductive life of mammals. In mice, ductal outgrowth from the nipple across the fat pad results in an intricate, well spaced ductal tree that further ramifies and develops alveolar structures during pregnancy. Ductal morphogenesis is regulated by the concerted action of circulating steroid and polypeptide hormones, and local epithelial-mesenchymal inductive signals. Transforming growth factor (TGF)-beta1-3 and hepatocyte growth factor (HGF)/scatter factor (SF) are important components of this latter signaling pathway. TGF-beta1 and TGF-beta3 have roles in both promotion and inhibition of branching morphogenesis that are dependent on concentration and context. HGF/SF promotes ductal outgrowth and tubule formation in the mammary gland. These data suggest that these two growth factors have complementary roles in promoting mammary ductal morphogenesis and in maintaining ductal spacing. In addition, TGF-beta3 triggers apoptosis in the alveolar epithelia, which is a necessary component of mammary gland involution and return of the ductal structure to a virgin-like state after lactation.

Topics: Animals; Apoptosis; Breast; Breast Neoplasms; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Isomerism; Lactation; Mice; Mice, Knockout; Morphogenesis; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

MUC4 is a one of the membrane mucins of the mucin gene (MUC) family, characterized by mucin tandem repeat domains and a transmembrane domain which associates it with the cell plasma membrane. Although MUC4 is encoded by a single gene, it is produced by epithelial cells as a heterodimer through a proteolytic cleavage mechanism. This heterodimer is found in both membrane and soluble forms associated with epithelia. Functionally, MUC4 is proposed to provide a protective mechanism for vulnerable epithelia, such as those of the airway, eye, female reproductive tract and mammary gland. The protective mechanism(s) may be highjacked by some carcinomas, such as those of the breast, to increase tumor progression. Two mechanisms are proposed to contribute to the MUC4 functions. First, MUC4 acts as an anti-adhesive or anti-recognition barrier at epithelial or tumor cell surfaces. Second, MUC4 can bind the receptor tyrosine kinase ErbB2 and alter its cellular signaling. Expression of MUC4 in mammary gland is repressed by posttranscriptional mechanisms involving basement membrane and TGF-beta, which are relieved during pregnancy to permit secretion of MUC4 into milk. These mechanisms are also abrogated in some breast cancers, providing a scenario for promotion of tumor progression. These observations imply important functions for MUC4 in both normal mammary function and in breast cancer.

Topics: Animals; Antigens, Surface; Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Adhesion; Disease Progression; Female; Humans; Mucin-4; Mucins; Receptor, ErbB-2; Transforming Growth Factor beta

Tamoxifen (TAM) has been used in the treatment of breast cancer for over a decade. The observed clinical efficacy of TAM has been attributed to both growth arrest and induction of apoptosis within the breast cancer cells. Although the primary mechanism of action of TAM is believed to be through the inhibition of estrogen receptor (ER), research over the years has indicated that additional, non-ER-mediated mechanisms exist. These include modulation of signaling proteins such as protein kinase C (PKC), calmodulin, transforming growth factor-beta (TGFbeta), and the protooncogene c-myc. Recent studies, including those from our laboratory, have implicated the role of caspases and mitogen-activated protein kinases (MAPK), including c-Jun N-terminal kinase (JNK) and p38 in TAM-induced apoptotic signaling. Oxidative stress, mitochondrial permeability transition (MPT), ceramide generation as well as changes in cell membrane fluidity may also play important roles in TAM-induced apoptosis. These various signaling pathways underlying TAM-induced apoptosis will be reviewed in this article.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Calcium; Calmodulin; Ceramides; Female; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Oxidative Stress; Protein Binding; Protein Kinase C; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Signal Transduction; Tamoxifen; Transforming Growth Factor beta

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS) already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene, and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies of in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the known in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TGFbeta. We propose that overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Cells, Cultured; Cellular Senescence; Disease Models, Animal; DNA-Binding Proteins; Epithelial Cells; Humans; Mammary Glands, Human; Models, Biological; Phenotype; Telomerase; Time Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Topics: Breast Neoplasms; Female; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Breast carcinoma commonly metastasizes to the skeleton in patients with advanced disease to cause bone destruction and the associated pain, hypercalcemia, fracture, and nerve-compression syndromes. In this scenario, the bone destruction is mediated by the osteoclast. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process. Transforming growth factor beta (TGFbeta), which is abundant in bone matrix and is released as a consequence of osteoclastic bone resorption, may promote breast carcinoma osteolysis by stimulating PTHrP production by tumor cells.. Stable breast carcinoma MDA-MB-231 cell lines were constructed that expressed mutant TGFbeta receptors, Smad proteins, or estrogen receptor (ER)-alpha and were used to determine the role of TGFbeta in modulating tumor production of PTHrP. These stable cell lines were applied to a mouse model of human breast carcinoma metastases to the bone to dissect the molecular mechanisms responsible for osteolytic bone metastases.. TGFbeta promoted the development and progression of osteolytic bone metastases by inducing tumor production of PTHrP, the effect of which was mediated through the Smad signaling pathway. PTHrP stimulated osteoclastic bone resorption by increasing osteoblast production of the receptor activator of nuclear factor K B (RANK) ligand and decreasing osteoblast production of osteoprotegerin (OPG). A constitutively active ER-alpha mutation (Tyr537Asn), identified from a human bone metastases, when it was expressed in human breast carcinoma cells, caused increased production of PTHrP. TGFbeta significantly enhanced the ER-alpha-mediated transcriptional activity induced by ER-alpha (Tyr537Asn), and this resulted in further stimulation of PTHrP production.. These data indicate a central role for TGFbeta in the pathogenesis of osteolytic bone metastases from breast carcinoma by 1) the induction of PTHrP through the Smad signaling pathway and 2) the potentiation of ER-alpha-mediated transcription induced by a constitutively active ER-alpha. Understanding the mechanisms of osteolysis at a molecular level will generate more effective therapeutic agents for patients with this devastating complication of cancer.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; DNA-Binding Proteins; Female; Humans; Mice; Osteolysis; Parathyroid Hormone-Related Protein; Proteins; Receptors, Estrogen; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta

Breast cancer is the most commonly diagnosed cancer in American women. The underlying mechanisms that cause aberrant cell proliferation and tumor growth involve conserved pathways, which include components of the cell cycle machinery. Proto-oncogenes, growth factors, and steroids have been implicated in the pathogenesis of breast cancer. Surgery, local irradiation, and chemotherapy have been the mainstay of treatment for early and advanced stage disease. Potential targets for selective breast cancer therapy are herein reviewed. Improved understanding of the biology of breast cancer has led to more specific "targeted therapies" directed at biological processes that are selectively deregulated in the cancerous cells. Examples include tamoxifen for estrogen receptor positive tumors and imunoneutralizing antibodies such as trastuzumab for Her2/neu overexpressing tumors. Other novel anticancer agents such as paclitaxel, a microtubule binding molecule, and flavopiridol, a cyclin dependent kinase inhibitor, exert their anticancer effects by inhibiting cell cycle progression.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Databases, Factual; Enzyme Inhibitors; Flavonoids; Humans; Microtubule-Associated Proteins; Neovascularization, Pathologic; Oncogenes; Paclitaxel; Piperidines; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta; Trastuzumab; Tumor Suppressor Proteins

In patients with advanced disease, several cancer types frequently metastasize to the skeleton, where they cause bone destruction. Osteolytic metastases are incurable and cause pain, hypercalcemia, fracture, and nerve compression syndromes. It was proposed over a century ago that certain cancers, such as that of the breast, preferentially metastasize to the favorable microenvironment provided by bone. Bone matrix is a rich store of immobilized growth factors that are released during bone resorption. Histological analysis of osteolytic bone metastases indicates that the bone destruction is mediated by the osteoclast rather than directly by the tumor cells. These observations suggest a vicious cycle driving the formation of osteolytic metastases: tumor cells secrete factors stimulating osteoclasts through adjacent bone marrow stromal cells; osteoclastic resorption in turn releases growth factors from the bone matrix; finally, locally released growth factors activate the tumor cells. This vicious cycle model has now been confirmed at the molecular level. In particular, transforming growth factor beta (TGF3beta) is abundant in bone matrix and released as a consequence of osteoclastic bone resorption. Bone-derived TGFbeta plays an integral role in promoting the development and progression of osteolytic bone metastases by inducing tumor production of parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption. In breast cancer cells TGFbeta appears to stimulate PTHrP secretion by a posttranscriptional mechanism through both Smad and p38 mitogen activated protein (MAP) kinase signaling pathways. Osteolytic metastases can be suppressed in vivo by inhibition of bone resorption, blockade of TGFbeta signaling in tumor cells, and by neutralization of PTHrP. Other factors released from bone matrix may also act on tumor cells in bone, which in turn may produce other factors that stimulate bone resorption, following the vicious cycle paradigm established for TGFbeta and PTHrP. An understanding at the molecular level of the mechanisms of osteolytic metastasis will result in more effective therapies for this devastating complication of cancer.

Topics: Bone and Bones; Bone Neoplasms; Breast Neoplasms; Calcium; Gonadal Steroid Hormones; Humans; Lymphocytes; Neovascularization, Pathologic; Osteoclasts; Osteolysis; Parathyroid Hormone-Related Protein; Proteins; Transforming Growth Factor beta

Topics: Breast; Breast Neoplasms; Female; Gene Expression; Humans; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 is a pluripotent cytokine that profoundly inhibits epithelial proliferation, induces apoptosis, and influences morphogenesis by mediating extracellular matrix deposition and remodeling. The physiologic roles of the action of TGF-beta in mammary gland, indeed in most tissues, are poorly understood. In order to understand the actions of TGF-beta, we need to take into account the complexity of its effects on different cell types and the influence of context on cellular responses. This task is further compounded by multiple mechanisms for regulating TGF-beta transcription, translation, and activity. One of the most significant factors that obscures the action of TGF-beta is that it is secreted as a stable latent complex, which consists of the 24-kDa cytokine and the 80-kDa dimer of its prepro region, called latency-associated peptide. Latency imposes a critical restraint on TGF-beta activity that is often overlooked. The extracellular process known as activation, in which TGF-beta is released from the latent complex, is emphasized in the present discussion of the role of TGF-beta in mammary gland development. Definition of the spatial and temporal patterns of latent TGF-beta activation in situ is essential for understanding the specific roles that TGF-beta plays during mammary gland development, proliferation, and morphogenesis.

Topics: Animals; Breast; Breast Neoplasms; Cell Division; Female; Humans; Mice; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is a tumor suppressor, the function of which is compromised in many types of human cancer, including breast cancer. The tumor suppressive effects of TGF-beta are caused by potent inhibition of cell proliferation due to cell cycle arrest in the G1 phase. Such antiproliferative responses are mediated by a signaling system that includes two types of cell surface receptors and intracellular signal transducers, the SMAD proteins. Different molecular mechanisms can lead to loss of antiproliferative TGF-beta responses in tumor cells, including mutations in components of the signaling system and inhibition of the SMAD signaling pathway by aberrant activities of various regulatory molecules. Some of these mechanisms will be discussed, with emphasis on their potential involvement in breast tumorigenesis.

Topics: Breast Neoplasms; Cell Differentiation; Cell Line; DNA-Binding Proteins; Female; Gene Expression Regulation, Enzymologic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Signal Transduction; Smad2 Protein; Smad3 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Altered responsiveness to extracellular signals and cell cycle dysregulation are hallmarks of cancer. The cell cycle is governed by cyclin-dependent kinases (cdks) that integrate mitogenic and growth inhibitory signals. Transforming growth factor (TGF)-beta mediates G1 cell cycle arrest by inducing or activating cdk inhibitors, and by inhibiting factors required for cdk activation. Mechanisms that lead to cell cycle arrest by TGF-beta are reviewed. Loss of growth inhibition by TGF-beta occurs early in breast cell transformation, and may contribute to breast cancer progression. Dysregulation of cell cycle effectors at many different levels may contribute to loss of G1 arrest by TGF-beta. Elucidation of these pathways in breast cancer may ultimately lead to novel and more effective treatments for this disease.

Topics: Breast Neoplasms; Cell Cycle; Cyclins; Female; Genes, myc; Genes, ras; Humans; Phosphorylation; Transforming Growth Factor beta

The transforming growth factor (TGF)-betas are potent growth inhibitors of normal epithelial cells. In established tumor cell systems, however, the preponderant experimental evidence suggests that TGF-betas can foster tumor-host interactions that indirectly support the viability and/or progression of cancer cells. The timing of this 'TGF-beta switch' during the progressive transformation of epithelial cells is not clear. More recent evidence also suggests that autocrine TGF-beta signaling is operative in some tumor cells, and can also contribute to tumor invasiveness and metastases independent of an effect on nontumor cells. The dissociation of antiproliferative and matrix associated effects of autocrine TGF-beta signaling at a transcriptional level provides for a mechanism(s) by which cancer cells can selectively use this signaling pathway for tumor progression. Data in support of the cellular and molecular mechanisms by which TGF-beta signaling can accelerate the natural history of tumors will be reviewed in this section.

Topics: Breast Neoplasms; Female; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The soy isoflavone genistein attenuates growth factor- and cytokine-stimulated proliferation of both normal and cancer cells. This article reviews our current understanding of the potential mechanisms of action of genistein. In membrane preparations from mammalian cells, genistein is a potent and specific inhibitor of tyrosine autophosphorylation of the epidermal growth factor (EGF) receptor. However, in several cell systems in which it inhibits growth, genistein does not alter tyrosine phosphorylation of the EGF receptor or other tyrosine kinase substrates thought to be involved in signal transduction pathways, suggesting that other mechanisms may be responsible for its action. Alternatives include inhibition of DNA topoisomerase II activity, regulation of cell cycle checkpoints, and antiangiogenic and antioxidant activity. Experiments in our laboratory suggest a new concept, that genistein may inhibit cell growth by modulating transforming growth factor (TGF) beta1 signaling pathways. Such a link between genistein action and TGFbeta1 function is supported by preliminary results of studies in patients with hereditary hemorrhagic telangiectasia (a genetic disorder involving mutations in proteins that regulate TGFbeta receptor complex formation and signaling) in which several patients had dramatic attenuation of their symptoms after 1 wk of ingesting soy-based beverages. These preclinical studies in combination with our cell culture data suggest that the mechanism of genistein involves, if not requires, TGFbeta1-signaling.

Topics: Animals; Anticarcinogenic Agents; Breast Neoplasms; Cardiovascular Diseases; Cell Division; Female; Genistein; Humans; Male; Prostatic Neoplasms; Protein-Tyrosine Kinases; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Transforming Growth Factor-beta (TGF beta) is the most potent known inhibitor of the progression of normal mammary epithelial cells through the cell cycle. During the early stages of breast cancer development, the transformed epithelial cells appear to still be sensitive to TGF beta-mediated growth arrest, and TGF beta can act as an anti-tumor promoter. In contrast, advanced breast cancers are mostly refractory to TGF beta-mediated growth inhibition and produce large amounts of TGF beta, which may enhance tumor cell invasion and metastasis by its effects on extracellular matrix. We postulate that this seemingly paradoxical switch in the responsiveness of tumor cells to TGF beta during progression is the consequence of the activation of the latent TGF beta that is produced and deposited into the tumor microenvironment, thereby driving the clonal expansion of TGF beta-resistant tumor cells. While tumor cells themselves may activate TGF beta, recent observations suggest that environmental tumor promoters or carcinogens, such as ionizing radiation, can cause stromal fibroblasts to activate TGF beta by epigenetic mechanisms. As the biological effects of the anti-estrogen tamoxifen may well be mediated by TGF beta, this model has a number of important implications for the clinical uses of tamoxifen in the prevention and treatment of breast cancer. In addition, it suggests a number of novel approaches to the treatment of advanced breast cancer.

Topics: Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Female; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Parathyroid hormone-related protein (PTH-rP) was purified and cloned 10 years ago as a factor responsible for the hypercalcemia associated with malignancy. Clinical evidence supports another important role for PTH-rP in malignancy as a mediator of the bone destruction associated with osteolytic metastasis. Patients with PTH-rP positive breast carcinoma are more likely to develop bone metastasis. In addition, breast carcinoma metastatic to bone expresses PTH-rP in >90% of cases, compared with only 17% of metastasis to nonbone sites. These observations suggest that PTH-rP expression by breast carcinoma cells may provide a selective growth advantage in bone due to its ability to stimulate osteoclastic bone resorption. Furthermore, growth factors such as transforming growth factor-beta (TGF-beta), which are abundant in bone matrix, are released and activated by osteoclastic bone resorption and may enhance PTH-rP expression and tumor cell growth. To investigate the role of PTH-rP in the pathophysiology of breast carcinoma metastasis to bone, the human breast carcinoma cell line MDA-MB-231 was studied in a murine model of human breast carcinoma metastasis to bone. A series of experiments were performed in which 1) PTH-rP secretion was altered, 2) the effects of PTH-rP were neutralized, or 3) the responsiveness to TGF-beta was abolished in MDA-MB-231 cells. Cultured MDA-MB-231 cells secreted low amounts of PTH-rP that increased fivefold in response to TGF-beta. Tumor cells inoculated into the left cardiac ventricle of nude mice caused osteolytic metastasis similar to that observed in humans with breast carcinoma. When PTH-rP was overexpressed in the tumor cells, bone metastases were increased. MDA-MB-231 cells transfected with the cDNA for human preproPTH-rP secreted a tenfold greater amount of PTH-rP and caused significantly greater bone metastases when inoculated into the left cardiac ventricle of female nude mice compared with parental cells. In contrast, when the biologic effects of PTH-rP were neutralized or its production was suppressed, such metastases were decreased. Treatment of mice with a neutralizing monoclonal antibody to human PTH-rP resulted in a decrease in the development and progression of bone metastasis due to the parental MDA-MB-231 cells. Similar results were observed when mice were treated with dexamethasone, a potent glucocorticoid that suppresses production of PTH-rP by the MDA-MB-231 cells in vitro. The role of bone-derived TGF-beta in

Topics: Animals; Antineoplastic Agents, Hormonal; Bone Neoplasms; Bone Remodeling; Bone Resorption; Breast Neoplasms; Cytokines; Dexamethasone; Disease Models, Animal; Female; Humans; Hypercalcemia; Mice; Neoplasm Proteins; Osteoclasts; Parathyroid Hormone-Related Protein; Proteins; Transforming Growth Factor beta

The transforming growth factor-beta s are potent growth inhibitors of normal and transformed breast epithelial cells in culture. In vivo, these peptides modulate the development of the mouse mammary gland. Tissue-specific overexpression of mature TGF-beta 1 in transgenic mice results in mammary gland atrophy and prevention of carcinogen-induced breast tumorigenesis. However, the inhibitory effect of endogenous or exogenous TGF-beta s on established tumor cells is less clear. Several published circumstantial and more direct data argue that, in some cases, the tumor cell TGF-beta s may contribute to the maintenance and/or progression of tumor cells in an intact host by modulating their interaction with host factors. This differential role of the TGF-beta s on mammary cells as determined by their normal or transformed phenotype as well as the biological and clinical implications of these data are discussed.

Topics: Animals; Breast; Breast Neoplasms; Cell Division; Female; Humans; Transforming Growth Factor beta

Endocrine therapy of breast cancer consists of a variety of both medical and surgical ablative treatment modalities, but ablative therapy is increasingly replaced by medical treatment. Most endocrine therapies have more than one endocrine effect, frequently together with direct growth inhibitory actions via receptors. Endocrine therapy can be effective in all phases of the disease, but curative only in early disease while in advanced cancer it can only prolong survival. In the past decade the number of available endocrine agents has been drastically increased. Novel approaches in the endocrine therapy of breast cancer are application of new antiestrogens, antiprogestins, new potent aromatase inhibitors, analogues of luteinizing hormone-releasing hormone (LHRH-A) and somatostatin, inhibitors of prolactin secretion, vitamin A and D analogues, bisphosphonates, growth factor antagonists, tyrosine protein kinase inhibitors, protease inhibitors, inhibitors of angiogenesis, radiolabeled hormones and monoclonal antibodies. New cell biological factors such as oncogenes and suppressorgenes, secretory proteins and membrane receptors can be used not only as prognostic factors but also for prediction of type of response to endocrine and chemotherapy. Thus, these cell biological parameters can be used to select high and low risk patients, type of systemic treatment, and can also be used as targets for new treatment modalities. Future studies on treatment of all stages of disease will increasingly focus on promising combined treatment modalities.

Topics: Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Enzyme Inhibitors; Estrogen Antagonists; Gonadotropin-Releasing Hormone; Humans; Insulin-Like Growth Factor I; Neoplasm Metastasis; Neoplasms, Hormone-Dependent; Neovascularization, Pathologic; Progesterone Congeners; Progestins; Prolactin; Retinoids; Somatostatin; Transforming Growth Factor beta; Vitamin D

Transforming growth factor beta (TGF-beta) receptor expression and signal transduction in human breast cancer are reviewed as a function of estrogen receptor (ER) expression. ER+ breast cancer cells are generally resistant to the inhibitory effects of TGF-beta. The only known exception appears to be MCF-7 early passage cells which are initially sensitive to TGF-beta, but gain resistance after long-term passage in tissue culture. A number of studies have shown that loss of sensitivity is due to inadequate TGF-beta type II (TGFRII) receptor expression. Stable transfection of TGFRII into ER+ breast cancer cell lines results in the acquisition of TGF-beta sensitivity and reversion of malignancy. Although there are exceptions, ER- breast cancer cells usually express TGFRII, but nevertheless show a low level of sensitivity to TGF-beta. Thus resistance in these cells implies a postreceptor mechanism. Given the frequency with which loss of TGF-beta sensitivity has been associated with loss of TGFRII, the ER- breast cancer cell lines may represent valuable models for identifying postreceptor mechanisms of resistance.

Topics: Animals; Breast Neoplasms; Female; Humans; Mammary Neoplasms, Experimental; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Growth inhibition by the TGF-beta s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF-beta mediated growth regulation is a critical event in breast tumorigenesis and several lines of in vitro and in vivo data support this hypothesis. However, a positive association between the expression of TGF-beta s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies in in vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF-beta include immunosuppression mediated by tumor TGF-beta s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF-beta on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.

Topics: Animals; Breast Neoplasms; Female; Humans; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Transforming Growth Factor beta

Transforming growth factor beta (TGF beta) has potent inhibitory effects upon epithelial proliferation and malignant progression may be associated with breakdown of the autocrine and paracrine inhibitory loops in which TGF beta participates. The therapeutic effecs of anti-estrogens may be partially attributable to boosting of local endogenous levels of TGF beta. This article reviews the evidence in support of TGF beta being a proximate effector in mediation of the anti-neoplastic effects of anti-estrogens. Both the conventional estrogen receptor (ER) dependent and ER independent mechanisms of action are likely to be involved. Evidence for preferential stromal induction of TGF beta by anti-estrogens is emphasized, together with the therapeutic potential of this strategy for improving outcome in early breast cancer irrespective of ER status.

Topics: Autocrine Communication; Breast Neoplasms; Drug Resistance, Neoplasm; Estrogen Antagonists; Estrogens; Female; Humans; Neoplasms, Hormone-Dependent; Paracrine Communication; Transforming Growth Factor beta

This review considers the various roles of the TGF-beta system in mammary carcinogenesis, tumor progression, and cellular responses to therapeutic measures. The paradigm that has evolved from the work of many investigators suggests that loss of tumor cell responsiveness to the effects of TGF-beta can result in a crucial shift in the net effect of TGF-beta within the context of the tumor-host interaction. Principal elements of host-tumor interactions in which this shift may play out, including immune suppression, angiogenesis, and modification of the surrounding extracellular matrix by tumor cells, are potentially amenable to manipulation. Additional effects of TGF-beta, such as suggested by reports of its ability to alter the drug resistance of tumor cells and the drug sensitivity of normal tissues, suggest that appropriate molecular intervention designed to affect the TGF-beta system might constitute an effective therapeutic strategy.

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Topics: Breast Neoplasms; Cell Adhesion; Cell Adhesion Molecules; Cell Cycle; Cell Division; Extracellular Matrix; Female; Gastrointestinal Neoplasms; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Neoplasms; Prostatic Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Bone and Bones; Breast Neoplasms; Carrier Proteins; Female; Gene Expression Regulation; Gonadal Steroid Hormones; Humans; Male; Prostate; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Active cell death is a genetically encoded self-destruction of a cell. There occur morphologically different types of active cell death, e.g. apoptosis in the liver or autophagic cell death in human mammary carcinoma cells after tamoxifen treatment (Pre)neoplastic lesions in rat liver exhibit enhanced rates of apoptosis, which tend to increase with increasing malignancy. Tumor promoters and non-genotoxic carcinogens inhibit active cell death, thereby increasing the accumulation of (pre)neoplastic cells and accelerating the development of cancer. On the other hand promoter withdrawal, fasting or application of negative growth signals such as transforming growth factor beta 1 (TGF beta 1) enhance apoptosis and can lead to selective regression of preneoplastic lesions or tumors.

Topics: Animals; Apoptosis; Breast Neoplasms; Humans; Liver Neoplasms; Neoplasms; Rats; Transforming Growth Factor beta

A number of lines of evidence suggest that IGFs are important mitogens in human breast cancer: (1) IGFs are the most potent growth factor in human breast cancer cells; (2) estrogen stimulates expression of IGF-II and the type 1 IGF receptor; and (3) stromal cells express IGFs, which may act in a paracrine manner. Numerous studies have demonstrated that IGFBPs modulate the mitogenic effects of IGFs in the local environment. In particular, we have recently demonstrated that IGFBP-3 inhibits the growth of Hs578T and MDA-MB-231 human breast cancer cells in an IGF-independent manner. Further studies revealed the existence of cell surface-associated IGFBP-3 receptors. Receptor binding and the subsequent antiproliferative action of IGFBP-3 was inhibited by IGFs, owing to the formation of an IGF-IGFBP-3 complex that prevents the binding of IGFBP-3 to its receptors. In addition, exogeneously added soluble heparin or heparan sulfate inhibited the binding of IGFBP-3 to the cell surface in a dose-dependent manner. However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically remove, IGFBP-3 binding was only minimally affected. These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting receptor binding of IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of IGFBP-3. Additionally, the role of IGFBP-3 in the antiproliferative effects of transforming growth factor (TGF)-beta and retinoic acid (RA) is supported by our observations that: (1) inhibition of IGFBP-3 gene expression using an IGFNBP-3 antisense oligodeoxynucleotide not only blocks TGF-beta and RA simulation of IGFBP-3 production by up to 90%m but also inhibits their antiproliferative effects by 40-60%; and (2) treatment with IGF-II and IGF-II analogs diminish TGF-beta effects by blocking TGF-beta induced binding of IGFBP-3 to the cell surface. Taken together, our results support the hypothesis that IGFBP-3 is an important antiproliferative factor in human breast cancer, acting in an IGF-independent manner in addition to its ability to modulate the binding of IGF peptides to IGF receptors.

Topics: Breast Neoplasms; Cell Division; Endopeptidases; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Models, Biological; Oligonucleotides, Antisense; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The development and differentiation of the epithelial component of glandular tissues such as the breast is regulated by two apparently unrelated processes. One of these is presumed to be epithelial cell collective autonomous, that is, it is mediated by gene products which act directly on the epithelial cells. An important component of autonomous regulation is the functional expression of homotypic cell-cell adhesion molecules such as cadherins. The second process is non-autonomous and involves an inductive effect of the neighboring mesenchymal cell collective. An important component of non-autonomous regulation is the aggregation/condensation of mesenchyme closely associated with the epithelium. We propose that molecular alterations in autonomous and non-autonomous pathways are important causes and indicators respectively of breast cancer progression and that these two fundamental regulators of epithelial collective organization are in fact inter-dependent. For example, we show that the expression of hepatocyte growth factor (HGF), an epithelially targeted mesenchymally derived morphogenic factor is regulated by mesenchymal cell density (condensation) and by factors released from epithelial cells. Breast epithelial cells produce factors which inhibit and stimulate HGF expression. The inhibitory factor is transforming growth factor beta (TGF-beta) and the activation state of TGF-beta is a crucial element in HGF homeostasis. The balance of negative and positive HGF regulators is markedly affected by the growth conditions and differentiation state of the epithelial cells. The expression of the HGF receptor, met, is high in normal breast epithelial cells and in dedifferentiated (ER negative) tumor cells but is reduced or lost in ER positive well differentiated epithelial cells. Our results indicate that the expression of at least one epithelial morphogen, HGF, is inter-dependently regulated by mesenchymal condensation and by factors released by neighboring epithelial cells.

Topics: Breast Neoplasms; Cell Adhesion; Cell Communication; Cell Differentiation; Epithelium; Fibroblasts; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Mesoderm; Models, Biological; Neoplasm Proteins; Neovascularization, Pathologic; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor beta

Tamoxifen is the most often prescribed non steroidal antioestrogenic agent in the world for breast cancer. Worldwide collaboration. has centralized the results, of different trials throughout the world on oral adjuvant therapy in the early stages of breast cancer. A significative regression of the tumour was observed in most cases. Moreover, recent epidemiological studies suggest that tamoxifen could prevent new contralateral primary tumours. The risk of the disease should thus be reduced by the prophylactic use of antioestrogens such as tamoxifen. Investigations using a variety of models have evaluated the effect of tamoxifen on tumour promotion and cell growth. Tamoxifen-induced growth inhibition is associated with major changes in biochemical events in cultured human breast cancer cells including cell proliferation or growth factor production. Growth inhibition of oestrogen-responsive human breast cancer cells is associated with an induced secretion of autoinhibitory polypeptides (TGF beta) and an antagonistic effect on the synthesis of proliferative proteins (TGF alpha,...). The first step in the mechanism of action of the drug is binding of tamoxifen to the oestrogen receptors. Development of resistance to tamoxifen treatment is a great problem in treatment of breast cancer patients and the mechanism of resistance will require further study: under the influence of the drug, tumours could become remodelled as selected subpopulations emerge resistant-tamoxifen. The fact that some breast cancers which are oestrogen receptor-negative respond to antioestrogen suggests that parallel but separate pathways for oestrogen and antioestrogen action may exist. This paper summarizes the results of the most recent studies concerning this promising drug.

Topics: Breast Neoplasms; Cell Division; Clinical Trials as Topic; Drug Resistance; Female; Growth Inhibitors; Humans; Neoplasms, Second Primary; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured

Acquisition of the antiestrogen resistance by breast cancer cells in vivo may result from a variety of mechanisms. The main pathway appears to involve loss of estrogen receptor (ER) expression or selection for ER negative cells among heterogenous population of tumor cells. However, clinical data suggest that, in about 30% of the cases, antiestrogen resistance arises even in the presence of estrogen receptors. Postulated mechanisms leading to the latter phenotype include selection for variant receptor forms during treatment, development of novel metabolic pathways for the drug, loss of nuclear co-factors, or activation of signal transduction pathway that cross activate ER signals. We have used an in vitro experimental system utilizing LY-2 cell line, an ER positive and antiestrogen resistant MCF-7 cell variant, to study the mechanism of antiestrogen resistance in the presence of functional ER. Result from a complementation experiment suggests that LY-2 phenotype is a recessive trait. Cloning of the genetic defect in the LY-2 cells would provide further insight for the mechanism of antiestrogen resistance in ER positive breast cancer cells.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Drug Resistance; Estrogen Antagonists; Estrogens; Glucocorticoids; Humans; Hybrid Cells; Mammary Neoplasms, Experimental; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured

The molecular mechanism of action of anti-oestrogens such as tamoxifen appears to be a complex mixture of antagonism of the mitogenic action of oestradiol at the level of the oestrogen receptor, plus a range of other activities from enzyme inhibition to growth factor modulation. This article will concentrate on two specific areas: 1) the inhibition of protein kinase C and calmodulin-dependent cAMP phosphodiesterase; and 2) the regulation by tamoxifen of peptide regulators of breast cancer epithelial cell growth such as insulin-like growth factor I (IGF I) and transforming growth factor beta (TGF-beta). The elucidation of these mechanisms is potentially important in the treatment and chemoprevention of breast cancer-the quantitative contribution of each individual mechanism of the overall antineoplastic action of anti-oestrogens is central to developing new and possibly more effective anti-oestrogens and optimizing strategies for their use.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Breast Neoplasms; Cell Division; Cyclic Nucleotide Phosphodiesterases, Type 1; Estradiol; Estrogen Antagonists; Female; Humans; Insulin-Like Growth Factor I; Protein Kinase C; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta

The new antiestrogen Droloxifene has a 10-60-fold higher binding affinity to the estrogen receptor (ER) compared to the related compound Tamoxifen. A similar relationship was found in growth inhibition studies which showed that Droloxifene inhibited the different ER positive human breast cancer cells more effectively than Tamoxifen, predominantly in drug concentrations which are found in humans during therapy. As another consequence of the high stability of the complex formed by Droloxifene binding to the ER, intermittent exposures with clinically relevant concentrations of Droloxifene brought about effective growth inhibition of human ER positive tumor cells even after short-term application. Droloxifene was found, like Tamoxifen, to block human breast cancer cells in G1-phase of the cell cycle. Moreover, cell-cycle data confirmed the superior growth-inhibiting potency of Droloxifene compared to Tamoxifen. Droloxifene was also found to effectively induce expression of the negative growth factor TGF-beta, to inhibit IGF-I stimulated cell growth and to prevent estrogen-stimulated proto-oncogene c-myc expression. Unlike Tamoxifen, Droloxifene is a potent inhibitor of protein biosynthesis in ER-positive breast cancer cells at physiologically relevant concentrations. Lower estrogenic and higher antiestrogenic effects on immature rat uterus indicate a higher therapeutic index for Droloxifene compared to Tamoxifen. In vivo, Droloxifene displayed increased growth inhibition of different tumors of animal (R3230AC and 13762) and human origin (T61). Furthermore, it was found that the two structurally similar drugs differ in their toxicologic characteristics in the following important respects: Droloxifene is devoid of any in vivo or in vitro carcinogenic or mutagenic effects, whereas Tamoxifen causes liver tumors in rats, induces DNA adduct formation in rats and hamsters and shows transforming activity in SHE-cells (Syrian hamster embryo fibroblasts). Considerably less toxicity and a lower level of intrinsic estrogenicity was observed even after maximum long-term exposure of different animal species to Droloxifene, in comparison with Tamoxifen. Therefore, it can be assumed that Droloxifene may represent an important step forward in the treatment of mammary carcinomas in women through its better tolerability and increased efficacy compared with Tamoxifen. For long-term adjuvant or preventive treatment of breast cancer, Droloxifene may well be the safer choice.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Drug Evaluation, Preclinical; Estrogen Antagonists; Female; Humans; Insulin-Like Growth Factor I; Proto-Oncogene Mas; Rats; Rats, Inbred Strains; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta

Topics: Breast Neoplasms; ErbB Receptors; Female; Growth Substances; Humans; Prognosis; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, IGF Type 1; Receptors, Cell Surface; Receptors, Somatostatin; Survival Rate; Transforming Growth Factor beta

The transforming growth factors beta are a family of peptides which are involved in the regulation of cell growth and differentiation. It has been suggested that the loss of sensitivity to growth inhibition by endogenous TGF-beta may contribute to the process of carcinogenesis in epithelial systems. However, many breast cancer cells remain sensitive to the growth inhibitory effects of these peptides, suggesting that the local induction of TGF-beta could provide a pharmacological approach to chemoprevention. Triphenylethylene anti-oestrogens, synthetic progestins and retinoids all offer potential as chemopreventative agents. A common feature of their mechanism of action is the ability to locally increase the production of the transforming growth factors beta.

Topics: Antineoplastic Agents; Breast Neoplasms; Humans; Progestins; Retinoids; Tamoxifen; Transforming Growth Factor beta

The fields of endocrinology and peptide growth factors can no longer be studied independently, nor can hypotheses about the modes of action of hormones and growth factors be constrained by outmoded classic definitions. Although much is still to be learned about the mechanisms of induction of TGF-beta activity by steroids/retinoids, about the involvement of specific nuclear receptors and about the factors regulating the differential effects of steroids/retinoids on the various isoforms of TGF-beta, it is clear that the identification of the interrelationships between these two superfamilies of multi-functional effectors has heralded a new era of discovery of the underlying regulatory events controlling cellular proliferation and differentiation.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cell Differentiation; Cell Division; Epithelium; Female; Gene Expression Regulation; Humans; Mice; Rats; Receptors, Retinoic Acid; Receptors, Steroid; Retinoids; Steroids; Transforming Growth Factor beta

The polypeptide growth factor transforming growth factor-beta (TGF-beta) is a multifunctional regulator of basic cellular functions: proliferation, differentiation, cell adhesion and interactions with the extracellular matrix. TGF-beta is part of a regulatory network of which our knowledge is still incomplete, together with other substances such as steroid hormones, oncogene products and integrins. Five isoforms for TGF-beta and five different TGF-beta receptors have been described. TGF-beta exhibits an antiproliferative effect in vitro and in vivo on many cells of epthelial, myeloid, lymphoid and mesenchymal origin together with a growth-stimulating effect on various cells like endothelial cells and epidermal keratinocytes. Production of TGF-beta and receptors for TGF-beta has been found in many cell types, both normal and malignant. Nevertheless the amount of in vivo data is too limited to identify possibilities for therapeutic intervention in the physiological and patophysiological functions of TGF-beta.

Topics: Breast Neoplasms; Cell Adhesion; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Female; Humans; Lung Neoplasms; Receptors, Cell Surface; Transforming Growth Factor beta

Topics: Antibodies, Monoclonal; Breast Neoplasms; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Prognosis; Receptors, Cell Surface; Receptors, Somatomedin; Transforming Growth Factor alpha; Transforming Growth Factor beta

Topics: Animals; Breast; Breast Neoplasms; Cell Differentiation; Cell Division; Epithelial Cells; Epithelium; Feedback; Female; Gene Expression Regulation; Hormones; Humans; Immunity, Cellular; Immunologic Factors; Mammary Glands, Animal; Mice; Models, Biological; Multigene Family; Neoplasm Proteins; Pregnancy; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Androgen Antagonists; Androgens; Breast Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Estrogens; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Flutamide; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Platelet-Derived Growth Factor; Prostatic Neoplasms; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta

Breast cancer patients who acquire tamoxifen resistance may respond to second-line hormonal therapy or progress to true endocrine resistance. The biological basis for these processes are poorly understood. Following successful therapy with tamoxifen there is little evidence at relapse for change in either the host endocrine environment or drug metabolic profile to account for the development of acquired resistance. Many tamoxifen resistant tumours still retain a structurally and functionally normal oestrogen receptor (ER) and yet will grow independent of oestrogen. The oestrogen-regulated molecular events which normally govern the growth of hormone-sensitive breast cancer involve a complex autocrine and paracrine interaction between several peptide growth factors (including TGF alpha, IGF-1 and TGF beta), their receptors and signal transduction pathways. Evidence now exists that constitutive activity of many of these mediators of the mitogenic signal can bypass the cell's dependence on oestrogen and provide a mechanism for hormone-independent growth. Research into these molecular mechanisms may result in a better understanding of how to overcome the clinical problem of tamoxifen resistance.

Topics: Breast Neoplasms; Drug Resistance; ErbB Receptors; Female; Humans; Insulin-Like Growth Factor I; Oncogenes; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta

In this review I summarize the experimental data in favor of the notion that control of epidermal growth factor (EGF) receptor (R) and/or c-erbB-2 protooncogene expression by specific autocrine growth factors and certain classical endocrine hormones serves as a transducer of extracellular signals that ultimately lead to growth responses in breast carcinoma cells. I summarize some new results on the role of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and TGF beta in the control of EGF-R protooncogene expression in human breast carcinoma cells. Furthermore, the data embracing the hypothesis that the growth actions of hormone receptors that are homologous to the v-erbA oncogene (estrogens, progesterone, thyroid hormones, retinoic acid, and vitamin D) are mediated, in part, by modulating EGF-R and/or c-erbB-2 protooncogene transcription are reviewed. Finally, I develop the theme that cooperation of certain c-erb-A-related, c-erbB-2 and/or EGF-R gene products contribute to the uncontrolled growth of human mammary carcinoma cells. From the evidence reviewed, one can infer that elucidation of the molecular control of EGF-R/c-erbB-2 gene expression by c-erbA-related gene products may lead to both a better understanding of breast carcinogenesis and a new therapeutic approach directed at controlling the transcriptional responses of EGF-R/c-erbB-2 genes.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cholecalciferol; Epidermal Growth Factor; ErbB Receptors; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Hydrocortisone; Progesterone; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tretinoin; Triiodothyronine

Estrogen receptor-negative human breast cancer is usually an aggressive, hormone independent tumour. Recent studies show that growth of these tumours could be influenced by anti-estrogens. Anti-estrogens appear to stimulate the production of Transforming Growth Factor-beta (TGF-beta) in hormone dependent as well as independent cell lines suggesting as a result inhibition of cellular proliferation of these cell lines by TGF-beta through an autocrine/paracrine mechanism.

Topics: Breast Neoplasms; Estrogen Antagonists; Female; Humans; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Cells, Cultured

We hypothesize that early events in the development of at least some human breast cancers involve faulty epithelial-mesenchymal interactions and that the stromal cells themselves play an active role in this abnormal process. In contrast, later events accelerating breast tumor progression may occur in association with genetic changes involving only the malignant epithelial cells. These conclusions arise from a review of the literature, our comparative studies of HA metabolism in fibroblasts cultured from either normal or malignant breast tissues, and from molecular-genetic studies performed on sequential specimens from a single patient and on a wide variety of human breast tumor samples. HA is a proteoglycan component of the ECM which is known to stimulate epithelial cell detachment and motility and is most abundant in fetal and rapidly growing tissues. We find that many breast cancer-derived fibroblasts are stimulated to produce HA in response to TGF-beta under conditions where HA accumulation by normal tissue fibroblasts is almost uniformly inhibited. In a single patient, we had the opportunity to examine three malignant effusions that occurred sequentially to identify genetic changes associated with the later stages of breast cancer progression. Although, common cytogenetic abnormalities were found in all the effusion samples, only the last effusion exhibited a loss of heterozygosity at the c-Ha-ras locus. In this case, the allelic loss correlated with improved growth in vitro of the primary cells and with ability to become a permanently established cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Breast Neoplasms; Cell Division; Cell Movement; Epithelium; Fibroblasts; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Hyaluronic Acid; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Oncogenes; Phenotype; Skin; Transforming Growth Factor beta

Breast cancer is characterized by hormonal regulation. The current article reviews the role of estrogen and polypeptide growth factors in control of proliferation and basement membrane invasion of breast cancer cells in vitro. The role of antiestrogens to regulate proliferation, invasion, and growth factor secretion is further highlighted. Finally, the use of in vitro cultures of breast cancer cells to model steps in the malignant progression of the disease is emphasized. The availability of hormone dependent and independent breast cancer cell lines should allow screening for better antiestrogens, antimetastatic drugs, and antagonists of local action of growth factors.

Topics: Animals; Basement Membrane; Breast Neoplasms; Cell Division; Estradiol; Estrogen Antagonists; Estrogens; Female; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Estrogen Antagonists; Estrogens; Female; Genes, ras; Humans; Neoplasms, Hormone-Dependent; Oncogenes; Phenotype; Platelet-Derived Growth Factor; Receptors, Estrogen; Somatomedins; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Cells, Cultured

We examined gene expression, germline variant, and somatic mutation features associated with pathologic response to neoadjuvant durvalumab plus chemotherapy in basal-like triple-negative breast cancer (bTNBC).. Germline and somatic whole-exome DNA and RNA sequencing, programmed death ligand 1 (PD-L1) IHC, and stromal tumor-infiltrating lymphocyte scoring were performed on 57 patients. We validated our results using 162 patients from the GeparNuevo randomized trial.. Gene set enrichment analysis showed that pathways involved in immunity (adaptive, humoral, innate), JAK-STAT signaling, cancer drivers, cell cycle, apoptosis, and DNA repair were enriched in cases with pathologic complete response (pCR), whereas epithelial-mesenchymal transition, extracellular matrix, and TGFβ pathways were enriched in cases with residual disease (RD). Immune-rich bTNBC with RD was enriched in CCL-3, -4, -5, -8, -23, CXCL-1, -3, -6, -10, and IL1, -23, -27, -34, and had higher expression of macrophage markers compared with immune-rich cancers with pCR that were enriched in IFNγ, IL2, -12, -21, chemokines CXCL-9, -13, CXCR5, and activated T- and B-cell markers (GZMB, CD79A). In the validation cohort, an immune-rich five-gene signature showed higher expression in pCR cases in the durvalumab arm (P = 0.040) but not in the placebo arm (P = 0.923) or in immune-poor cancers. Independent of immune markers, tumor mutation burden was higher, and PI3K, DNA damage repair, MAPK, and WNT/β-catenin signaling pathways were enriched in germline and somatic mutations in cases with pCR.. The TGFβ pathway is associated with immune-poor phenotype and RD in bTNBC. Among immune-rich bTNBC RD, macrophage/neutrophil chemoattractants dominate the cytokine milieu, and IFNγ and activated B cells and T cells dominate immune-rich cancers with pCR.

Topics: Albumins; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Cyclophosphamide; Doxorubicin; Female; Humans; Neoadjuvant Therapy; Paclitaxel; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

The most effective way to control severity and mortality rate of the novel coronavirus disease (COVID-19) is through sensitive diagnostic approaches and an appropriate treatment protocol. We aimed to identify the effect of adding corticosteroid and Tocilizumab to a standard treatment protocol in treating COVID-19 patients with chronic disease through hematological and lab biomarkers.. This study was performed retrospectively on 68 COVID-19 patients with chronic disease who were treated by different therapeutic protocols. The patients were categorized into four groups: control group represented the patients' lab results at admission before treatment protocols were applied; group 1 included patients treated with anticoagulants, Hydroxychloroquine, and antibiotics; group 2 comprised patients treated with Dexamethasone; and group 3 included patients treated with Dexamethasone and Tocilizumab.. The study paves the way into the effectiveness of combining Dexamethasone with Tocilizumab in treatment COVID-19 patients with chronic diseases.. La forma más eficaz de controlar la gravedad y la tasa de mortalidad de la enfermedad del nuevo coronavirus (COVID-19) es mediante enfoques de diagnóstico sensibles y un protocolo de tratamiento adecuado. Nuestro objetivo fue identificar el efecto de agregar corticosteroides y tocilizumab a un protocolo de tratamiento estándar en el tratamiento de pacientes con COVID-19 con enfermedad crónica a través de biomarcadores hematológicos y de laboratorio.. Este estudio se realizó de forma retrospectiva en 68 pacientes COVID-19 con enfermedad crónica que fueron tratados por diferentes protocolos terapéuticos. Los pacientes se clasificaron en cuatro grupos: el grupo de control representaba los resultados de laboratorio de los pacientes en el momento de la admisión antes de que se aplicaran los protocolos de tratamiento; el grupo 1 incluyó a pacientes tratados con anticoagulantes, hidroxicloroquina y antibióticos; el grupo 2 estaba compuesto por pacientes tratados con dexametasona; y el grupo 3 incluyó a pacientes tratados con dexametasona y tocilizumab.. El estudio allana el camino hacia la eficacia de la combinación de dexametasona con tocilizumab en el tratamiento de pacientes con COVID-19 con enfermedades crónicas.. The Child-Mother Index constitutes a potential useful risk factor indicator for statistical analyses on data after birth. The value of the Child-Mother Index based on the estimated fetal weight before birth deserves evaluation.. Six ceria supports synthesized by various synthesis methodologies were used to deposit cobalt oxide. The catalysts were thoroughly characterized, and their catalytic activity for complete methane oxidation was studied. The supports synthesized by direct calcination and precipitation with ammonia exhibited the best textural and structural properties as well as the highest degree of oxidation. The remaining supports presented poorer textural properties to be employed as catalytic supports. The cobalt deposited over the first two supports presented a good dispersion at the external surface, which induced a significant redox effect that increased the number of Co. Some studies show that children with obesity are more likely to receive a diagnosis of depression, anxiety, or attention-deficit hyperactivity disorder (ADHD). But this does not necessarily mean obesity causes these conditions. Depression, anxiety, or ADHD could cause obesity. A child's environment, including family income or their parents' mental health, could also affect a child's weight and mental health. Understanding the nature of these relationships could help scientists develop better interventions for both obesity and mental health conditions. Genetic studies may help scientists better understand the role of the environment in these conditions, but it's important to consider both the child's and their parents’ genetics in these analyses. This is because parents and children share not only genes, but also environmental conditions. For example, families that carry genetic variants associated with higher body weight might also have lower incomes, if parents have been affected by biases against heavier people in society and the workplace. Children in these families could have worse mental health because of effects of their parent’s weight, rather than their own weight. Looking at both child and adult genetics can help disentangle these processes. Hughes et al. show that a child's own body mass index, a ratio of weight and height, is not strongly associated with the child’s mental health symptoms. They analysed genetic, weight, and health survey data from about 41,000 8-year-old children and their parents. The results suggest that a child's own BMI does not have a large effect on their anxiety symptoms. There was also no clear evidence that a child's BMI affected their symptoms of depression or ADHD. These results contradict previous studies, which did not account for parental genetics. Hughes et al. suggest that, at least for eight-year-olds, factors linked with adult weight and which differ between families may be more critical to a child's mental health than a child’s own weight. For older children and adolescents, this may not be the case, and the individual’s own weight may be more important. As a result, policies designed to reduce obesity in mid-childhood are unlikely to greatly improve the mental health of children. On the other hand, policies targeting the environmental or societal factors contributing to higher body weights, bias against people with higher weights, and poor child mental health directly may be more beneficial.. The development of an efficient photocatalyst for C2 product formation from CO. Оценка антиастенического эффекта последовательной терапии левокарнитином (ЛК) и ацетилкарнитином (АЛК) пациентов с артериальной гипертензией и/или ишемической болезнью сердца (ИБС) с астеническим синдромом (АС).. В открытое сравнительное исследование были включены 120 пациентов в возрасте 54—67 лет с артериальной гипертензией и/или ИБС с АС. Пациенты 1-й группы (. У больных 1-й группы отмечено статистически значимое уменьшение различных проявлений АС. Отличия носили достоверный характер по сравнению как с исходным уровнем, так и со 2-й группой. Установлено эндотелийпротективное действие ЛК и АЛК.. Полученные результаты свидетельствуют, что у таких коморбидных пациентов использование ЛК и АЛК уменьшает выраженность проявлений АС, а установленные эндотелиотропные свойства препаратов позволяют рекомендовать их в составе комплексной персонифицированной терапии пациентов с сердечно-сосудистыми заболеваниями.. Naproxen sodium 440 mg/diphenhydramine 50 mg combination demonstrated improvement in sleep maintenance (WASO) vs. naproxen sodium 550 mg and higher efficiency in average daily pain reduction compared with the comparison groups. The treatment was well tolerated There were no serious or unexpected adverse events reported in the study.. Сравнительный анализ эффективности и безопасности новой комбинации напроксена натрия и дифенгидрамина у пациентов с неспецифическим болевым синдромом в пояснично-крестцовом отделе спины (M54.5 «Боль внизу спины») и нарушением сна (G47.0 «Нарушения засыпания и поддержания сна [бессонница]»).. Проведено проспективное многоцентровое рандомизированное открытое сравнительное в параллельных группах клиническое исследование. Пациенты были рандомизированы в 3 группы. Больные 1-й группы получали напроксен натрия (440 мг) и дифенгидрамин (50 мг), 2-й — напроксен натрия (550 мг), 3-й — парацетамол (1000 мг) и дифенгидрамин (50 мг). Исследуемые препараты пациенты принимали однократно перед сном в течение 3 дней. Все пациенты также принимали 275 мг (1 таблетка) напроксена натрия в качестве препарата фоновой терапии. Первичным критерием эффективности было общее время бодрствования после наступления сна (WASO), измеряемое методом актиграфии. Также использовались критерии оценки продолжительности и качества сна и выраженности боли.. Анализ эффективности проведен для ITT популяции (. Применение комбинации напроксена натрия (440 мг) и дифенгидрамина (50 мг) характеризовалось более выраженным поддержанием сна по сравнению с напроксеном натрия 550 мг и более высокой эффективностью в отношении снижения интенсивности боли по сравнению со 2-й и 3-й группами. Отмечена хорошая переносимость препарата, серьезных нежелательных явлений зарегистрировано не было.

Topics: Acetaminophen; Acetylcarnitine; Acetylcholinesterase; Acids; Acinetobacter baumannii; Acinetobacter Infections; Adaptation, Psychological; Adolescent; Adsorption; Adult; Aged; Alcohol Drinking; Alzheimer Disease; Amikacin; Ammonia; Anaerobiosis; Animals; Anorexia; Anti-Bacterial Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Anxiety; Aptamers, Nucleotide; Asthenia; Attention Deficit Disorder with Hyperactivity; Bacterial Proteins; Beryllium; beta-Lactamases; Biofuels; Biomass; Biosensing Techniques; Bismuth; Blister; Body Mass Index; Body Surface Area; Boronic Acids; Brain; Breast Neoplasms; Butyrylcholinesterase; Cannabis; Carbapenems; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carboxylic Acids; Carcinoma, Hepatocellular; Cardiovascular Diseases; Carnitine; Case-Control Studies; Catalysis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Child; China; Cholinesterase Inhibitors; Clarithromycin; Clostridioides; Clostridioides difficile; Clostridium Infections; Cohort Studies; Colistin; Colitis; Colon; Coloring Agents; Coronary Artery Bypass; Creatinine; Crystalloid Solutions; Cytokines; Depression; Dextran Sulfate; Dextrans; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Diarrhea; Dietary Supplements; Diphenhydramine; Disease Models, Animal; Disease Outbreaks; Double-Blind Method; Doxorubicin; Drosophila; Drug Tapering; Dysbiosis; Electrons; Escherichia coli; Extracellular Vesicles; Fatigue; Female; Fermentation; gamma-Cyclodextrins; Gastrointestinal Microbiome; Glucose; Graft Survival; Graft vs Host Disease; Head and Neck Neoplasms; Heart Arrest, Induced; Hematopoietic Stem Cell Transplantation; High-Intensity Interval Training; Hippocampus; Humans; Hydrogen-Ion Concentration; Hypertension; Incidence; Interferon-gamma; Italy; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Lab-On-A-Chip Devices; Lactoferrin; Larva; Length of Stay; Lignin; Liver; Liver Neoplasms; Liver Transplantation; Living Donors; Low Back Pain; Lung; Lung Volume Measurements; Macrophages; Male; Melphalan; Men; Mendelian Randomization Analysis; Meropenem; Methane; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Mitochondrial Proteins; Molecular Docking Simulation; Molecular Structure; Mothers; Motivation; Mycoplasma; Mycoplasma hominis; Mycoplasma Infections; NAD; Nanocomposites; Nanoparticles; Nanotubes, Carbon; Naproxen; Neovascularization, Pathologic; Neurons; Nitrates; Nucleolin; Opuntia; Paratyphoid Fever; Phenotype; Phosphatidylinositol 3-Kinases; Phytochemicals; Plant Extracts; Pregnancy; Prevalence; Prospective Studies; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Wistar; Resveratrol; Retrospective Studies; Rifampin; Risk Factors; RNA, Messenger; Selenium; Sleep; Social Behavior; Soil; Soil Pollutants; Squamous Cell Carcinoma of Head and Neck; Staphylococcus aureus; Structure-Activity Relationship; Suicidal Ideation; Suicide; Superoxide Dismutase-1; Surveys and Questionnaires; Swimming; Syndrome; Tannins; Temperature; Transforming Growth Factor beta; Transplantation Conditioning; Treatment Outcome; Triple Negative Breast Neoplasms; Troponin T; Tumor Microenvironment; United Kingdom; Ureaplasma; Ureaplasma urealyticum; Urinary Tract Infections; Viscum; Waste Disposal Facilities; Wastewater; Water; Water Pollutants, Chemical; Wolfiporia; Young Adult

Breast cancer-related lymphedema (BCRL) is a treatment-related inflammatory complication in breast cancer survivors (BCSs). This study was aimed to evaluate the effect of synbiotic supplementation on serum concentrations of IL-10, TGF-β, VEGF, adiponectin, and edema volume among overweight or obese BCSs with lymphedema following a low-calorie diet (LCD).. In a randomized double-blind, controlled clinical trial, 88 obese and overweight BCSs women were randomized to synbiotic supplement (. There were no significant differences among participants in the baseline, except for IL-10 and adiponectin. Post-intervention, no significant differences were observed regarding the anti-inflammatory markers, including IL-10, VEGF, adiponectin, and TGF-β between the groups. After 10 weeks of intervention edema volume significantly decreased in the synbiotic group; additionally, anthropometric measurements (body weight, BMI, body fat percent, and WC) decreased in both groups significantly (. Synbiotic supplementation coupled with an LCD in a 10-week intervention had beneficial effects on increasing the serum TGF-β, IL-10, and adiponectin levels in women with BCRL. It also reduced arm lymphedema volume. Therefore, synbiotic supplementation can be effective in improving health status in BCRL patients.

Topics: Adiponectin; Anti-Inflammatory Agents; Biomarkers; Breast Neoplasms; Caloric Restriction; Cancer Survivors; Double-Blind Method; Edema; Female; Humans; Interleukin-10; Lymphedema; Obesity; Overweight; Synbiotics; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP).. The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression.. At baseline, a significantly higher level of expression (p < 0.05) of PD-L1 was identified in patient monocytes compared to HD. TCR modulation revealed dysfunction of circulating T-cells in patient baseline samples as compared to HD, and this was more pronounced in PD-1. Functional T cell analysis suggests that baseline T cell functionality is hampered in metastatic breast cancer patients, at least in part mediated by the PD-1 signaling pathway. These preliminary data support the rationale for investigating the possible beneficial effects of adding PD-1 blockade to improve responses to TGFβ blockade and radiotherapy.. NCT01401062 .

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; B7-H1 Antigen; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Female; Humans; Middle Aged; Programmed Cell Death 1 Receptor; Receptors, Antigen, T-Cell; Transforming Growth Factor beta; Young Adult

Inflammation affects tumor immune surveillance and resistance to therapy. Here, we show that production of IL1β in primary breast cancer tumors is linked with advanced disease and originates from tumor-infiltrating CD11c

Topics: Animals; Breast Neoplasms; Capecitabine; CD11c Antigen; Cell Line, Tumor; Cell Membrane; Female; Furans; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-1beta; Ketones; Leukocytes, Mononuclear; Macrophages; Mice; Mice, SCID; Myeloid Cells; Neoplasm Metastasis; Neoplasm Transplantation; Paclitaxel; Pilot Projects; Transcription, Genetic; Transforming Growth Factor beta

Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) have been involved in tumor growth and metastasis. Sevoflurane may promote angiogenesis, whereas propofol can present an anti-angiogenic effect. In this study, we compared the effects of propofol/remifentanil-based total intravenous anesthesia (TIVA) and sevoflurane-based inhalational anesthesia on the release of VEGF-C and TGF-β, as well as recurrence- free survival (RFS) rates in the patients undergoing breast cancer surgery.. Eighty female patients undergoing breast cancer resection were enrolled and randomized to receive either sevoflurane-based inhalational anesthesia (SEV group) or propofol/remifentanil-based TIVA (TIVA group). The serum concentrations of VEGF-C and TGF-β before and 24 h after surgery were measured and RFS rates over a two-year follow-up were analyzed in both groups. The postoperative pain scores assessed using a visual analogue scale (VAS) and the use of perioperative opioids were also evaluated.. Although VAS scores at 2 h and 24 h after surgery were comparable between the two groups, there were more patients receiving postoperative fentanyl in the TIVA group (16[40%]) compared with the SEV group (6[15%], p = 0.023). VEGF-C serum concentrations increased after surgery from 105 (87-193) pg/ml to174 (111-281) pg/ml in the SEV group (P = 0.009), but remained almost unchanged in the TIVA group with 134 (80-205) pg/ml vs.140(92-250) pg/ml(P = 0.402). The preoperative to postoperative change for VEGF-C of the SEV group (50 pg/ml) was significantly higher than that of the TIVA group (12 pg/ml) with a difference of 46 (- 11-113) pg/ml (P = 0.008). There were also no significant differences in the preoperative and postoperative TGF-β concentrations between the two groups. The two-year RFS rates were 78% and 95% in the SEV and TIVA groups (P = 0.221), respectively.. In comparison with sevoflurane-based inhalational anesthesia, propofol/remifentanil -based total intravenous anesthesia can effectively inhibit the release of VEGF-C induced by breast surgery, but didn't seem to be beneficial in the short-term recurrence rate of breast cancer.. Chictr.org.cn ChiCTR1800017910 . Retrospectively Registered (Date of registration: August 20, 2018).

Topics: Adult; Anesthesia, Inhalation; Anesthesia, Intravenous; Biomarkers; Breast Neoplasms; Drug Therapy, Combination; Female; Follow-Up Studies; Humans; Middle Aged; Prognosis; Propofol; Prospective Studies; Remifentanil; Sevoflurane; Transforming Growth Factor beta; Treatment Outcome; Vascular Endothelial Growth Factor C

To best define biomarkers of response, and to shed insight on mechanism of action of certain clinically important agents for early breast cancer, we used a brief-exposure paradigm in the preoperative setting to study transcriptional changes in patient tumors that occur with one dose of therapy prior to combination chemotherapy. Tumor biopsies from breast cancer patients enrolled in two preoperative clinical trials were obtained at baseline and after one dose of bevacizumab (HER2-negative), trastuzumab (HER2-positive) or nab-paclitaxel, followed by treatment with combination chemo-biologic therapy. RNA-Sequencing based PAM50 subtyping at baseline of 46 HER2-negative patients revealed a strong association between the basal-like subtype and pathologic complete response (pCR) to chemotherapy plus bevacizumab (p ≤ 0.0027), but did not provide sufficient specificity to predict response. However, a single dose of bevacizumab resulted in down-regulation of a well-characterized TGF-β activity signature in every single breast tumor that achieved pCR (p ≤ 0.004). The TGF-β signature was confirmed to be a tumor-specific read-out of the canonical TGF-β pathway using pSMAD2 (p ≤ 0.04), with predictive power unique to brief-exposure to bevacizumab (p ≤ 0.016), but not trastuzumab or nab-paclitaxel. Down-regulation of TGF-β activity was associated with reduction in tumor hypoxia by transcription and protein levels, suggesting therapy-induced disruption of an autocrine-loop between tumor stroma and malignant cells. Modulation of the TGF-β pathway upon brief-exposure to bevacizumab may provide an early functional readout of pCR to preoperative anti-angiogenic therapy in HER2-negative breast cancer, thus providing additional avenues for exploration in both preclinical and clinical settings with these agents.

Topics: Angiogenesis Inhibitors; Bevacizumab; Breast Neoplasms; Cell Hypoxia; Female; Humans; Receptor, ErbB-2; Sequence Analysis, RNA; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with the capability to act as tumour suppressor or tumour promoter depending on the cellular context. TGF-beta receptor type-2 (TGFBR2) is the ligand-binding receptor for all members of the TGF-β family. Data from mouse model experiments demonstrated that loss of Tgfbr2 expression in mammary fibroblasts was linked to tumour initiation and metastasis. Using a randomised tamoxifen trial cohort including in total 564 invasive breast carcinomas, we examined TGFBR2 expression (n=252) and phosphorylation level of downstream target SMAD2 (pSMAD2) (n=319) in cancer-associated fibroblasts (CAFs) and assessed links to clinicopathological markers, prognostic and treatment-predictive values. The study revealed that CAF-specific TGFBR2 expression correlated with improved recurrence-free survival. Multivariate analysis confirmed CAF-TGFBR2 to be an independent prognostic marker (multivariate Cox regression, hazard ratio: 0.534, 95% (CI): 0.360-0.793, P=0.002). CAF-specific pSMAD2 levels, however, did not associate with survival outcome. Experimentally, TGF-β signalling in fibroblasts was modulated using a TGF-β ligand and inhibitor or through lentiviral short hairpin RNA-mediated TGFBR2-specific knockdown. To determine the role of fibroblastic TGF-β pathway on breast cancer cells, we used cell contact-dependent cell growth and clonogenicity assays, which showed that knockdown of TGFBR2 in CAFs resulted in increased cell growth, proliferation and clonogenic survival. Further, in a mouse model transfected CAFs were co-injected with MCF7 and tumour weight and proportion was monitored. We found that mouse xenograft tumours comprising TGFBR2 knockdown fibroblasts were slightly bigger and displayed increased tumour cell capacity. Overall, our data demonstrate that fibroblast-related biomarkers possess clinically relevant information and that fibroblasts confer effects on breast cancer cell growth and survival. Regulation of tumour-stromal cross-talk through fibroblastic TGF-β pathway may depend on fibroblast phenotype, emphasising the importance to characterise tumour microenvironment subtypes.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Differentiation; Cell Proliferation; Cell Survival; Disease-Free Survival; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; MCF-7 Cells; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Predictive Value of Tests; Premenopause; Prognosis; Proportional Hazards Models; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study aimed to investigate the effect and mechanism of trauma flap healing promoted by the Zhikang capsule after radical breast cancer surgery. The enrolled breast cancer patients were randomly divided into two groups: treatment and observation. The patients in the treatment group were treated with the Zhikang capsule in addition to the conventional dressing changes, while patients in the observation group underwent only the regular dressing changes. Serum samples of 98 breast cancer patients (with complete clinical data) who underwent modified radical mastectomy were collected and analyzed for expressions of transforming growth factor beta (TGF-β) and basic fibroblast growth factor (bFGF). The drainage fluid amount and tissue necrosis rate were found to be lower in the treatment group than in the observation group. Moreover, bFGF expression in peripheral blood was higher in the treatment group than in the observation group. However, no significant difference was found between the two groups in the expression of TGF-β in peripheral blood. In conclusion, Zhikang capsule is effective in promoting flap healing after radical breast cancer surgery, and the increase of bFGF expression in peripheral blood may be the underlying mechanism.

Topics: Adult; Aged; Breast Neoplasms; Drugs, Chinese Herbal; Female; Fibroblast Growth Factor 2; Gene Expression; Humans; Mastectomy, Modified Radical; Middle Aged; Necrosis; Surgical Flaps; Transforming Growth Factor beta; Wound Healing

Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β-regulated "metastamir" that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.

Topics: Animals; Anoikis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Inbred BALB C; MicroRNAs; Neoplasm Metastasis; Proto-Oncogene Proteins; RNA, Neoplasm; Transforming Growth Factor beta; Up-Regulation

TGF-β acts as a suppressor of primary tumor initiation but has been implicated as a promoter of the later malignant stages. Here associations with risk of invasive breast cancer are assessed for single-nucleotide polymorphisms (SNP) tagging 17 genes in the canonical TGF-β ALK5/SMADs 2&3 and ALK1/SMADs 1&5 signaling pathways: LTBP1, LTBP2, LTBP4, TGFB1, TGFB2, TGFB3, TGFBR1(ALK5), ALK1, TGFBR2, Endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, and SMAD7 [Approved Human Gene Nomenclature Committee gene names: ACVRL1 (for ALK1) and ENG (for Endoglin)].. Three-hundred-fifty-four tag SNPs (minor allele frequency > 0.05) were selected for genotyping in a staged study design using 6,703 cases and 6,840 controls from the Studies of Epidemiology and Risk Factors in Cancer Heredity (SEARCH) study. Significant associations were meta-analyzed with data from the NCI Polish Breast Cancer Study (PBCS; 1,966 cases and 2,347 controls) and published data from the Breast Cancer Association Consortium (BCAC).. Associations of three SNPs, tagging TGFB1 (rs1982073), TGFBR1 (rs10512263), and TGFBR2 (rs4522809), were detected in SEARCH; however, associations became weaker in meta-analyses including data from PBCS and BCAC. Tumor subtype analyses indicated that the TGFB1 rs1982073 association may be confined to increased risk of developing progesterone receptor negative (PR(-)) tumors [1.18 (95% CI: 1.09-1.28), 4.1 × 10(-5) (P value for heterogeneity of ORs by PR status = 2.3 × 10(-4))]. There was no evidence for breast cancer risk associations with SNPs in the endothelial-specific pathway utilizing ALK1/SMADs 1&5 that promotes angiogenesis.. Common variation in the TGF-β ALK5/SMADs 2&3 signaling pathway, which initiates signaling at the cell surface to inhibit cell proliferation, might be related to risk of specific tumor subtypes.. The subtype specific associations require very large studies to be confirmed.

Topics: Aged; Breast Neoplasms; Case-Control Studies; Disease Susceptibility; Female; Humans; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Polymorphism, Single Nucleotide; Prognosis; Prospective Studies; Signal Transduction; Transforming Growth Factor beta

Regulatory T cells (T(Reg)), CD4(+)CD25(+)FOXP3(+), are implicated in suppressing tumor immune responses. We analyzed peripheral blood lymphocytes (PBL) from breast cancer patients receiving a modified HLA class II HER2/neu peptide (AE37) vaccine for T(Reg) cells and correlated their levels with vaccine-specific immune responses. The mean CD4(+)CD25(+)FOXP3(+) T(Reg) cells decreased in patients with vaccination with no significant difference in serum TGF-β levels. IFN-γ ELISPOT and DTH increased after vaccination with a good correlation between T(Reg) cell reduction and size of DTH to AE37. The T(Reg) cell reduction and associated immune response suggest that AE37 may be clinically useful.

Topics: Breast Neoplasms; Cancer Vaccines; Female; Genes, MHC Class II; Humans; Interferon-gamma; Leukocytes, Mononuclear; Receptor, ErbB-2; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

In breast cancer, vascular endothelial growth factor C, transforming growth factor β, placental growth factor, and fibroblast growth factor (acidic and basic) promote angiogenesis and metastases. We tested the hypothesis that a propofol-paravertebral anesthetic (PPA) technique would attenuate postoperative changes in these angiogenic factors to a greater extent than balanced general anesthesia (GA) and morphine analgesia in women undergoing surgery for primary breast cancer.. Forty women with primary breast cancer undergoing surgical excision were randomized to receive either standard GA or PPA technique. Venous blood was sampled before and at 24 h after surgery and serum analyzed. The primary endpoint was a preoperative versus postoperative change in vascular endothelial growth factor C and transforming growth factor β concentrations.. Using a visual analog scale (median [25-75% interquartile range]), PPA patients (1 [0-2]) had less pain at 2 h (P = 0.02) than did GA patients (3 [2-5]). The mean postoperative change in vascular endothelial growth factor C concentrations among GA patients was 733 versus 27 pg/ml for PPA patients (difference, 706 [97.5% CI, 280-1,130] pg/ml, P = 0.001). In contrast, the mean postoperative change in transforming growth factor β concentration among GA patients was -163 versus 146 pg/ml for PPA patients (difference, 309 [97.5% CI, -474 to -143] pg/ml, P = 0.005). Concentrations of placental growth factor and fibroblast growth factor, both acidic and basic, were undetectable in serum.. Anesthetic technique influences serum concentrations of factors associated with angiogenesis in primary breast cancer surgery.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Anesthesia; Anesthesia, General; Anesthesia, Intravenous; Biomarkers; Breast Neoplasms; Female; Humans; Middle Aged; Propofol; Prospective Studies; Transforming Growth Factor beta; Vascular Endothelial Growth Factor C; Young Adult

The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer.. Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected.. Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached.. Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cancer Vaccines; Combined Modality Therapy; Fatigue; Female; Headache; Humans; Kaplan-Meier Estimate; Middle Aged; Molecular Sequence Data; Neoplasm Metastasis; Pain; Peptides; Receptor, ErbB-2; T-Lymphocytes; Transforming Growth Factor beta; Trastuzumab; Treatment Outcome; Vaccination

To evaluate the modifications of circulating angiogenic factors, metalloproteinases and acute-phase cytokines after the first single zoledronic acid (ZA) intravenous infusion.. Eighteen consecutive breast cancer patients with bone metastases were evaluated for circulating levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), metalloproteinase 1 (MMP-1), metalloproteinase 2 (MMP-2), interleukins 1beta, 6 and 8 (IL-1beta, IL-6, IL-8), interferon gamma, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta1 just before and 2 and 7 days after ZA infusion.. The MMP-2 basal value showed a statistically significant decrease 48 h after ZA (p = 0.01), being at 7 days higher than the day 2 value (p = 0.03). The VEGF basal value showed a statistically significant decrease 48 h after ZA infusion (p = 0.03), increasing above the basal level at 7 days (p = 0.07). The bFGF basal level almost significantly decreased 2 days after infusion (p = 0.06), being at 7 days higher than the basal value (p = 0.09). Comparing the day 2 values with basal ones, the linear regression model showed a significant positive correlation between IL-8 and bFGF (p = 0.02), IL-8 and TNF-alpha (p < 0.0001), bFGF and TNF-alpha (p = 0.01), MMP-1 and TNF-alpha (p = 0.02).. ZA could exert an antiangiogenic activity and inhibition of tumor cell bone invasiveness by a transient reduction of VEGF, bFGF and MMP-2 circulating levels after infusion.

Topics: Aged; Aged, 80 and over; Angiogenesis Inducing Agents; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cytokines; Diphosphonates; Female; Fibroblast Growth Factor 2; Humans; Imidazoles; Interferon-gamma; Interleukins; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Metalloproteases; Middle Aged; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Zoledronic Acid

Allogeneic blood stem-cell donors demonstrate more vigorous mobilization of CD34(+) cells to the circulation in response to cytokine administration than do autologous donors. Transforming growth factor (TGF-beta1) has been implicated as a mobilization inhibitor. A study was designed to determine whether plasma TGF-beta1 levels are elevated in cytokine-mobilized autologous cancer donors compared with cytokine-mobilized normal donors.. Plasma collected from 29 autologous cancer donors and 33 normal allogeneic stem-cell donors following administration of mobilizing cytokines just prior to the first collection was assayed for TGF-beta1 using a sandwich-type ELISA. Plasma from three volunteers not treated with cytokine was also analyzed. Comparisons were made using the Student's t test on log-transformed data.. Average TGF-beta1 levels in the plasma of cancer patients were significantly higher than in allogeneic stem-cell donors (4.4 ng/mL versus 7.2 ng/mL; p = 0.038). The allogeneic donors required fewer collections to harvest greater numbers of CD34(+) cells and colony-forming unit granulocyte-macrophage (CFU-GM) than autologous donors. Plasma from three untreated volunteers had mean TGF-beta1 levels of 0.36 ng/mL, with all three levels below the 25th percentile for allogeneic donors and the 5th percentile for cancer patients.. Cytokine administration was associated with increased plasma TGF-beta1 levels. The levels were higher in cancer patients being mobilized for stem-cell collection than in allogeneic blood stem-cell donors. These differences could underlie the increased number of apheresis procedures required to harvest autologous graft products from cancer patients.

Topics: Antigens, CD34; Blood Donors; Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Humans; Lymphoma, Non-Hodgkin; Neoplasms; Transforming Growth Factor beta; Transplantation, Autologous; Transplantation, Homologous

In addition to suppressing breast cancer cell growth, retinoids potentiate growth inhibition in human breast cancer when tested in vitro and in vivo with tamoxifen and/or interferon. The purpose of this study was to ascertain the biologic effects of all-trans-retinoic acid (ATRA) administered alone and with tamoxifen +/- interferon and to identify the relationship between ATRA plasma concentrations and optimal biological dose (the lowest dose that produces a biological response). Three consecutive groups of 15 patients with locally advanced operable breast cancer were treated, in accordance with good clinical practice (GCP) requirements, with ATRA at 3 dose levels alone or with tamoxifen +/- alpha-interferon 2a at flat doses. After 3 weeks, the tumors were surgically removed. Biological parameters measured at the beginning (in biopsy tissue) and end (in surgical tissue) of the study were compared. The optimal biological dose for ATRA was 15 mg/m2/day. Treatments influenced tumor grade but not cell cycle kinetics (G0-G1 phase) or proliferation (Ki67 levels). ATRA induced progesterone receptors independent of dose level and co-administered drugs, but did not induce estrogen receptors when administered alone. Retinoic acid receptor (RAR)-alpha was not affected by treatment and RAR-alpha was moderately influenced whereas RAR-beta (concomitantly with transforming growth factor-beta) was induced in 33% of patients by ATRA alone. ATRA pharmacokinetics were dose- and time-dependent. Neither the ATRA + tamoxifen nor the ATRA + tamoxifen + interferon combinations potentiated the ATRA-induced biological changes. Future studies evaluating the role of RAR-beta as a biological marker of retinoid activity are warranted.

Topics: Aged; Aneuploidy; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Bone Marrow Diseases; Breast Neoplasms; Carcinoma; Drug Administration Schedule; Drug Interactions; Female; Follow-Up Studies; Headache; Humans; Hypercholesterolemia; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Mastectomy; Middle Aged; Neoplasm Proteins; Receptors, Retinoic Acid; Receptors, Steroid; Recombinant Proteins; Safety; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome; Tretinoin

The purpose of this study was to establish the safety and tolerability of recombinant transforming growth factor-beta3 (TGF-beta3; CGP 46614) mouthwashes intended for prevention of chemotherapy-induced mucositis. Local effects were especially analyzed by objective and subjective measurements of mucositis. Secondary aims were analysis of potential systemic exposure and development of anti-TGF-beta3-antibodies. Eleven breast cancer patients received chemotherapy with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. (n = 8) or 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days (n = 3). TGF-beta3 mouthwashes (10 ml; provided by Novartis, Basel, Switzerland) were administered for 4 days, four times a day, starting 1 day before chemotherapy. The dose was escalated in following patients from 25 microg/ml (n = 3) to 50 microg/ml (n = 3) and 100 microg/ml (n = 5). Clinically, the mucosa was scored objectively and according to WHO criteria. The percentage of viable oral epithelial cells was determined by trypan blue dye exclusion. Morphology of cells was assessed in buccal smears. Plasma samples were collected for determination of TGF-beta3 levels and anti-TGF-beta3-antibodies. Adverse events were recorded by the patient in a diary. Mouthwashes with TGF-beta3 were well tolerated. Three patients scored for mucositis > grade 0 (WHO grading criteria). The percentage of viable oral epithelial cells in patients treated with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. was stable, whereas in patients treated with 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days, an increase was observed. The morphology of buccal cells showed a transient shift from mature to immature cells in the first week. Neither systemic absorption of TGF-beta3 nor development of TGF-beta3-antibodies was observed. TGF-beta3 mouthwashes were well tolerated and deserve further study in preventing chemotherapy-induced mucositis.

Topics: Administration, Oral; Adult; Antibodies; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Middle Aged; Mouth Mucosa; Mouthwashes; Stomatitis; Transforming Growth Factor beta

Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factor-beta (TGF-beta) levels and serum lipids was also examined.. Thirty-one patients were treated with tamoxifen (20 mg p.o. daily), and fenretinide (400 mg p.o. daily with a 3-day drug holiday each month). Plasma TGF-beta testing was performed using isoform specific sandwich ELISA.. Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ER-negative patients, and three hormone therapy naive ER-positive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of -13.5 mg/dl; p = 0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18 mg/dl, p = 0.0001, a 35% increase) with tamoxifen and fenretinide therapy. TGF-beta1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated.. Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400 mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.

Topics: Adult; Aged; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease Progression; Female; Fenretinide; Humans; Lipids; Middle Aged; Pilot Projects; Tamoxifen; Transforming Growth Factor beta; Treatment Outcome

The relationship between plasma transforming growth factor-beta 1 (TGF-beta 1) and second primary breast cancer was explored in a prevention trial of the synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide; 4-HPR). Plasma concentrations of TGF-beta 1 were measured by radioimmunoassay in plasma obtained at randomisation and after approximately 1 year of intervention in 28 women treated with 4-HPR and 27 untreated controls with stage I breast cancer. Baseline and 1 year growth factor concentrations were not significantly different between treated and control groups. After a median follow-up of 65 months, an increase in TGF-beta 1 over 1 year was the only significant, independent predictor of a shorter survival free from secondary primary breast cancer. Moreover, the change in TGF-beta 1 levels had a tendency to influence the treatment effect on second breast cancer incidence. Our data suggest that the role of plasma TGF-beta 1 as a surrogate endpoint of breast carcinogenesis should be assessed further.

Topics: Adult; Aged; Antineoplastic Agents; Breast Neoplasms; Disease-Free Survival; Female; Fenretinide; Follow-Up Studies; Humans; Middle Aged; Neoplasms, Second Primary; Radioimmunoassay; Transforming Growth Factor beta

Tamoxifen is an anti-estrogen with proven efficacy and low toxicity in the treatment of breast cancer. However, tamoxifen has been shown to exert a number of nonhormonal as well as hormonal effects. One nonhormonal effect of tamoxifen is the induction of transforming growth factor-beta (TGF-beta) secretion. TGF-beta has been implicated in the pathogenesis of radiation-induced fibrosis.. We investigated the development of lung fibrosis in breast cancer patients who were treated after mastectomy with radiotherapy, with or without simultaneous adjuvant treatment with tamoxifen.. Data from 196 women were included in the analysis. Eighty-four women were postmenopausal patients who participated in a randomized trial testing tamoxifen as an adjuvant to postmastectomy radiotherapy. The radiotherapy technique employed an 8-MV photon field covering the axillary and the infraclavicular and supraclavicular regions of the affected side of the chest; the chest wall was treated with an abutted electron field. Optical density changes in pretreatment and post-treatment chest x-ray films were used to monitor the development of lung fibrosis; lung reactions were assessed in the photon-irradiated field only. Logistic regression analysis was used to explore relationships between radiation dose and the development of lung fibrosis in patients either receiving or not receiving tamoxifen. All P values are from two-sided tests.. Among the 84 women who participated in the randomized trial of radiotherapy plus tamoxifen (n = 38) versus radiotherapy alone (n = 46), there was a significant association between tamoxifen treatment and the incidence of marked lung fibrosis (relative risk = 2.0; 95% confidence interval [CI] = 1.2-3.5; P = .01). When logistic regression analysis was used to evaluate data from all 196 patients, a highly significant relationship was found between the incidence of lung fibrosis and total radiation dose (P = .0005). In the full analysis, an increased risk of marked lung fibrosis was found again for patients who received tamoxifen simultaneously with radiotherapy (with patients receiving radiotherapy alone as the referent, odds ratio = 2.9; 95% CI = 1.3-6.3; P = .007). Patient age and menopausal status did not significantly influence the results.. Tamoxifen treatment during postmastectomy radiotherapy enhances the risk of radiation-induced lung fibrosis.. In view of pre-existing data, we hypothesize that tamoxifen mediates the enhancement of radiation-induced lung fibrosis through the induction of TGF-beta secretion. If this hypothesis is correct, new strategies might be devised for preventing or reducing radiation-induced fibrosis. Because we studied a relatively small portion of the irradiated lung, we cannot recommend changes in current therapeutic measures; however, we strongly encourage additional studies of lung fibrosis in patients receiving tamoxifen and radiotherapy.

Topics: Aged; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chemotherapy, Adjuvant; Female; Humans; Mastectomy; Middle Aged; Pulmonary Fibrosis; Radiotherapy Dosage; Radiotherapy, Adjuvant; Tamoxifen; Transforming Growth Factor beta

Fenretinide (4-HPR), a synthetic derivative of retinoic acid, has proven effective at inhibiting in vitro breast cancer cell growth and preventing the progression of chemically induced mammary carcinoma in rodents. Our group has made a particular effort with regard to this molecule in clinical studies aimed at evaluating its pharmacology, toxicity, and efficacy in breast cancer prevention. We have demonstrated that 4-HPR blood levels remain constant during administration for as long as 5 years, that the drug accumulates in the human breast, and that it induces a significant decline of plasma insulin-like growth factor-I (IGF-I) levels. To date, 2,972 Stage I breast cancer patients have been randomized to evaluate the efficacy of a 5-year administration of 4-HPR to prevent new contralateral primary breast cancers. Compliance to protocol and treatment is high and tolerability of the drug is good; only 51 women out of 1,397 (3.6%) had to interrupt drug intake due to toxicity. The only potential limitation to the extensive use of 4-HPR is diminished dark adaptation, which occurs in about one-fourth of the patients and is dependent on the decline of plasma retinol below the threshold level of 100 ng/ml. Plasma levels of (4-methoxyphenyl)retinamide (4-MPR), the principal metabolite of 4-HPR, which are higher in elderly women with a high percentage of adipose tissue, are the major determinants of the retinol decrease. However, about 50% of the patients with altered dark-adaptometry are asymptomatic and the alterations are promptly reversible upon drug discontinuation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Breast; Breast Neoplasms; Drug Therapy, Combination; Drug Tolerance; Female; Fenretinide; Humans; Insulin-Like Growth Factor I; Middle Aged; Pilot Projects; Research Design; Tamoxifen; Transforming Growth Factor beta; Treatment Outcome; Vitamin A

Except for biochemical effects, suspension state (Sus) is proved to induce epithelial-mesenchymal transition (EMT) of circulating tumor cells (CTCs) mechanically. However, the difference between the effects of the mechanical microenvironment in capillaries (simplified as shear stress (SS) and Sus) and single Sus on EMT is unclear, nor the underlying mechanism. Here, breast tumor cells (BTCs) were loaded with Sus and SS to mimic the situation of CTCs stimulated by these two kinds of mechanics. It was demonstrated that the EMT of BTCs was enhanced by Sus and SS and the mechanotransductor yes-associated protein (YAP) was partially cytoplasmic stored with microRNA (miR)-29b decreased, which was detected by miR sequencing. Though it couldn't possess a feedback regulation, YAP promoted miR-29b expression and posttranscriptionally regulated BTCs EMT through miR-29b, where transforming growth factor β involved. Analysis of clinical database showed that high miR-29b expression was beneficial to high survival rate stabilizing its role of tumor suppressor. This study discovers the mechanism that Sus and SS promote BTCs EMT by YAP through miR-29b posttranscriptionally and highlight the potential of YAP and miR-29b in tumor therapy. The combination of suspension state and shear stress promotes transforming growth factor β involved epithelial-mesenchymal transition by yes-associated protein through microRNA-29b.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; MicroRNAs; Transforming Growth Factor beta; Tumor Microenvironment; YAP-Signaling Proteins

Topics: Animals; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cytokines; Female; Fibroblasts; Humans; Interleukin-6; Mice; Transforming Growth Factor beta

Karyopherin-α3 (KPNA3), a karyopherin- α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMTrelated genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-β or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-β-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type. [BMB Reports 2023; 56(2): 120-125].

Topics: alpha Karyopherins; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

The low sensitivity of tumor cells and immunosuppressive microenvironments lead to unsatisfactory efficacy of natural killer (NK) cell immunotherapy. In this work, we developed a safe and effective combination treatment strategy by integrating a selenadiazole derivative (PSeD)-loaded metal azolate framework (PSeD@MAF-4(R)) with NK cells derived from cancer patients against a xenograft human breast tumor model. Intriguingly, it was found that only PSeD@MAF-4(R) pretreatment on tumor cells exhibited synergistic effects with NK cells in inhibiting tumor cell growth by up-regulating NKG2D and its ligands to maximize the interactions between NK and MCF-7 cells. Moreover, PSeD@MAF-4(R) pretreatment could significantly enhance the degranulation of NK cells and regulate their secretions of pro- or anti-inflammatory cytokines (

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Immunotherapy; Killer Cells, Natural; Transforming Growth Factor beta; Tumor Microenvironment

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-β signaling by directly interacting with TGF-β type I receptor (TβRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TβRI in the ER. By decreasing cell surface TβRI levels, USP19-ER inhibits TGF-β/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-β-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Endopeptidases; Epithelial-Mesenchymal Transition; Female; Humans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Breast cancer subtypes and their phenotypes parallel different stages of the mammary epithelial cell developmental hierarchy. Discovering mechanisms that control lineage identity could provide novel avenues for mitigating disease progression. Here we report that the transcriptional corepressor TLE3 is a guardian of luminal cell fate in breast cancer and operates independently of the estrogen receptor. In luminal breast cancer, TLE3 actively repressed the gene-expression signature associated with highly aggressive basal-like breast cancers (BLBC). Moreover, maintenance of the luminal lineage depended on the appropriate localization of TLE3 to its transcriptional targets, a process mediated by interactions with FOXA1. By repressing genes that drive BLBC phenotypes, including SOX9 and TGFβ2, TLE3 prevented the acquisition of a hybrid epithelial-mesenchymal state and reduced metastatic capacity and aggressive cellular behaviors. These results establish TLE3 as an essential transcriptional repressor that sustains the more differentiated and less metastatic nature of luminal breast cancers. Approaches to induce TLE3 expression could promote the acquisition of less aggressive, more treatable disease states to extend patient survival.. Transcriptional corepressor TLE3 actively suppresses SOX9 and TGFβ transcriptional programs to sustain the luminal lineage identity of breast cancer cells and to inhibit metastatic progression.

Topics: Breast Neoplasms; Cell Differentiation; Co-Repressor Proteins; Humans; Neoplasms; Receptors, Estrogen; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF-β induces pleckstrin-2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA-MB-231 and MCF-7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin-2 localizes to the cell membrane and cell protrusions following TGF-β treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF-β- and PLEK2-induced cell invasion. The carboxyl-terminal PH domain of PLEK2 is critical for TGF-β- and PLEK2-induced Akt activation and plays an important role in cell invasion. Pleckstrin-2 interacts with PPM1B and promotes its ubiquitin-dependent degradation. The PLEK2-PPM1B axis utilizes nuclear factor-κB signaling to promote cell migration and invasion. Our data implicate the TGF-β-STAT3/Smad3-PLEK2-PPM1B signaling cascade in TGF-β-induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Membrane Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Phosphatase 2C; Transforming Growth Factor beta; Transforming Growth Factors

Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancer-related deaths in women worldwide. Cancer-associated fibroblasts (CAFs) are one of the fundamental cellular components of the tumor microenvironment and play a critical role in the initiation, progression, and therapy resistance of breast cancer. However, the detailed molecular mechanisms of CAFs activation from normal fibroblasts (NFs) are still not well understood. In the present study, we reported that ZNF32 expression in breast cancer cells was negatively correlated with CAF-related markers (FSP1, α-SMA, and FAP) in stromal fibroblasts, and loss of ZNF32 promoted the activation of CAFs, as evidenced by the enhanced proliferation and contractility of CAFs. ZNF32 deficiency-mediated fibroblast activation promoted the growth and metastasis of breast cancer cells in vitro and in vivo. Mechanistically, we demonstrated that ZNF32 inhibited TGFB1 transcription by directly binding to the -1968/-1962 region of the TGFB1 promoter, leading to the prevention of fibroblast activation. Altogether, our findings reveal an important mechanism by which ZNF32 suppression increases the transcription of the TGFB1 gene in breast cancer cells, and subsequently, elevated levels of secretory TGF-β stimulate NFs transformation into CAFs, which in turn facilitates the malignant progression of breast cancer. Our data implicated ZNF32 as a potential therapeutic strategy against breast cancer.

Topics: Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Proliferation; Female; Fibroblasts; Humans; Kruppel-Like Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

MicroRNA-9 (miR-9) has previously been described as a dual-functional RNA during breast cancer progression and its roles need to be clarified thoroughly.. A miR-9 knockout mode of mouse breast cancer, the MMTV-PyMT model (PyMT-miR-9. MiR-9 is ectopically expressed in breast cancer and its level is negatively correlated with the prognosis, especially in basal-like breast cancer patients. Additionally, miR-9 is essential for breast cancer progression by promoting the expansion and activity of tumour-initiating cells (TICs) in preneoplastic glands, established tumours and xenograft modes. Mechanistically, the activity of TICs hinges on a positive TGF-β/miR-9 regulatory loop mediated by the STARD13/YAP axis.. These findings demonstrate that miR-9 is an oncogenic miRNA rather than a tumour-suppressor in breast cancer, calling for rectification of the model for this conserved and highly abundant miRNA.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Neoplastic Stem Cells; Transforming Growth Factor beta

Topics: Breast Neoplasms; Female; Humans; Neoplasm Staging; Transforming Growth Factor beta

Apelin/APJ axis plays a critical role in cancer progression, thus its targeting inhibits tumor growth. However, blocking of Apelin/APJ axis in combination with immunotherapeutic approaches may be more effective. This study aimed to investigate the effects of APJ antagonist ML221 in combination with a DC vaccine on angiogenic, metastatic and apoptotic-related factors in a breast cancer (BC) model. Four groups of female BALB/c mice with 4T1-induced BC were treated with PBS, APJ antagonist ML221, DC vaccine, and "ML221 + DC vaccine". After completion of the treatment, the mice were sacrificed and the serum levels of IL-9 and IL-35 as well as the mRNA expression of angiogenesis (including VEGF, FGF-2, and TGF-β), metastasis (including MMP-2, MMP-9, CXCR4) and apoptosis-related markers (Bcl-2, Bax, Caspase-3) in tumor tissues were determined using ELISA and real-time PCR, respectively. Angiogenesis was also evaluated by co-immunostaining of tumor tissues with CD31 and DAPI. Primary tumor metastasis to the liver was analyzed using hematoxylin-eosin staining. The efficiency of combination therapy with "ML221 + DC vaccine" was remarkably higher than single therapies in preventing liver metastasis compared to the control group. In comparison with the control group, combination therapy could significantly reduce the expression of MMP-2, MMP-9, CXCR4, VEGF, FGF-2, and TGF-β in tumor tissues (P < 0.05). It also decreased the serum level of IL-9 and IL-35 compared with the control group (P < 0.0001). Moreover, vascular density and vessel diameter were significantly reduced in the combination therapy group compared with the control group (P < 0.0001). Overall, our findings demonstrate that combination therapy using a blocker of the apelin/APJ axis and DC vaccine can be considered a promising therapeutic program in cancers.

Topics: Animals; Apelin; Apelin Receptors; Breast Neoplasms; Dendritic Cells; Female; Fibroblast Growth Factor 2; Interleukin-9; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Transforming Growth Factor beta; Vaccine Efficacy; Vascular Endothelial Growth Factor A

Tumor metastasis contributes to the low overall survival of tumor patients, while transforming growth factor-β (TGFβ) has been recognized as a prominently promoting factor in the development of tumor metastasis. Platelets reserve abundant TGFβ, which will be secreted to peripheral blood after activation, and they are the dominant source of circulating TGFβ. Therefore, downregulation of platelet-derived TGFβ is expected to inhibit the metastasis of circulating tumor cells. Here, unfolded human serum albumin (HSA)-coated perfluorotributylamine (PFTBA) nanoparticles were constructed to display a favorable platelet delivery and an antiplatelet effect to downregulate platelet-derived TGFβ

Topics: Albumins; Animals; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Mice; Nanoparticles; Neoplasm Metastasis; Serum Albumin, Human; Transforming Growth Factor beta

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; GTPase-Activating Proteins; Humans; Integrins; Mice; Neoplasm Metastasis; Talin; Transforming Growth Factor beta

We investigated a prognostic impact of radiotherapy-induced lymphopenia (RIL) in breast cancer patients treated with breast-conservative surgery (BCS). We included 531 breast cancer patients who were treated with BCS and adjuvant radiotherapy. None of these received (neo)adjuvant chemotherapy. Pre- and post- absolute lymphocyte counts (ALC) were reviewed before and after radiotherapy. The primary endpoint was to evaluate recurrence-free survival (RFS) according to the pre-to-post ALC ratio. Binary logistic regression model was used to identify risk factors for RIL. Either continuous or categorical (> 2.4) pre-to-post ALC ratio was associated with RFS. In 531 patients receiving whole breast irradiation (WBI) and regional nodal irradiation (RNI), RFS was significantly reduced in the patients with high pre-to-post ALC ration (> 2.4). In multivariable analysis, low pre-to-post post ALC ratio was significantly related to decreased RFS in the multivariable analysis (HR 2.293, 95% CIs 1.110-4.735, P = 0.025). In 452 patients treated with WBI alone, high pre-to-post ALC ratio was still significantly associated with decreased RFS in the multivariable analysis (HR 2.708, 95% CIs 1.016-7.218, P = 0.046). In binary logistic regression analysis, RNI was only significant risk factor for clinically meaningful RIL. Our findings show that a markedly decrease in ALC during radiotherapy has a negative prognostic impact.

Topics: Breast Neoplasms; Female; Humans; Lymphocyte Count; Lymphopenia; Prognosis; Radiation Oncology; Transforming Growth Factor beta

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-β (TGF-β)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-β-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-β-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-β-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-β-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-β signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Topics: Breast Neoplasms; Carcinoma; Female; Fibroblasts; Glutamine; Histones; Humans; Mechanistic Target of Rapamycin Complex 1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors; Tumor Microenvironment

Luminal-B type human breast cancer cell line (BT-474) to assess the synergistic effects of ozone applied after chemotherapeutic treatment with various dosages of doxorubicin, and compare the results with the effects on L929 fibroblast cell line.. Doxorubicin (1-50 M) was added to each cell lines and left to sit for 24 h at 37 °C. Then, as combination groups, half of the groups were incubated with 30 g/mL ozone for 25 min. Tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and matrix metalloproteinase-2 and - 9 (MMP-2 and MMP-9) levels were measured using the MTT test, flow cytometry, and immunocytochemistry, respectively.. When compared to simply doxorubicin-applied cells without ozone treatment, each dose of doxorubicin + ozone treatment considerably boosted L929 viability but significantly decreased BT-474 viability. Additionally, the combination increased the apoptotic impact of doxorubicin on BT-474 but not L929 (P < 0.001). TGF-, MMP-2, and MMP-9 levels of L929 after combination were substantially higher than those of the other groups (P < 0.01). Doxorubicin's effect on BT-474's protein levels, which had significantly decreased in comparison to those of the other groups, was reversed by the combination treatment (P < 0.05).. Doxorubicin's anti-proliferative and apoptotic effects were enhanced by ozone treatment in BT-474 cells, but it also repaired and healed healthy fibroblast cells that had been harmed by the cytotoxicity of the chemotherapy drug. If doxorubicin and ozone treatment are coupled, BT-474 cells may develop resistance to it through expressions of TNF-α, TGF-β, MMP-2, and MMP-9.

Topics: Apoptosis; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.. We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.. In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix  that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.. Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Topics: Alternative Splicing; Animals; Breast Neoplasms; Female; Fibronectins; Humans; Lung Neoplasms; Mice; Protein Isoforms; Serine-Arginine Splicing Factors; Transforming Growth Factor beta

TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape.. In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections.. A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland.. We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.

Topics: Animals; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Neoplasms, Animal; Mice; Proteasome Endopeptidase Complex; Proteomics; Signal Transduction; Transforming Growth Factor beta

Metastasis is the leading driver of cancer-related death. Tumor cell plasticity associated with the epithelial-mesenchymal transition (EMT), an embryonic program also observed in carcinomas, has been proposed to explain the colonization of distant organs by the primary tumor cells. Many studies have established correlations between EMT marker expression in the primary tumor and metastasis in vivo. However, the longstanding model of EMT-transitioned cells disseminating to secondary sites is still actively debated and hybrid states are presently considered as more relevant during tumor progression and metastasis. Here, we describe an unexplored role of EMT on the tumor microenvironment by controlling tumor innervation. Using in vitro and in vivo breast tumor progression models, we demonstrate that TGFβ-mediated tumor cell EMT triggers the expression of the embryonic LincRNA Platr18 those elevated expression controls the expression of the axon guidance protein semaphorin-4F and other neuron-related molecules such as IGSF11/VSIG-3. Platr18/Sema4F axis silencing abrogates axonogenesis and attenuates metastasis. Our observations suggest that EMT-transitioned cells are also locally required in the primary tumor to support distant dissemination by promoting axonogenesis, a biological process known for its role in metastatic progression of breast cancer.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Fibronectins; HSP90 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Mammary Neoplasms, Experimental; Mice; Microfilament Proteins; Osteoblasts; Osteoclasts; Osteogenesis; Osteolysis; Proteome; Secretome; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

Transforming growth factor-β (TGF-β) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-β signaling by binding to the inhibitory SMAD7 and inducing its proteasomal degradation. How RNF12 activity is regulated and its exact role in cancer is incompletely understood. Here we report that RNF12 was overexpressed in invasive breast cancers and its high expression correlated with poor prognosis. RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in zebrafish and murine xenograft models. RNF12 levels were positively associated with the phosphorylated AKT/protein kinase B (PKB) levels, and both displayed significant higher levels in the basal-like subtype compared with the levels in luminal-like subtype of breast cancer cells. Mechanistically, AKT-mediated phosphorylation induced the nuclear localization of RNF12, maintained its stability, and accelerated the degradation of SMAD7 mediated by RNF12. Furthermore, we demonstrated that RNF12 and AKT cooperated functionally in breast cancer cell migration. Notably, RNF12 expression strongly correlated with both phosphorylated AKT and phosphorylated SMAD2 levels in breast cancer tissues. Thus, our results uncovered RNF12 as an important determinant in the crosstalk between the TGF-β and AKT signaling pathways during breast cancer progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Nucleus; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Prognosis; Protein Stability; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Zebrafish

KDM6A, an X chromosome-linked histone lysine demethylase, was reported to be frequently mutated in many tumor types including breast and bladder cancer. However, the functional role of KDM6A is not fully understood. Using MCF10A as a model of non-tumorigenic epithelial breast cells, we found that silencing KDM6A promoted cell migration and transformation demonstrated by the formation of tumor-like acini in three-dimensional culture. KDM6A loss reduced the sensitivity of MCF10A cells to therapeutic agents commonly used to treat patients with triple-negative breast cancer and also induced TGFβ extracellular secretion leading to suppressed expression of cytotoxic genes in normal human CD8+ T cells in vitro. Interestingly, when cells were treated with TGFβ, de novo synthesis of KDM6A protein was suppressed while TGFB1 transcription was enhanced, indicating a TGFβ/KDM6A-negative regulatory axis. Furthermore, both KDM6A deficiency and TGFβ treatment promoted disorganized acinar structures in three-dimensional culture, as well as transcriptional profiles associated with epithelial-to-mesenchymal transition and metastasis, suggesting KDM6A depletion and TGFβ drive tumor progression.. Our study provides the preclinical rationale for evaluating KDM6A and TGFβ in breast tumor samples as predictors for response to chemo and immunotherapy, informing personalized therapy based on these findings.

Topics: Antineoplastic Agents; Breast Neoplasms; Epithelial Cells; Female; Histone Demethylases; Humans; Transforming Growth Factor beta; Urinary Bladder Neoplasms

Doxorubicin (DOX) resistance remains a major challenge for adriamycin-based treatment of breast cancer (BC). Transforming growth factor β (TGF-β) has been reported to contribute to drug resistance. Although the role of long noncoding RNAs (LncRNAs) in cancer progression has been widely studied, its effect on TGF-β-induced resistance remains limited. This study aimed to investigate the role of LncRNA on the regulation of TGF-β-induced drug resistance.. Cell counting kit-8 (CCK-8) and an EdU assay were used to evaluate cell viability and proliferation. The level of LncRNA mRNA expression in BC tissues and cells was examined by quantitative real-time PCR. Changes in epithelial-mesenchymal transition (EMT) and cell apoptosis were quantified by Western blot and immunofluorescence.. TGF-β induced EMT and promoted DOX resistance. LncRNA urothelial carcinoma-associated 1(lncRNA UCA1) associated with TGF-β was upregulated in BC cells and tissues. LncRNA UCA1 silencing enhanced sensitivity to DOX decreased cellular proliferation and increased apoptosis in BC cells. The effect of TGF-β on EMT and DOX resistance disappeared following a lncRNA UCA1 knockdown.. These findings suggest that lncRNA-UCA1, a mediator of TGF-β signaling, could predispose BC patients to EMT and DOX resistance.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation

TGFβ is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFβ stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFβ signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFβ1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFβ signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFβ signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFβ signaling targets, consistent with its extracellular binding to TGFβ. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFβ1 and attenuates (but importantly does not eliminate) TGFβ signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

Topics: Actins; Breast Neoplasms; Cells, Cultured; Cicatrix; Collagen; Collagen Type III; Cysteine; Female; Fibroblasts; Humans; Myofibroblasts; Transforming Growth Factor beta; Tumor Microenvironment

Acquired metastasis and invasion of cancer cells during radiotherapy are in part due to induction of epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, which are mediated by TGF-β signaling. Here we evaluated the anti-metastatic therapeutic potential of vactosertib, an orally bioavailable TGF-β type I receptor (activin receptor-like kinase 5, ALK5) inhibitor, via suppression of radiation-induced EMT and CSC properties, oxidative stress generation, and breast to lung metastasis in a breast cancer mouse model and breast cancer cell lines.. Co-treatment of vactosertib with radiation was investigated in the 4T1-Luc allografted BALB/c syngeneic mouse model and in 4T1-Luc and MDA-MB-231 cells. The anti-metastatic therapeutic potential of vactosertib in breast cancer was investigated using fluorescence immunohistochemistry, real-time quantitative reverse transcription-polymerase chain reaction, western blotting, wound healing assay, mammosphere formation assay, and lung metastasis analysis. Radiation induced TGF-β signaling, EMT markers (Vimentin, Fibronectin, Snail, Slug, Twist, and N-cadherin), CSC properties (expression of pluripotent stem cell regulators, mammosphere forming ability), reactive oxygen species markers (NOX4, 4-HNE), and motility of breast cancer cells. These results indicate that inhibition of TGF-β signaling with vactosertib in breast cancer patients undergoing radiotherapy would be an attractive strategy for the prevention of cancer metastasis and recurrence.

Topics: Aniline Compounds; Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Mice; Protein Kinase Inhibitors; Receptor, Transforming Growth Factor-beta Type I; Transforming Growth Factor beta; Triazoles

Ovo-like transcriptional repressor 1 (OVOL1) is a key mediator of epithelial lineage determination and mesenchymal-epithelial transition (MET). The cytokines transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMP) control the epithelial-mesenchymal plasticity (EMP) of cancer cells, but whether this occurs through interplay with OVOL1 is not known. Here, we show that OVOL1 is inversely correlated with the epithelial-mesenchymal transition (EMT) signature, and is an indicator of a favorable prognosis for breast cancer patients. OVOL1 suppresses EMT, migration, extravasation, and early metastatic events of breast cancer cells. Importantly, BMP strongly promotes the expression of OVOL1, which enhances BMP signaling in turn. This positive feedback loop is established through the inhibition of TGF-β receptor signaling by OVOL1. Mechanistically, OVOL1 interacts with and prevents the ubiquitination and degradation of SMAD family member 7 (SMAD7), which is a negative regulator of TGF-β type I receptor stability. Moreover, a small-molecule compound 6-formylindolo(3,2-b)carbazole (FICZ) was identified to activate OVOL1 expression and thereby antagonizing (at least in part) TGF-β-mediated EMT and migration in breast cancer cells. Our results uncover a novel mechanism by which OVOL1 attenuates TGF-β/SMAD signaling and maintains the epithelial identity of breast cancer cells.

Topics: Breast Neoplasms; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Invasiveness; Receptor, Transforming Growth Factor-beta Type I; Transcription Factors; Transforming Growth Factor beta

Bone metastases occur in 50-70% of patients with late-stage breast cancers and effective therapies are needed. The expression of enhancer of zeste homolog 2 (EZH2) is correlated with breast cancer metastasis, but its function in bone metastasis hasn't been well-explored. Here we report that EZH2 promotes osteolytic metastasis of breast cancer through regulating transforming growth factor beta (TGFβ) signaling. EZH2 induces cancer cell proliferation and osteoclast maturation, whereas EZH2 knockdown decreases bone metastasis incidence and outgrowth in vivo. Mechanistically, EZH2 transcriptionally increases ITGB1, which encodes for integrin β1. Integrin β1 activates focal adhesion kinase (FAK), which phosphorylates TGFβ receptor type I (TGFβRI) at tyrosine 182 to enhance its binding to TGFβ receptor type II (TGFβRII), thereby activating TGFβ signaling. Clinically applicable FAK inhibitors but not EZH2 methyltransferase inhibitors effectively inhibit breast cancer bone metastasis in vivo. Overall, we find that the EZH2-integrin β1-FAK axis cooperates with the TGFβ signaling pathway to promote bone metastasis of breast cancer.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enhancer of Zeste Homolog 2 Protein; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin beta1; Signal Transduction; Transforming Growth Factor beta

The epithelial mesenchymal transition (EMT) is a process by which cells lose their adhesive nature and gain the migratory properties associated with mesenchymal cells. This transition allows cells to migrate away from a primary tumor while maintaining their newly acquired invasive behavior, suggesting that there is a bistable switch between the epithelial and mesenchymal phenotypes. In recent experimental work, we found evidence of this bistability in the MCF7 breast carcinoma cell line (Gasior et al., 2019). Underlying the complex processes governing EMT, we identify a feedback loop between E-cadherin, a protein involved in cellular adhesion, and Slug, a transcription factor that is upregulated during EMT. Here, we present a simple mathematical model that examines the relationship between E-cadherin and Slug in response to pro-epithelial and pro-mesenchymal factors, cell-cell contact and TGF-β, respectively. We hypothesize that cell-cell contact is a critical component in the transition from the epithelial to the mesenchymal phenotype and that it is possible to initiate EMT with the loss of cell-cell contact or the activation of the TGF-β signaling pathway. We propose a reversible bistable switch in response to a loss of cell-cell contact but an irreversible bistable switch when the cell is exposed to TGF-β. Taken together, this model shows that acquiring and retaining invasive behavior by cells with high levels of cell-cell contact is not impossible but, instead, depends on the cooperation between the two switches. The predictions of this model for E-cadherin and Slug levels were compared against relative gene expression data from our recent experiments with MCF7 cells (Gasior et al., 2019). Our model works well to predict E-cadherin and Slug mRNA expression in low confluence experiments, while also highlighting issues that arise when comparing experimental results to theoretical predictions.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Transforming Growth Factor beta

We aimed to examine the potential anticancer effects of ozone applied after chemotherapeutic treatment with different concentrations of doxorubicin in Luminal-A subtype of human breast cancer cell line (MCF-7) and compare the results with effects on L929 fibroblast cell line.. Both cell lines were incubated with increasing doses of doxorubicin (1-50 μM) for 24 h at 37°C. Then, half of groups were incubated with 30 μg/mL ozone for 25 min as combination groups. Cell viability was analyzed by MTT assay, apoptosis by flow cytometry, and levels of tumor necrosis factor alpha, transforming growth factor beta, and matrix metalloproteinase-2 and MMP-9 by immunocytochemistry.. Doxorubicin + ozone treatment enhanced viability of L929 (p<0.01) but reduced viability of MCF-7 compared to only doxorubicin-applied cells without ozone treatment (p<0.001). This combined treatment also enhanced apoptotic effect of doxorubicin on MCF-cells (p<0.001), but not on L929. It significantly increased all protein levels of L929 compared with those of other groups (p<0.05 for tumor necrosis factor alpha and MMP-2; p<0.01 for transforming growth factor beta and MMP-9). This treatment reversed the effect of doxorubicin on tumor necrosis factor alpha levels and considerably reduced MMP-2 and MMP-9 levels of MCF-7 compared with those of control group (p<0.01 and p<0.001, respectively).. Ozone treatment potentiated the apoptotic and anticancer activities of doxorubicin in MCF-7 cells and showed repairing and healing effect on healthy fibroblast cells, which were damaged from cytotoxic effects of chemotherapeutic agent. MCF-7 cells may acquire sensitivity against the doxorubicin combined with ozone treatment through activating tumor necrosis factor alpha, MMP-2, and MMP-9 expressions.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Ozone; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Angiogenesis is one of the hallmarks of cancers that is involved in tumor progression. Angiogenic factors induce the formation of new blood vessels and tumor extension, and finally reduce the survival of patients. Intraoperative radiotherapy (IORT), in which radiation is delivered to the tumor bed can kill cells and change tumor microenvironment. Here, we compared the impact of IORT on the levels of angiogenic factors in the blood and surgical wound fluids (SWF) of the breast cancer patients.. Three hundred sixty patients, who had undergone breast-conserving surgery between 2013 and 2018, were enrolled in IORT and non-IORT groups non-randomly. Blood and drained wound fluid (WF) samples were collected from the patients before and after surgery, followed by quantification of the amounts of TGF-β, EGF, FGF, VEGF, and DLL4 in the patients using ELISA.. Our results were indicative of significant differences between the pre-surgery and post-surgery serum levels of EGF, DLL4, and VEGF. Furthermore, ROC analyses showed that TGF-β and DLL4 can differentiate of the early-stage from late-stage of the disease. Interestingly, the rate of the death and recurrence was reduced in IORT group.. In summary, IORT is a safe and effective treatment that can affect angiogenic factors and improve the overall- and recurrence-free survival of breast cancer patients.

Topics: Angiogenesis Inducing Agents; Breast Neoplasms; Epidermal Growth Factor; Female; Humans; Intraoperative Care; Mastectomy, Segmental; Radiotherapy, Adjuvant; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

The TGF-β-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-β pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

Topics: Animals; Breast Neoplasms; Chloride Channels; Female; Humans; Mice; Neoplasm Metastasis; Proteomics; Transforming Growth Factor beta; Tumor Microenvironment

Triple-negative breast cancer (TNBC) is a highly aggressive and invasive type of breast cancer. In addition, type 2 diabetes mellitus (T2DM) is recognized as a risk factor for cancer metastasis, which is associated with mortality in patients with breast cancer. Cancer-associated fibroblasts (CAFs) generated from adipose tissue-derived mesenchymal stem cells (AT-MSCs) play a vital role in the progression of TNBC. However, to date, whether T2DM affects the ability of AT-MSCs to differentiate into CAFs is still unclear. In this study, we found that in coculture with TNBC cells [breast cancer cells (BCCs)] under hypoxic conditions, AT-MSCs derived from T2DM donors (dAT-MSCs) were facilitated to differentiate into CAFs, which showed fibroblastic morphology and the induced expression of fibroblastic markers, such as fibroblast activation protein, fibroblast-specific protein, and vimentin. This was involved in the higher expression of transforming growth factor beta receptor 2 (TGFβR2) and the phosphorylation of Smad2/3. Furthermore, T2DM affected the fate and functions of CAFs derived from dAT-MSCs. While CAFs derived from AT-MSCs of healthy donors (AT-CAFs) exhibited the markers of inflammatory CAFs, those derived from dAT-MSCs (dAT-CAFs) showed the markers of myofibroblastic CAFs. Of note, in comparison with AT-CAFs, dAT-CAFs showed a higher ability to induce the proliferation and in vivo metastasis of BCCs, which was involved in the activation of the transforming growth factor beta (TGFβ)-Smad2/3 signaling pathway. Collectively, our study suggests that T2DM contributes to metastasis of BCCs by inducing the myofibroblastic CAFs differentiation of dAT-MSCs. In addition, targeting the TGFβ-Smad2/3 signaling pathway in dAT-MSCs may be useful in cancer therapy for TNBC patients with T2DM.

Topics: Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Diabetes Mellitus, Type 2; Female; Fibroblasts; Humans; Mesenchymal Stem Cells; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

This study aimed to investigate innovative targets in breast cancer patients by considering the interaction of the lncRNA-miR-mRNA network in response to low-dose aspirin. The candidate miRs were first taken from the GEO and TCGA databases. Then, the candidate network was constructed using the high-throughput sequencing data. The expression levels of candidate targets were finally measured using Real-Time PCR in luminal A breast cancer patients undergoing aspirin (80 mg daily for three months) and non-aspirin groups during chemotherapy after surgery. The expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins were measured using the ELISA technique. 5 lncRNAs, 12 miRs, and 10 genes were obtained in the bioinformatic phase. A significant expression increase of the candidate tumor suppressor lncRNAs, miRs, and genes and a substantial expression decrease of the candidate onco-lncRNAs, oncomiRs, and oncogenes were achieved after the aspirin consumption. Unlike the non-aspirin group, the expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins were significantly decreased following aspirin consumption. The Kaplan-Meier analysis indicated a longer overall survival rate in the patients after aspirin consumption. Our results showed that the lncRNA-miR-mRNA network might be a significant target for aspirin; their expression changes may be a new strategy with potential efficacy for cancer therapy or prevention.

Topics: Breast Neoplasms; Female; Gene Regulatory Networks; Humans; Interleukin-17; MicroRNAs; RNA, Long Noncoding; RNA, Messenger; Transforming Growth Factor beta

Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-β type II receptor (TβRII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-β signaling in recipient cells. Up-take of extracellular vesicle-TβRII (EV-TβRII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-TβRII delivered as cargo to CD8

Topics: Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Extracellular Vesicles; Female; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Parathyroid hormone-related protein (PTHrP) was discovered as the tumor product causing the humoral hypercalcemia of malignancy. Its structural similarity to the hormone, PTH, with 8 of the first 13 amino acids identical, was sufficient to explain the sharing by PTHrP and PTH of a common receptor, PTH1R, although the remainder of the sequences are unique. PTHrP has important roles in development of several organs, including breast and bone, and functions as a paracrine factor postnatally in these and other tissues. In addition to its hormonal role in cancer, PTHrP is produced by two thirds of primary breast cancers and 90% of bone metastases from breast cancer, leading to the concept that its production in bone by breast cancer cells promotes bone resorption, thus favoring tumor establishment and expansion, and an exit from tumor dormancy in bone through downregulation of leukemia inducing factor receptor (LIFR). Cancer production of PTHrP is increased by bone-derived growth factors, with particular attention paid to TGFβ, as well as by promoter-driven transcriptional effects, such as the hedgehog signaling factor, GLI2, and microenvironment effects including changes in underlying stiffness of substrates for cells. Although interest has been focused on PTHrP-induced bone resorption in bone metastasis, a mechanistically separate, protective effect against tumor progression has been proposed. Although there is conflicting mouse data, there are clinical studies suggesting that increased production of PTHrP by breast cancers confers upon them a less invasive phenotype, an effect distinct from the bone resorption-stimulating action that favors bone metastasis.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Hedgehog Proteins; Humans; Mice; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta; Tumor Microenvironment

Sentinel lymph nodes (SLNs) are both the first site where breast cancer (BC) metastases form and where anti-tumoral immunity develops. Despite being the most potent antigen-presenting cells, dendritic cells (DCs) located in a nodal tissue can both promote or suppress immune response against cancer in SLNs.. In SLNs excisions obtained from 123 invasive BC patients, we performed immunohistochemistry (IHC) for CD1a, CD1c, DC-LAMP, and DC-SIGN to identify different DCs populations. Then we investigated the numbers of DCs subsets in tumor-free, micrometastatic, and macrometastatic SLNs with the use of a light microscope.. We observed that CD1c+ and DC-SIGN+ DCs were more numerous in SLNs with a larger tumor size. More abundant intratumoral DC-LAMP+ population was related to a higher number of metastatic lymph nodes. Conversely, more abundant CD1a+ DCs were associated with a decreasing nodal burden in SLNs and a lower number of involved lymph nodes. Moreover, densities of the investigated DC populations differed with respect to tumor grade, HER2 overexpression, hormone receptor status, and histologic type of BC.. According to their subtype, DCs are associated with either lower or higher nodal burden in SLNs from invasive BC patients. These relationships appear to be dependent not only on the maturation state of DCs but also on the histological and biological characteristics of the tumor.

Topics: Breast Neoplasms; Dendritic Cells; Female; Humans; Lymph Nodes; Lymphadenopathy; Melanoma; Melanoma, Cutaneous Malignant; Sentinel Lymph Node; Sentinel Lymph Node Biopsy; Skin Neoplasms; Transforming Growth Factor beta

Metastatic breast cancer cannot be cured, and alteration of fatty acid metabolism contributes to tumor progression and metastasis. Here, we were interested in the elongation of very long-chain fatty acids protein 5 (Elovl5) in breast cancer. We observed that breast cancer tumors had a lower expression of Elovl5 than normal breast tissues. Furthermore, low expression of Elovl5 is associated with a worse prognosis in ER

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Fatty Acid Elongases; Female; Humans; Lipids; Lung Neoplasms; Mice; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Several mechanisms have been posited to play a role in the sleep and breast cancer association, including alterations in immune function, but evidence remains inconclusive. A closer look at how sleep quality traits affect the breast microenvironment may provide clues for molecular mechanisms underlying the link between sleep and breast cancer. We examined the association between sleep quality traits (sleep duration, sleep aids, and insomnia) and tissue-based protein levels and gene expression of several inflammatory markers associated with breast cancer.. Breast tissues (normal n = 165 and adipose n = 74) were surgically obtained from women diagnosed with breast cancer. Protein levels by immunohistochemistry were determined using the quickscore method for 11 inflammatory markers in the normal epithelial breast tissue (interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), cyclooxygenase-2 (COX-2), leptin, serum amyloid A1 (SAA1), lactoferrin, transforming growth factor-beta (TGF-β), and signal transducer and activator of transcription 3 markers (STAT3). Relative quantification of 4 genes (COX-2, IL-6, TNF-α and LEP) in the adipose breast tissue was carried out using qPCR. Patient characteristics and sleep traits (average sleep duration per night, taking sleeping aids in the past year, and the average number of insomnia episodes per month) were determined by telephone interview. Associations were tested using Spearman's rank correlation (r. TGF-β and CRP levels in normal epithelial breast tissue were positively correlated with sleep aids (ar. Our findings indicate that sleep duration, sleep aids, and insomnia may differently affect women's breast tissues depending on menopausal status. From a public health perspective, these results warrant further validation in larger studies. Since sleep is a modifiable factor, it may be an interesting approach for breast cancer prevention.

Topics: Biomarkers; Breast Neoplasms; C-Reactive Protein; Cyclooxygenase 2; Female; Humans; Interleukin-10; Interleukin-6; Interleukin-8; Lactoferrin; Leptin; Sleep Initiation and Maintenance Disorders; Sleep Quality; STAT3 Transcription Factor; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Necrosis Factor-alpha

The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFβ. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Plasticity; Epithelial-Mesenchymal Transition; Female; Humans; Receptors, Androgen; Snail Family Transcription Factors; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

MicroRNAs (miRNAs) are involved in the progression of diverse human cancers. This work aimed to delve into how microRNA-135a-5p (miR-135a-5p) affects the biological behaviors of Breast Cancer (BC) cells.. Gene Expression Omnibus (GEO) datasets were used to analyze the expression differences of miR-135a-5p in cancer tissues of BC patients. Quantitative real-time PCR and western blot were conducted to detect miR-135a-5p and Bcl-2 Associated Athanogene (BAG3) expression levels in BC tissues and cells, respectively. The proliferation, migration, invasion, and cell cycle of BC cells were detected by cell counting kit-8 assay, BrdU assay, wound healing assay, transwell assay, and flow cytometry. The targeted relationship between miR-135a-5p and BAG3 mRNA 3'UTR predicted by bioinformatics was further testified by a dual-luciferase reporter gene assay. Pearson's correlation analysis was adopted to analyze the correlation between miR-135a-5p expression and BAG3 expression. The downstream pathways of BAG3 were analyzed by the LinkedOmics database.. MiR-135a-5p was significantly down-regulated and BAG3 expression was significantly raised in BC tissues. MiR-135a-5p overexpression repressed the viability, migration and invasion of BC cells, and blocked cell cycle progression in G0/G1 phase while inhibiting miR-135a-5p worked oppositely. BAG3 was verified as a target of miR-135a-5p. Overexpression of BAG3 reversed the impacts of miR-135a-5p on the malignant biological behaviors of BC cells. The high expression of BAG3 was associated with the activation of the cell cycle, mTOR and TGF-β signaling pathways.. MiR-135a-5p regulates BAG3 to repress the growth, migration, invasion, and cell cycle progression of BC cells.

Topics: 3' Untranslated Regions; Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Breast Neoplasms; Bromodeoxyuridine; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Metastasis is the major cause of death and failure of cancer chemotherapy in patients with breast cancer (BC). Activation of TGF-β/lncRNA-MALAT1/miR-200c has been reported to play an essential role during the metastasis of BC cells. The present study aimed to validate the suppression of BC-cell migration and invasion by baicalin and explore its regulatory effects on the TGF-β/lncRNA-MALAT1/miR-200c signaling pathway. We found that baicalin treatment inhibited cell viability and migration and invasion. Mechanistically, baicalin treatment significantly downregulated the expression of TGF-β, ZEB1, and N-cadherin and upregulated E-cadherin on both mRNA and protein levels. Additionally, baicalin treatment significantly downregulated the expression of lncRNA-MALAT1 and upregulated that of miR-200c. Collectively, baicalin significantly suppresses cell viability, migration, and invasion of BC cells possibly by regulating the TGF-β/lncRNA-MALAT1/miR-200c pathway.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; MicroRNAs; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Co-enzyme nicotinamide adenine dinucleotide NAD(H) regulates hundreds of biochemical reactions within the cell. We previously reported that NAD(H) redox status may have prognostic value for predicting breast cancer metastasis. However, the mechanisms of NAD(H) involvement in metastasis remain elusive. Given the important roles of TGFβ signalling in metastatic processes, such as promoting the epithelial-to-mesenchymal transition, we aimed to investigate the involvement of the mitochondrial NAD(H) redox status in TGFβ receptor signalling. Here we present the initial evidence that NAD(H) redox status is responsive to TGFβ receptor signalling in triple-negative breast cancer cells in culture. The mitochondrial NAD(H) redox status was determined by the optical redox imaging (ORI) technique. Cultured HCC1806 (less aggressive) and MDA-MB-231 (more aggressive) cells were subjected to ORI after treatment with exogenous TGFβ1 or LY2109761, which stimulates or inhibits TGFβ receptor signalling, respectively. Cell migration was determined with the transwell migration assay. Global averaging quantification of the ORI images showed that 1) TGFβ1 stimulation resulted in differential responses between HCC1806 and MDA-MB-231 lines, with HCC1806 cells having a significant change in the mitochondrial redox status, corresponding to a larger increase in cell migration; 2) HCC1806 cells acutely treated with LY2109761 yielded immediate increases in ORI signals. These preliminary data are the first evidence that suggests the existence of a cell line-dependent shift of the mitochondrial NAD(H) redox status in the TGFβ receptor signalling induced migratory process of breast cancer cells. Further research should be conducted to confirm these results as improved understanding of the underlying mechanisms of metastatic process may contribute to the identification of prognostic biomarkers and therapeutic targets.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Mitochondria; NAD; Optical Imaging; Oxidation-Reduction; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

Cancer-associated fibroblasts (CAFs) represent one of the main components in the tumor stroma and play a key role in breast cancer progression. Transforming growth factor-β (TGF-β) has been established to mediate breast cancer metastasis by regulating the epithelial-mesenchymal transition (EMT) and stemness of cancer cells. Caveolin-1 (CAV-1) is a scaffold protein of caveolae that is related to the proliferation and metabolism of cancer cells. It is now well demonstrated that CAV-1 deficiency in the tumor stroma is positively correlated with distant metastasis, but the mechanism remains unclear. Here, we explore whether CAV-1-deficient fibroblasts play an essential role in the EMT and stemness of breast cancer cells (BCCs) through TGF-β signaling. We establish a specific small interfering RNA (siRNA) to inhibit CAV-1 expression in fibroblasts and coculture them with BCCs to investigate the effect of CAV‑1-deficient fibroblasts and the tumor microenvironment on breast cancer progression. This study refreshingly points out that CAV-1 deficiency in fibroblasts enhances TGF-β1 secretion and then activates the TGF-β1/Smad signaling pathway of BCCs, thus promoting the metastasis and stemness of BCCs. Collectively, our findings indicate an unexpected role of CAV-1 deficiency in fibroblasts and the tumor microenvironment as a permissive factor, which is regulated by the TGF-β1 signaling pathway in BCCs.

Topics: Breast Neoplasms; Caveolin 1; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Humans; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

Breast cancer is a highly heterogeneous disease with multiple drivers and complex regulatory networks. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. Postn has been shown to be involved in various processes of tumor development, such as angiogenesis, invasion, cell survival and metastasis. The expression of Postn in breast cancer cells has been correlated with a more aggressive phenotype. Despite extensive research, it remains unclear how epithelial cancer cells regulate Postn expression.. Using murine tumor models and human TMAs, we have assessed the proportion of tumor samples that have acquired Postn expression in tumor cells. Using biochemical approaches and tumor cell lines derived from Neu+ murine primary tumors, we have identified major regulators of Postn gene expression in breast cancer cell lines.. Here, we show that, while the stromal compartment typically always expresses Postn, about 50% of breast tumors acquire Postn expression in the epithelial tumor cells. Furthermore, using an in vitro model, we show a cross-regulation between FGFR, TGFβ and PI3K/AKT pathways to regulate Postn expression. In HER2-positive murine breast cancer cells, we found that basic FGF can repress Postn expression through a PKC-dependent pathway, while TGFβ can induce Postn expression in a SMAD-independent manner. Postn induction following the removal of the FGF-suppressive signal is dependent on PI3K/AKT signaling.. Overall, these results reveal a novel regulatory mechanism and shed light on how breast tumor cells acquire Postn expression. This complex regulation is likely to be cell type and cancer specific as well as have important therapeutic implications.

Topics: Animals; Breast Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Epithelial Cells; Female; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Humans; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Fibroblast Growth Factor; Signal Transduction; Transforming Growth Factor beta

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFβ is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFβ blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFβ blockade. This increase was mediated by upregulation of another TGFβ family member, activin A.

Topics: Activins; Animals; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Humans; Immunosuppression Therapy; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Neoplasm Recurrence, Local; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Tumour stromal cells support tumourigenesis. We report that Syndecan-2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan-2 modulated TGFβ signalling (SMAD7, PAI-1), migration and immunosuppression of patient-derived tumour-associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan-2 in TASCs significantly enhanced TGFβ signalling (SMAD7, PAI-1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFβ signalling (SMAD7, PAI-1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan-2-peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFβ-regulated immunosuppressive genes, such as CXCR4 and PD-L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan-2-peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan-2 within the TME inhibits tumour growth and metastasis due to decreased TGFβ signalling and increased immune control.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Immune Evasion; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Signal Transduction; Stromal Cells; Syndecan-2; Transforming Growth Factor beta; Tumor Microenvironment

Breast cancer frequently metastasizes to the skeleton causing significant morbidity. None of the therapeutic strategies used to manage breast cancer bone metastases are really curative. Here, we set-up a novel and advanced model by using fresh tissue from human vertebral bone metastasis from breast carcinoma patients able to retain the tumor microenvironment. The tissue model is based on an ex-vivo culture for up to 40 days and on a constant monitoring of tissue viability, gene expression profile (IL10, IL1b, MMP1, MMP7, PTH1R, PTH2R, TNF, ACP5, SPI1, VEGFA, CTSK, TGF-β) and histological and immunohistochemical analyses (CDH1/E-cadherin, CDH2/N-cadherin, KRT8/Cytokeratin 8, KRT18/Cytokeratin 18, Ki67, CASP3/Caspase 3, ESR1/Estrogen Receptor Alpha, CD68 and CD8). Results confirmed the development of a reliable, reproducible and cost-effective advanced model of breast cancer bone metastasis able to preserve and maintain long-term tissue viability, as well as molecular markers, tissue histomorphology, tissue micro-architecture and antigen expression. The study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumor subtypes so predicting patient specific responses.

Topics: Bone Neoplasms; Breast Neoplasms; Female; Humans; Transforming Growth Factor beta; Tumor Microenvironment

Two recent papers from the laboratory of Professor Ming Li demonstrate that inhibition of transforming growth factor β specifically in CD4

Topics: Breast Neoplasms; Female; Humans; Transforming Growth Factor beta

Our previous studies have shown that MCF-7 breast cancer cell line exposed to 6 Gy and allowed to recover for 7 days (D7-6G) developed radio-resistance. In this study, we have tested the ability of these cells to form tumors in severe combined immunodeficiency (SCID) mice and characterized these tumors by proteomic analyses. Untreated (MCF-C) and D7-6G cells (MCF-R) were injected s.c. in SCID mice and tumor growth monitored. On Day 18, the mice were killed and tumor tissues were fixed in formalin or RNA later. Expression of genes was assessed by reverse transcription-polymerase chain reaction and proteins by enzyme-linked immunosorbent assay/antibody labeling and flow cytometry. Label free proteomic analyses was carried out by liquid chromatography-mass spectrometry. Metabolic analysis was carried out using Seahorse analyzer. MCF-R cells had a shorter latency and formed larger tumors. These tumors were characterized by an increased expression of transforming growth factor β (TGF-β) isoforms; its downstream genes pSMAD3, Snail-1, Zeb-1, HMGA2; hybrid epithelial/mesenchymal phenotype; migration, enrichment of cancer stem cells and radioresistance following challenge dose of radiation. Proteomic analysis of MCF-7R tumors resulted in identification of a total of 649 differentially expressed proteins and pathway analyses using protein annotation through evolutionary relationship indicated enrichment of genes involved in metabolism. Data are available via ProteomeXchange with identifier PXD022506. Seahorse analyzer confirmed increased metabolism in these cells with increased oxidative phosphorylation as well as glycolysis. Increased uptake of 2-NBDG further confirmed increased glycolysis. In summary, we demonstrate that radioresistant breast cancer cells had an enrichment of TGF-β signaling and increased metabolism.

Topics: Animals; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice; Mice, SCID; Radiation Tolerance; Transcriptome; Transforming Growth Factor beta

Metastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of the immunosuppressive tumor environment is required to improve its efficacy. The effects of this "metastatic" tumor environment on NK cells, however, remain largely unknown. Previous studies, including our own, have demonstrated that metastasis-associated macrophages (MAMs) are one of the most abundant immune cell types in the metastatic tumor niche in mouse models of metastatic breast cancer. We thus investigated the effects of MAMs on antitumor functions of NK cells in the metastatic tumor microenvironment.. MAMs were isolated from the tumor-bearing lung of C57BL/6 mice intravenously injected with E0771-LG mouse mammary tumor cells. The effects of MAMs on NK cell cytotoxicity towards E0771-LG cells were evaluated. MAMs isolated from the metastatic lung suppressed NK cell-induced tumor cell apoptosis. This study demonstrates that MAMs are a main negative regulator of NK cell function within the metastatic tumor niche, and MAM targeting is an attractive strategy to improve NK cell-based immunotherapy for metastatic breast cancer.

Topics: Adoptive Transfer; Animals; Antigens, Ly; Breast Neoplasms; Female; Gene Knockout Techniques; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred C57BL; Natural Cytotoxicity Triggering Receptor 1; Neoplasm Transplantation; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Transforming Growth Factor beta; Tumor-Associated Macrophages

Natural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output.. Single-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controls RESULTS: In addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-β (TGFβ) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFβ processing pathway, was increased on NK cells from some patients. Blocking the GARP-TGFβ axis recapitulated the effects of TGFβ neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time.. TGFβ contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFβ and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

Topics: Breast Neoplasms; Case-Control Studies; Coculture Techniques; Cytotoxicity, Immunologic; Energy Metabolism; Female; Glycolysis; Humans; Interferon-gamma; K562 Cells; Killer Cells, Natural; Membrane Proteins; Microscopy, Confocal; Middle Aged; Mitochondria; Neoplasm Metastasis; Oxidative Phosphorylation; Signal Transduction; Single-Cell Analysis; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signaling plays a critical role in promoting epithelial-to-mesenchymal transition (EMT), cell migration, invasion, and tumor metastasis. ΔNp63α, the major isoform of p63 protein expressed in epithelial cells, is a key transcriptional regulator of cell adhesion program and functions as a critical metastasis suppressor. It has been documented that the expression of ΔNp63α is tightly controlled by oncogenic signaling and is frequently reduced in advanced cancers. However, whether TGF-β signaling regulates ΔNp63α expression in promoting metastasis is largely unclear. In this study, we demonstrate that activation of TGF-β signaling leads to stabilization of E3 ubiquitin ligase FBXO3, which, in turn, targets ΔNp63α for proteasomal degradation in a Smad-independent but Erk-dependent manner. Knockdown of FBXO3 or restoration of ΔNp63α expression effectively rescues TGF-β-induced EMT, cell motility, and tumor metastasis in vitro and in vivo. Furthermore, clinical analyses reveal a significant correlation among TGF-β receptor I (TβRI), FBXO3, and p63 protein expression and that high expression of TβRI/FBXO3 and low expression of p63 are associated with poor recurrence-free survival (RFS). Together, these results demonstrate that FBXO3 facilitates ΔNp63α degradation to empower TGF-β signaling in promoting tumor metastasis and that the TβRI-FBXO3-ΔNp63α axis is critically important in breast cancer development and clinical prognosis. This study suggests that FBXO3 may be a potential therapeutic target for advanced breast cancer treatment.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; HaCaT Cells; HEK293 Cells; Humans; Neoplasm Metastasis; Protein Isoforms; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

A large number of breast cancer patients perishes due to metastasis instead of primary tumor, but molecular mechanisms contributing towards cancer metastasis remain poorly understood. Therefore, prompting development of novel treatment is inevitable. A vast variety of plant derived natural substance possesses several therapeutically active constituents, e.g. alkaloids, flavonoids, tannins, resins, terpenoids etc. that exhibit various pharmacological properties e.g. anti-inflammatory, anti-microbial and anti-cancer properties. Sanguinarine (SAN) alkaloid found its place among such naturally occurring substances that exerts several pharmacological activities, including anti-cancer effects.. Until now, role of SAN not only against epithelial-mesenchymal transition (EMT) but also against metastasis progression in breast cancer remains indistinct. Thus, aim of the present study was to investigate effects of SAN on EMT process and cancer metastasis in animal model.. MTT assay was performed to assess SAN effects on proliferation in breast cancer. Scratch assay was performed to evaluate effects of SAN on migration in breast cancer. Colony formation assay was performed to determine effects of SAN on colonization characteristics of breast cancer. Western blotting was performed to measure EMT regulating protein expression as well as major pathway protein expression induced against TGF-β treatment in breast cancer. Tail vein method of injecting breast cancer cells in bulb/c mice was conducted to study metastasis progression and thereafter assessing effects of SAN against metastasis in mice.. In vivo results: MTT assay performed, demonstrated dose dependent inhibition of cell proliferation in breast cancer. Scratch assay results showed, SAN played a major role as migration inhibitor in estrogen receptor positive (ER+) breast cancer. Colony forming assay results demonstrated that SAN constrains ability of breast cancer to develop into well-defined colonies. Western blotting results for EMT regulating protein expression, after TGF-β treatment showed, SAN inhibited cadherin switch in ER+ breast cancer. Moreover, expression of pathway proteins involved in EMT process after TGF-β treatment i.e. Smad, PI3K/Akt and MAP kinase were significantly masked against SAN treatment.. The appearance of metastatic nodules in lung tissues of mice model, helps to study the effects of SAN against metastasis in bulb/c mice. The obtained results have confirmed that SAN impeded lung metastasis. The macroscopic examination has confirmed metastasis inhibitory role of SAN in breast cancer. The Hematoxylin and eosin (H&E) staining results further advocate anti-metastatic characteristics of SAN, presented by fewer metastatic nodule and lesions appearance in SAN treated mice compared to untreated metastasis mice.. In summary, SAN displayed prominent anti-metastatic effects in animal model and anti-proliferation effects together with significant inhibitory potential on EMT regulating protein expression against TGF-β treatment in ER+ breast cancer. So, overall findings of our study highlighted the pre-clinical significance of SAN in animal model therefore, further studies in humans as a part of clinical trial will be needed to establish pharmacokinetics and other effects of SAN, so that it can be a potential candidate for future treatment of metastatic breast cancer (MBC).

Topics: Animals; Benzophenanthridines; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Isoquinolines; Lung Neoplasms; Mice; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Transforming Growth Factor beta

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

Topics: Breast Neoplasms; Cell Line, Tumor; Extracellular Matrix; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Microtubule-Associated Proteins; Neoplasm Proteins; Podosomes; Proteolysis; Transforming Growth Factor beta

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFβ/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFβ-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis.

Topics: Animals; Antibodies, Heterophile; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; Discoidin Domain Receptors; Disease Models, Animal; Enzyme Activation; Female; Gene Knockdown Techniques; Humans; Mice; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Neoplasms; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Male; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Invasiveness; Nuclear Proteins; Prostatic Neoplasms; Transforming Growth Factor beta; Wnt Signaling Pathway; Wnt3A Protein

Topics: Biomarkers, Tumor; Breast Neoplasms; CD4-Positive T-Lymphocytes; Cohort Studies; Gene Expression Regulation, Neoplastic; Humans; Interferon-alpha; Interferon-gamma; Neoplasm Recurrence, Local; Prognosis; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Biglycan; Breast Neoplasms; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; Humans; Mice; MicroRNAs; Signal Transduction; Transforming Growth Factor beta

To investigate the auto-induction of transforming growth factor-b1 (TGF-β1) in breast cancer stem cells (BCSCs) and its effect on cell viability and stemness.. Human BCSCs (aldehyde dehydrogenase positive; ALDH+) were grown in serum-free Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) and treated for periods of 1, 2 and 4 hours with 0.1 ng/ml recombinant human TGF-β1 protein (rhTGF-β1). The medium was then replaced with serum-free DMEM/F12 without rhTGF-β1 for 24 hours. Cell viability was determined using a trypan blue exclusion assay. Type 1 TGF-β receptor (TβR1), TGF-β1, octamer-binding transcription factor 4 (OCT4) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) messenger RNA (mRNA) expression levels were analysed using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR). The TGF-β protein level in the culture medium was determined using an enzyme-linked immunosorbent assay (ELISA).. The expression levels of rhTGF-β1, TGF-β1 and TβR1 mRNA significantly increased in BCSCs compared to control after treatment for 1 and 2 hours but decreased after 4 hours. This is in line with alteration of stemness gene, OCT4 and ALDH1A1 mRNA expressions. However, the secretion of newly synthesised TGF-β1 significantly increased after 2 hours. In contrast, viable BCSCs decreased after 1 hour and then gradually increased 2.7 times compared to control after 4 hours.. TGF-β1 treatment in low concentration and for short period of time triggers its auto-induction in BCSCs, leading to increased cell viability and stemness gene expression via autocrine signalling.

Topics: Breast Neoplasms; Humans; Neoplastic Stem Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors

Topics: Axilla; Breast Neoplasms; Female; Humans; Sentinel Lymph Node Biopsy; Transforming Growth Factor beta

Metastasis is the main cause of breast cancer mortality. Recent studies have proved that lipid metabolic reprogramming plays critical roles in breast cancer carcinogenesis and metastasis. We aim to identify critical lipid metabolism genes in breast cancer metastasis.. We designed and cloned a CRISPR pooled library containing lipid metabolic gene guide RNAs and performed a genetic screen in vivo. Transwell assay and animal experiments were used to evaluate cell metastatic ability in vitro or in vivo, respectively. We performed immunohistochemistry with breast cancer tissue microarray to study the clinical significance of NSDHL.. We identified a cholesterol metabolic enzyme, NSDHL, as a potential metastatic driver in triple-negative breast cancer. NSDHL was highly expressed in breast cancer tissues and predicted a poor prognosis. NSDHL knockdown significantly suppressed cell proliferation and migration. Mechanistically, NSDHL activated the TGFβ signaling pathway by inhibiting the endosomal degradation of TGFβR2. In addition, blocking the upstream metabolism of NSDHL with ketoconazole rescued cancer metastasis and TGFβR2 degradation. However, the inactivation of NSDHL (Y151X) did not rescue the migration ability and the TGFβR2 protein expression.. Taken together, our findings established that NSDHL serves as a metastatic driver, and its function depends on its enzyme activity in cholesterol biosynthesis and is mediated by the NSDHL-TGFβR2 signal pathway. Our study indicated that NSDHL and steroid biosynthesis may serve as new drug targets for patients with advanced breast cancer.

Topics: 3-Hydroxysteroid Dehydrogenases; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholesterol; Female; Humans; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

While tumoral Smad-mediated transforming growth factor β (TGFβ) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFβ signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E

Topics: Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Osteoclasts; Osteolysis; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Immune checkpoint blockade (ICB) has become one of the most promising approaches to activating antitumor immunity. However, only a small subset of patients with breast cancer benefit from ICB treatment. To improve the therapeutic effect in the clinic, precision immunotherapy is proposed to accurately eliminate cancer stem cells that contribute to local recurrence or metastasis, but currently little is known about their immunological properties. In this study, breast cancer-specific datasets in The Cancer Genome Atlas were collected and analyzed by using multiple open-access web servers. We found that the immunophenotype of breast cancer was characterized by a hypoactive immune microenvironment and a low response to immune checkpoint blockade. The innate immune checkpoint CD200 and the adaptive immune checkpoint CD276, respectively, exhibited a strong correlation with basal/stem gene signature and invasiveness gene signature, both of which represent breast cancer stem cells. Wnt, TGF-β, and Hedgehog signaling, which are recognized as stemness-related pathways, showed a significant association with the expression of CD200 and CD276, suggesting cancer stem cell-specific immune checkpoints could be downregulated by inhibiting these pathways. Of note, levels of CD200 and CD276 expression were higher in TGF-β dominant breast cancer than in other immune types of breast cancer. We also identified gene signatures that represent Wnt, TGF-β, and Hedgehog signaling-related CD200 and CD276 expression in breast cancer stem cells. For the luminal A subtype, the patient group with a high level of these gene signatures plus a low infiltration of CD8

Topics: Antigens, CD; B7 Antigens; Breast Neoplasms; Female; Hedgehog Proteins; Humans; Immune Checkpoint Inhibitors; Immunophenotyping; Immunotherapy, Adoptive; Neoplasm Invasiveness; Neoplastic Stem Cells; Precision Medicine; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Proteins

This study aimed to investigate the effect of the new ciprofloxacin chalcone [7-(4-(N-substituted carbamoyl methyl) piperazin-1 yl)] on the proliferation, migration, and metastasis of MCF-7 and MDA-MB-231 breast cancer cell lines.. Cell viability, colony formation and cell migration abilities were analysed. Cell cycle distribution and apoptosis were examined by flow cytometry. The molecular mechanism underlying chalcone's activity was investigated using qRT-PCR and western blotting.. This new ciprofloxacin chalcone significantly inhibited proliferation, colony formation, and cell migration abilities of both cancer cell lines. Furthermore, it initiated apoptosis and caused cell cycle arrest at G2/M and S phase in MCF-7 and MDA-MB-231 cell lines, respectively. In addition, it up-regulated the expression of pro-apoptotic factors, p53, PUMA and NOXA, and down-regulated the expression of anti-apoptotic factors, MDM2 and MDM4. At the same time, it inhibited epithelial-mesenchymal transition by increasing the expression of E-cadherin and decreasing the expression of TGF-β1, SNAI1, TWIST1, MMP2, and MMP9.. This new ciprofloxacin chalcone exhibited promising apoptotic and anti-metastatic activities against MCF-7 and MDA-MB-231 breast cancer cell lines, and, therefore, is an attractive molecule for drug development in the treatment of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cadherins; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Survival; Chalcone; Ciprofloxacin; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Molecular Structure; Proteins; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

Topics: Axilla; Breast Neoplasms; Female; Humans; Lymphedema; Transforming Growth Factor beta

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell-matrix adhesion through β1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of β1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Topics: Acrylic Resins; Animals; Breast Neoplasms; Cell Culture Techniques; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Focal Adhesions; Integrin beta1; Kinesins; Mammary Glands, Animal; Mice; Protein Serine-Threonine Kinases; Septins; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-β, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coculture Techniques; Disease Models, Animal; Female; Interferon-gamma; Interleukin-2; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucin-1; Myeloid-Derived Suppressor Cells; Pancreatic Neoplasms; Proto-Oncogene Proteins c-myc; Signal Transduction; Spleen; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Antigens, CD; Benzodioxoles; Breast Neoplasms; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Culture Media, Conditioned; Female; Humans; Imidazoles; Immunohistochemistry; Mesenchymal Stem Cells; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Real-Time Polymerase Chain Reaction; Receptor, Transforming Growth Factor-beta Type I; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Microenvironment

It remains challenging to treat tumor metastasis currently in the light of multiple cascade processes of tumor metastasis. Additionally, multiple clinical drugs for metastasis have quite limited therapeutic potential and even facilitate metastasis in preclinical models. Thus, potential metastasis targets and novel metastasis-directed drugs are urgently needed to be further developed. Herein, transforming growth factor-β (TGF-β) is verified to contribute to lung metastasis in a context-dependent manner in the 4T1 orthotopic tumor-bearing mice model, which induces epithelial-mesenchymal-transition (EMT) to promote tumor dissemination from the primary site and dampens the anti-tumor response of neutrophils to support tumor colonization at the metastatic niche. In view of neutrophils' superior tropism towards both inflammatory primary tumor and metastatic niche, SB525334, a TGF-β receptor inhibitor, is loaded into cationic liposome (SBLP) which is subsequently incorporated into neutrophils to yield the cyto-pharmaceuticals (SBLP/NE). The systemically infused SBLP/NE can simultaneously migrate into both primary and metastatic sites, then release SB525334 in response to tumor stimuli, and contextually inhibit TGF-β-mediated-EMT and phenotype reversal of infiltrated neutrophils, showing substantial metastasis suppression efficacy without causing any detectable toxicities. This project shifts the paradigm for metastasis suppression therapy by simultaneous blockage of contextual TGF-β using metastatic-cascades-targeting neutrophil cyto-pharmaceuticals.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Neoplasm Metastasis; Pharmaceutical Preparations; Transforming Growth Factor beta

Polysaccharide hydrogels are promising candidate matrices for recapitulating the characteristics of extracellular matrix (ECM) in breast tumors in terms of their structure and composition. Herein, to obtain an ECM-mimetic matrix, hydroxyethyl chitosan (HECS) hydrogels were prepared through Schiff-base crosslinking reaction using dialdehyde hyaluronic acid as crosslinker. The obtained HECS hydrogels displayed a highly porous structure, a stiffness comparable to that of breast tissue, and a fast water-absorption speed. The amount of crosslinker had great effects on the swelling and rheological behaviors of the HECS hydrogels. Preliminary results from in vitro biological assessments confirmed that MCF-7 cells incubated within HECS hydrogels preferred to grow into three-dimensional spheroids. Importantly, the cells displayed enhanced migrative capability and upregulated expression levels of MMP-2, TGF-β and VEGF in comparison to two-dimension cultured cells. Hence, the HECS hydrogels show great promise as a biomimetic ECM in constructing breast tumor models.

Topics: Breast Neoplasms; Cell Movement; Cell Proliferation; Chitosan; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronic Acid; Hydrogels; Matrix Metalloproteinase 2; MCF-7 Cells; Porosity; Schiff Bases; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A

Flavonoids are compounds of interest in the search for new anti-cancer therapies. We have previously isolated the methoxyflavones 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone (8-methoxycirsilineol), 5,4'-dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol), and 5,4,'3'-trihydroxy-6,7,8-trimethoxyflavone (sideritoflavone) from Baccharis densiflora. Herein, we investigate the toxicity of these methoxyflavones in human breast-derived cell line. Our main aim was to focus on the cancer stem cell (CSC) sub-population of JIMT-1 breast cancer cells.. Initially, dose response experiments yielding inhibitory concentration 50 (IC. Altogether our data show that the methoxyflavone sideritoflavone has favourable anti-cancer effects that may be exploited for development to be used in combination with CSC specific compounds.

Topics: Antineoplastic Agents, Phytogenic; Baccharis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Flavones; Gene Expression Regulation, Neoplastic; Humans; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

Breast cancer poses a serious health risk for women throughout the world. Among the Asian population, Pakistani women have the highest risk of developing breast cancer. One out of nine women is diagnosed with breast cancer in Pakistan. The etiology and the risk factor leading to breast cancer are largely unknown. In the current study the risk factors that are most pertinent to the Pakistani population, the etiology, molecular mechanisms of tumor progression, and therapeutic targets of breast cancer are studied. A correlative, cross-sectional, descriptive, and questionnaire-based study was designed to predict the risk factors in breast cancer patients. Invasive Ductal Carcinoma (90%) and grade-II tumor (73.2%) formation are more common in our patient's data set. Clinical parameters such as mean age of 47.5 years (SD ± 11.17), disturbed menstrual cycle (> 2), cousin marriages (repeated), and lactation period (< 0.5 Y) along with stress, dietary and environmental factors have an essential role in the development of breast cancer. In addition to this in silico analysis was performed to screen the miRNA regulating the TGF-beta pathway using TargetScanHuman, and correlation was depicted through Mindjet Manager. The information thus obtained was observed in breast cancer clinical samples both in peripheral blood mononuclear cells, and biopsy through quantitative real-time PCR. There was a significant dysregulation (**P>0.001) of the TGF-β1 signaling pathway and the miRNAs (miR-29a, miR-140, and miR-148a) in patients' biopsy in grade and stage specifically, correlated with expression in blood samples. miRNAs (miR-29a and miR-140, miR-148a) can be an effective diagnostic and prognostic marker as they regulate SMAD4 and SMAD2 expression respectively in breast cancer blood and biopsy samples. Therefore, proactive therapeutic strategies can be devised considering negatively regulated cascade genes and amalgamated miRNAs to control breast cancer better.

Topics: Adult; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; MicroRNAs; Middle Aged; Pakistan; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

Accumulating evidence indicates that INHBA (Inhibin β-A, a member of the TGF-β superfamily) functions as an oncogene in cancer progression. However, little is known as to how INHBA regulates the progression and aggressiveness of breast cancer (BC). This study explored the function and underlying mechanism of INHBA in epithelial-mesenchymal transition (EMT) of BC cells. INHBA expression in BC cell lines was measured using RT-qPCR and Western blot. The would-healing and transwell migration assays were used to investigate the effect of INHBA overexpression or silencing on BC cell motility. Moreover, the expression levels of EMT-related genes were quantified after overexpressing or silencing of INHBA. Based on published dataset, INHBA was significantly upregulated in BC tissues compared to the adjacent normal tissues. A higher level of INHBA expression was also correlated with a poor survival in BC patients. In addition,

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Inhibin-beta Subunits; Mice; Signal Transduction; Transforming Growth Factor beta

TGFβs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFβ pathway are in early-phase oncology trials. However, TGFβs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored.. A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFβ antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms.. Heterogeneous responses to anti-TGFβ treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFβ antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFβ on the tumor cell. Applying transcriptomic signatures derived from treatment-naïve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFβ therapy.. Contrary to dogma, tumor-suppressive responses to TGFβ are retained in some advanced metastatic tumors. Safe deployment of TGFβ antagonists in the clinic will require good predictive biomarkers.

Topics: Animals; Antineoplastic Agents, Immunological; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta; Treatment Outcome

To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells.. Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA).. The tumor inducers Tβ4 and TGF-β1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tβ4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium.. F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.

Topics: Animals; Breast Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Cytokines; Endothelial Cells; Female; Gene Expression; Humans; Mice; Peptide Fragments; Protective Agents; Receptors, Cell Surface; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-β (TGF-β) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Breast Neoplasms; Cell Line, Tumor; Co-Repressor Proteins; Conserved Sequence; Epithelial-Mesenchymal Transition; Female; Gene Regulatory Networks; Humans; MCF-7 Cells; Mice; Mutation; Octamer Transcription Factor-1; Protein Interaction Domains and Motifs; Rats; Transforming Growth Factor beta

While several tools have been developed to map axes of variation among individual cells, no analogous approaches exist for identifying axes of variation among multicellular biospecimens profiled at single-cell resolution. For this purpose, we developed 'phenotypic earth mover's distance' (PhEMD). PhEMD is a general method for embedding a 'manifold of manifolds', in which each datapoint in the higher-level manifold (of biospecimens) represents a collection of points that span a lower-level manifold (of cells). We apply PhEMD to a newly generated drug-screen dataset and demonstrate that PhEMD uncovers axes of cell subpopulational variation among a large set of perturbation conditions. Moreover, we show that PhEMD can be used to infer the phenotypes of biospecimens not directly profiled. Applied to clinical datasets, PhEMD generates a map of the patient-state space that highlights sources of patient-to-patient variation. PhEMD is scalable, compatible with leading batch-effect correction techniques and generalizable to multiple experimental designs.

Topics: Algorithms; Animals; Antineoplastic Agents; Biopsy; Breast Neoplasms; Cluster Analysis; Cytophotometry; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Female; Humans; Image Interpretation, Computer-Assisted; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Pattern Recognition, Automated; Phenotype; Recombinant Proteins; Single-Cell Analysis; Software; Transforming Growth Factor beta

Radiotherapy is routinely used in the treatment of breast cancer. However, its efficiency is often limited by the development of radioresistance and metastasis. The cancer cells surviving irradiation show epithelial-mesenchymal transition (EMT) along with increased migration, invasion and metastasis. In this study, we have evaluated the role of α-lipoic acid in preventing the radiation-induced EMT and in sensitizing the breast cancer cells to radiation. The breast cancer cell lines, MCF-7 and MDA-MB-231 were pretreated with lipoic acid, irradiated and the changes associated with cell growth, clonogenicity, migration, matrix metalloproteinases (MMPs), EMT and TGFβ signaling were measured. Our data showed that lipoic acid pretreatment sensitized the breast cancer cells to the ionizing radiation and inhibited the radiation-induced migration and the release of MMP2 and MMP9. Lipoic acid also prevented the TGFβ1 release and inhibited the radiation-induced EMT in breast cancer cells. The inhibition of TGFβ signaling by lipoic acid is associated with the inhibition of radiation-induced activation and translocation of NF-κB. These results suggest that α-lipoic acid inhibits the radiation-induced TGFβ signaling and nuclear translocation of NF-κB, thereby inhibiting the radiation-induced EMT and sensitizing the breast cancer cells to ionizing radiation.

Topics: Breast Neoplasms; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MCF-7 Cells; NF-kappa B; Radiation Tolerance; Signal Transduction; Thioctic Acid; Transforming Growth Factor beta

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFβ pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

Topics: Adult; Aged; Aged, 80 and over; Axilla; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Proliferation; Cell Separation; Chemokine CXCL12; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Follow-Up Studies; Humans; Kaplan-Meier Estimate; Lymph Nodes; Lymphatic Metastasis; Middle Aged; Myofibroblasts; Neoplasm Invasiveness; Primary Cell Culture; Prognosis; Progression-Free Survival; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Chimeric antigen receptor (CAR)-modified T cell therapy evokes only modest antitumor responses in solid tumors. Meso-CAR-T cells are CAR-T cells targeted mesothelin, which are over-expressed in tumor tissues of breast cancer patients. To improve the therapeutic effects, we combined it with rAd.sT, a transforming growth factor β signaling-targeted oncolytic adenovirus, to therapy breast cancer. In subcutaneous MDA-MB-231 xenograft of NSG mice, both rAd.sT and meso-CAR-T inhibited tumor growth, however combination therapy produced stronger inhibitory effects. Interestingly, rAd.sT reduced tumor burden at initial stage following vector treatments, while meso-CAR-T cells decreased tumor burden at a later stage. Moreover, meso-CAR-T could target tumor microenvironments, and combination therapy could enhance cytokines production, such as interleukin (IL)-6 and IL-12 in tumor microenvironment. In conclusion, combination of rAd.sT with meso-CAR-T produced much more impressive antitumor responses to breast cancer and its metastasis, which could be developed as a promising therapeutic strategy.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Breast Neoplasms; Combined Modality Therapy; Female; GPI-Linked Proteins; Humans; Immunotherapy, Adoptive; Mesothelin; Mice; Oncolytic Virotherapy; Oncolytic Viruses; Receptors, Chimeric Antigen; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Disassembly of intercellular junctions is a hallmark of epithelial-mesenchymal transition (EMT). However, how the junctions disassemble remains largely unknown. Here, we report that E3 ubiquitin ligase Smurf1 targets p120-catenin, a core component of adherens junction (AJ) complex, for monoubiquitination during transforming growth factor β (TGFβ)-induced EMT, thereby leading to AJ dissociation. Upon TGFβ treatment, activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylates T900 of p120-catenin to promote its interaction with Smurf1 and subsequent monoubiquitination. Inhibition of T900 phosphorylation or ubiquitination of p120-catenin abrogates TGFβ-induced AJ dissociation and consequent tight junction (TJ) dissociation and cytoskeleton rearrangement, hence markedly blocking lung metastasis of murine breast cancer. Moreover, the T900 phosphorylation level of p120-catenin is positively correlated with malignancy of human breast cancer. Hence, our study reveals the underlying mechanism by which TGFβ induces dissociation of AJs during EMT and provides a potential strategy to block tumor metastasis.

Topics: Adherens Junctions; Animals; Breast Neoplasms; Catenins; Cell Line, Tumor; Cells, Cultured; Delta Catenin; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasms; Phosphorylation; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Ubiquitination

Extracellular signals such as TGF-β can induce epithelial-to-mesenchymal transition (EMT) in cancers of epithelial origin, promoting molecular and phenotypical changes resulting in pro-metastatic characteristics. We identified C/EBPα as one of the most TGF-β-mediated downregulated transcription factors in human mammary epithelial cells. C/EBPα expression prevents TGF-β-driven EMT by inhibiting expression of known EMT factors. Depletion of C/EBPα is sufficient to induce mesenchymal-like morphology and molecular features, while cells that had undergone TGF-β-induced EMT reverted to an epithelial-like state upon C/EBPα re-expression. In vivo, mice injected with C/EBPα-expressing breast tumor organoids display a dramatic reduction of metastatic lesions. Collectively, our results show that C/EBPα is required for maintaining epithelial homeostasis by repressing the expression of key mesenchymal markers, thereby preventing EMT-mediated tumorigenesis. These data suggest that C/EBPα is a master epithelial "gatekeeper" whose expression is required to prevent unwarranted mesenchymal transition, supporting an important role for EMT in mediating breast cancer metastasis.

Topics: Animals; Breast Neoplasms; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; Lung Neoplasms; Mammary Glands, Human; Mice, SCID; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-β stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.

Topics: Amino Acid Motifs; Animals; Breast Neoplasms; Carcinogenesis; Cell Proliferation; Chlorocebus aethiops; COS Cells; Female; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; HEK293 Cells; Humans; Membrane Proteins; Organoids; Phosphatidylinositol 3-Kinase; Protein Isoforms; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Despite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there is a need to develop novel approaches that could overcome mechanisms of tumor resistance to checkpoint inhibition. Emerging evidence has implicated the phenomenon of cancer plasticity or acquisition of mesenchymal features by epithelial tumor cells, as an immune resistance mechanism.. Two soluble factors that mediate tumor cell plasticity in the context of epithelial-mesenchymal transition are interleukin 8 (IL-8) and transforming growth factor beta (TGF-β). In an attempt to overcome escape mechanisms mediated by these cytokines, here we investigated the use of a small molecule inhibitor of the IL-8 receptors CXCR1/2, and a bifunctional agent that simultaneously blocks programmed death ligand 1 (PD-L1) and traps soluble TGF-β.. We demonstrate that simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 signaling synergizes to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells, significantly enhancing T-cell infiltration and activation in tumors, and leading to improved antitumor activity.. This study highlights the potential benefit of combined blockade of CXCR1/2 and TGF-β signaling for modulation of tumor plasticity and potential enhancement of tumor responses to PD-L1 blockade. The data provide rationale for the evaluation of this novel approach in the clinic.

Topics: Animals; Antineoplastic Agents; Apoptosis; B7-H1 Antigen; Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Patients with advanced breast cancer (BC) showed a higher incidence of regional and distant metastases. Sine oculis homeobox homolog 1 (SIX-1) has been confirmed to be a key tumorigenic and metastatic regulator in BC progression. Yet, molecular mechanisms behind SIX-1-induced BC metastases remain largely unknown. Here we found that SIX-1 was frequently up-regulated in BC and correlated with poor outcomes when tested in human BC tissue microarray. Then, we manipulated the expression of SIX-1 by via shRNA-mediated knockdown and lentivirus-mediated overexpression. Transwell assay in vitro and lung metastases model of nude mice in vivo showed that SIX-1 promoted BC cell invasion and migration in vitro, and facilitated metastases in vivo. Mechanistically, SIX-1 could promote the transcription of lncATB, which exerts critical pro-metastatic role in BC by directly binding to the miR-200 family, especially for miR-200c, to induce EMT and promote metastases. In conclusion, SIX-1 exerts its pro-metastatic role in BC through lncATB/miR-200s axis of EMT signalling pathway and could act as an important diagnostic marker as well as a significant therapeutic target for clinically advanced BC.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Mice, Nude; MicroRNAs; Middle Aged; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Treatment Outcome; Zinc Finger E-box-Binding Homeobox 1

New anticancer drugs are usually tested on cancer cells in culture in a standard medium. We stimulated immune polynuclear cells by lipopolysaccharides to obtain an enriched medium (EM) containing inflammatory cytokines more closely reflecting the tumor microenvironment and tested a rhenium-diselenium (Re-diSe) drug in this new model. Concentrations of cytokines were compared with a control medium (CM).. Human-derived breast cancer cells were grown in culture either in CM or EM with or without Re-diSe. Assays of tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), intereukin 1 beta (IL1β), transforming growth factor-beta (TGFβ), insulin growth factor 1 (IGF1) and vascular epidermal growth factor A (VEGFA) were performed by enzyme-linked immunosorbent assays. The production of reactive oxygen species (ROS) was determined by 2,7-dichlorofluorescein test. The cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests.. Concentrations of TNFα, IL6 and Il1β were observed to be significantly higher in EM than in CM. There was no difference for TGFβ, IGF1 and VEGFA. The cells were sensitive to Re-diSe, with reduced concentrations of TGFβ, IGF1, VEGFA and ROS, but the half-maximal inhibitory concentration was significantly higher in EM than in CM.. The efficacy of the Re-diSe drug was confirmed in this model of aggressive cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Primary Cell Culture; Reactive Oxygen Species; Rhenium; Selenium; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.

Topics: Adult; BRCA1 Protein; Breast Neoplasms; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mammary Glands, Human; Middle Aged; Organoids; p38 Mitogen-Activated Protein Kinases; Single-Cell Analysis; Stem Cells; Transforming Growth Factor beta; Young Adult

ΔNp63 is a transcription factor of the p53 family and has crucial functions in normal development and disease. The expression pattern of ΔNp63 in human cancer suggests dynamic regulation of this isoform during cancer progression and metastasis. Many primary and metastatic tumors express high levels of ΔNp63, while ΔNp63 loss is crucial for tumor dissemination, indicating an oscillatory expression of ΔNp63 during cancer progression. Here, we use genetically engineered orthotopic mouse models of breast cancer to show that while depletion of ΔNp63 inhibits primary mammary adenocarcinoma development, oscillatory expression of ΔNp63 in established tumors is crucial for metastatic dissemination in breast cancer. A TGFβ-regulated miRNA network acted as upstream regulators of this oscillatory expression of ΔNp63 during cancer progression. This work sheds light on the pleiotropic roles of ΔNp63 in cancer and unveils critical functions of TGFβ in the metastatic process. SIGNIFICANCE: This study unveils TGFβ signaling and a network of four miRNAs as upstream regulators of ΔNp63, providing key information for the development of therapeutic strategies to treat cancers that commonly overexpress ΔNp63.

Topics: Adenocarcinoma; Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Mutation; Prognosis; Spatio-Temporal Analysis; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Alterations in the composition and architecture of the extracellular matrix (ECM) can influence cancer growth and dissemination. During epithelial-mesenchymal transition (EMT), epithelial cells assume a mesenchymal cell phenotype, changing their adhesion profiles from cell-cell contacts to cell-matrix interactions, contributing to metastasis. Breast cancer cells present at different stages of differentiation, producing distinct ECMs in the same tumor mass. However, the contribution of ECM derived from metastatic tumor cells to EMT is unclear. Here, we showed the mechanisms involved in the interaction of MCF-7, a low-metastatic, epithelial breast cancer cell line, with the ECM produced by a high metastatic breast tumor cell, MDA-MB-231 (MDA-ECM). MDA-ECM induced morphological changes in MCF-7 cells, decreased the levels of E-cadherin, up-regulated mesenchymal markers, and augmented cell migration. These changes were accompanied by the activation of integrin-associated signaling, with increased phosphorylation of FAK, ERK, and AKT and activation canonical TGF-β receptor signaling, enhancing phosphorylation of SMAD2 and SMAD4 nuclear translocation in MCF-7 cells. Treatment with Kistrin (Kr), a specific ligand of integrin αvβ3 EMT induced by MDA-ECM, inhibited TGF-β receptor signaling in treated MCF-7 cells. Our results revealed that after interaction with the ECM produced by a high metastatic breast cancer cell, MCF-7 cells lost their characteristic epithelial phenotype undergoing EMT, an effect modulated by integrin signaling in crosstalk with TGF-β receptor signaling pathway. The data evidenced novel potential targets for antimetastatic breast cancer therapies.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Humans; Integrin alphaVbeta3; Protein Binding; Signal Transduction; Transforming Growth Factor beta

TGFβ promotes cancer aggressiveness in advanced stages. NK1R-Tr expression in advanced breast cancer has a pro-carcinogenic effect. In this study, we aimed to investigate the effect of the association of TGFβ with NK1R-Tr expression on the proliferation and apoptosis of breast cancer cells.. Immunohistochemical staining and Western blot analysis were used to detect TGFβ and NK1R-Tr in breast cancer and paracancerous tissue samples. MDA-MB-231 and BT549 cells were stimulated with TGFβ after NK1R knockdown or treated with the NK1R antagonist aprepitant, and the effects of TGFβ and NK1R-Tr on proliferation and apoptosis were detected by CCK-8, colony formation and flow cytometry assays. In vivo xenograft models were used to further verify the effects of NK1R-Tr and TGFβ. The regulatory effects of Smad4 on NK1R promoter activity were confirmed by ChIP and dual-luciferase reporter assays.. The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in adjacent tissues and were positively correlated in human breast cancer tissues. NK1R knockdown or aprepitant treatment in MDA-MB-231 and BT549 cells attenuated the effects of TGFβ on cell proliferation. The proportion of cells in G2/M phase significantly increased, the expression of cyclin B1 decreased, and the expression of P21 increased; these effects were weakened by TGFβ treatment. Apoptosis in breast cancer cells was significantly increased. In vivo xenograft models were used to further verify that NK1R-Tr and TGFβ promoted tumour growth. After TGFβ treatment, the binding capacity of Smad4 to the NK1R promoter, as well as luciferase activity, was enhanced.. The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in normal tissues, and both were correlated with a poor patient prognosis. TGFβ and NK1R-Tr promoted cell proliferation and inhibited apoptosis, and TGFβ regulated the expression of NK1R-Tr via Smad4.

Topics: Animals; Apoptosis; Aprepitant; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neurokinin-1 Receptor Antagonists; Receptors, Neurokinin-1; Smad4 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

Topics: Adenoviridae; Animals; Bone Neoplasms; Breast Neoplasms; Combined Modality Therapy; Female; Genetic Vectors; Humans; Immune Checkpoint Inhibitors; Mice; Mice, Nude; Middle Aged; Oncolytic Virotherapy; Protein Tyrosine Phosphatase, Non-Receptor Type 22; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The mesenchymal transcription factor forkhead box F2 (FOXF2) is a critical regulator of embryogenesis and tissue homeostasis. Our previous studies demonstrated that FOXF2 is ectopically expressed in basal-like breast cancer (BLBC) cells and that FOXF2 deficiency promotes the epithelial-mesenchymal transition and aggressiveness of BLBC cells. In this study, we found that FOXF2 controls transforming growth factor-beta (TGF-β)/SMAD signaling pathway activation through transrepression of TGF-β-coding genes in BLBC cells. FOXF2-deficient BLBC cells adopt a myofibroblast-/cancer-associated fibroblast (CAF)-like phenotype, and tend to metastasize to visceral organs by increasing autocrine TGF-β signaling and conferring aggressiveness to neighboring cells by increasing paracrine TGF-β signaling. In turn, TGF-β silences FOXF2 expression through upregulating miR-182-5p, a posttranscriptional regulator of FOXF2 and inducer of metastasis. In addition to mediating a reciprocal repression loop between FOXF2 and TGF-β through direct transrepression by SMAD3, miR-182-5p forms a reciprocal repression loop with FOXF2 that directly transrepresses MIR182 expression. Therefore, FOXF2 deficiency accelerates the visceral metastasis of BLBC through unrestricted increases in autocrine and paracrine TGF-β signaling, and miR-182-5p expression. Our findings provide novel mechanisms underlying the roles of TGF-β, miR-182-5p, and FOXF2 in accelerating BLBC dissemination and metastasis, and may facilitate the development of therapeutic strategies for aggressive BLBC.

Topics: Animals; Breast Neoplasms; Cell Movement; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice; Mice, SCID; MicroRNAs; Transforming Growth Factor beta

Soft tissue tumors, including breast cancer, become stiffer throughout disease progression. This increase in stiffness has been shown to correlate to malignant phenotype and epithelial-to-mesenchymal transition (EMT) in vitro. Unlike current models, utilizing static increases in matrix stiffness, our group has previously created a system that allows for dynamic stiffening of an alginate-matrigel composite hydrogel to mirror the native dynamic process. Here, we utilize this system to evaluate the role of matrix stiffness on EMT and metastasis both in vitro and in vivo. Epithelial cells were seen to lose normal morphology and become protrusive and migratory after stiffening. This shift corresponded to a loss of epithelial markers and gain of mesenchymal markers in both the cell clusters and migrated cells. Furthermore, stiffening in a murine model reduced tumor burden and increased migratory behavior prior to tumor formation. Inhibition of FAK and PI3K in vitro abrogated the morphologic and migratory transformation of epithelial cell clusters. This work demonstrates the key role extracellular matrix stiffening has in tumor progression through integrin signaling and, in particular, its ability to drive EMT-related changes and metastasis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Progression; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Humans; Hydrogels; In Vitro Techniques; Integrins; Mammary Neoplasms, Animal; Mice; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phenotype; Signal Transduction; Transforming Growth Factor beta

Drug resistance is a major problem for breast cancer patients. Docetaxel is an anti-mitotic agent that serves as first line of treatment in metastatic breast cancer, however it is susceptible to cellular drug resistance. Drug-resistant cells are able to spread during treatment, leading to treatment failure and eventually metastasis, which remains the main cause for cancer-associated death. In previous studies, we used single-cell technologies and identified a set of genes that exhibit increased expression in drug-resistant cells, and they are mainly regulated by Lef1. Furthermore, upregulating Lef1 in parental cells caused them to become drug resistant. Therefore, we hypothesized that inhibiting Lef1 could resensitize cells to docetaxel. Here, we confirmed that Lef1 inhibition, especially on treatment with the small molecule quercetin, decreased the expression of Lef1 and resensitized cells to docetaxel. Our results demonstrate that Lef1 inhibition also downregulated ABCG2, Vim, and Cav1 expression and equally decreased Smad-dependent TGF-β signaling pathway activation. Likewise, these two molecules worked in a synergetic manner, greatly reducing the viability of drug-resistant cells. Prior studies in phase I clinical trials have already shown that quercetin can be safely administered to patients. Therefore, the use of quercetin as an adjuvant treatment in addition to docetaxel for the treatment of breast cancer may be a promising therapeutic approach.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; Breast Neoplasms; Caveolin 1; Cell Line, Tumor; Cell Proliferation; Cell Survival; Docetaxel; Down-Regulation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Lymphoid Enhancer-Binding Factor 1; MCF-7 Cells; Neoplasm Metastasis; Neoplasm Proteins; Quercetin; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Vimentin

The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a β-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFβ-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFβ-dependent regulatory loops conferring cellular plasticity and invasive behavior.

Topics: Adaptor Proteins, Vesicular Transport; beta Catenin; Breast Neoplasms; Cadherins; Endocytosis; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Transcription Factor 4; Transforming Growth Factor beta

Baoyuan Jiedu (BYJD for short) decoction, a traditional Chinese medicine formula, is composed of Astragalus, Ginseng, Aconite root, Honeysuckle, Angelica, Licorice, which has the functions of nourishing qi and blood, enhancing immune function, improving quality of life and prolonging survival time of tumor patients. The present study aimed to investigate the effect and mechanism of BYJD decoction on reversing the pre-metastatic niche. We showed that BYJD decoction could prolong the survival time of 4T1 tumor-bearing mice. Moreover, we found that the BYJD decoction inhibited the formation of lung pre-metastatic niche and inhibited recruitment of myeloid derived suppressor cells (MDSCs) in the lung. Mechanistically, we showed that the proteins and genes expression of TGF-β, Smad2, Smad3, p-Smad2/3, Smad4, CCL9 in the TGF-β/CCL9 signaling pathway were suppressed by BYJD decoction. In line with the above findings, our results confirm that BYJD decoction inhibits the accumulation of MDSC in pre-metastatic niche of lung via TGF-β/CCL9 pathway.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Chemokines, CC; Drugs, Chinese Herbal; Female; Gene Expression Regulation, Neoplastic; Lung; Lung Neoplasms; Macrophage Inflammatory Proteins; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Tumor Microenvironment

The tumor microenvironment (TME) is a crucial factor in cancer progression. In breast cancer, cancer-associated fibroblasts (CAFs) and the derived stromal components have been recognized as comprising the majority of the pathological structure of the TME. In this study, we show that metformin (Met), a diabetes drug, transforms CAFs in the TME. Met disrupts tumor-stromal cross talk by preventing breast cancer cell transforming growth factor-β (TGF-β) signaling and the production of stromal-derived factor-1 (SDF-1) and interleukin-8 (IL-8) by CAFs. The suppression of bidirectional signaling between tumor cells and CAFs by Met is attributed to increased phospho-AMP kinase (p-AMPK) levels. By upregulating p-AMPK in CAFs, Met induces prolyl hydroxylases (PHDs), leading to the degradation of hypoxia-inducible factor-1α (HIF-1α) in CAFs. Moreover, interruption of HIF-1α-driven SDF-1 signaling in CAFs by Met leads to decreased breast cancer cell invasion. These findings suggest that Met may be used to target tumor-promoting signaling between CAFs and breast cancer cells in the TME.

Topics: Adenylate Kinase; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Chemokine CXCL12; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; MCF-7 Cells; Metformin; Neoplasm Invasiveness; Prolyl Hydroxylases; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation

Primary tumor metastasis remains to be a tough obstacle for clinical breast cancer treatment. Since evidences have shown that mitochondria play a crucial role in tumor metastasis, we designed a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to simultaneously inhibit breast cancer progression and metastasis. A novel mitochondrial targeted hybrid peptide R8MTS, which consists of a cell penetrating peptide octaarginine (R8) and a mitochondrial targeting sequence ALD5

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Female; Humans; Matrix Metalloproteinase 2; Methacrylates; Mice; Mitochondria; Neoplasm Metastasis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Paired box 6 (PAX6) is a transcription factor that has oncogenic features. In breast cancer, PAX6 facilitates tumor progression; however, the underlying mechanism is largely unknown. The majority of breast cancer‑related mortalities are associated with metastasis of cancer cells. Therefore, the present study aimed to investigate the role of PAX6 in breast tumor metastasis. PAX6 was stably overexpressed in breast cancer cells to perform tumor migration and metastasis assays in vitro and in vivo. In addition, the expression of PAX6 and transforming growth factor β (TGF‑β)‑SMAD signaling associated proteins on human breast cancer tissue array, as well as key factors involved in epithelial‑mesenchymal transition (EMT) were assayed to explore the mechanism underlying metastasis of breast cancer cells. The expression levels of PAX6 were demonstrated to be increased in human breast cancer tissues and associated with poor clinical outcomes. Overexpression of PAX6 markedly promoted metastasis. Further investigation revealed that PAX6 overexpression increased TGF‑β‑SMAD signaling pathway and induced EMT. These results suggested that highly expressed PAX6 led to EMT through TGF‑β‑SMAD signaling pathway, thereby promoting cell metastasis and ultimately affecting survival in patients with breast cancer. Taken together, findings indicated that PAX6 may serve as a therapeutic target for the clinical treatment of breast cancer and the underlying mechanism could be used to overcome metastasis of cancer cells.

Topics: Animals; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Datasets as Topic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Neoplasm Metastasis; PAX6 Transcription Factor; Prognosis; Signal Transduction; Smad Proteins; Tissue Array Analysis; Transforming Growth Factor beta; Xenograft Model Antitumor Assays; Zebrafish

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.

Topics: Animals; Ataxin-1; Breast Neoplasms; Cell Line, Tumor; Cell Plasticity; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Neoplastic Stem Cells; Receptor, ErbB-2; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and β1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and β1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated β1 with TNC in lysates. The knockdown of αv and β1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvβ1 heterodimer levels to activate transforming growth factor-β signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues.

Topics: Breast Neoplasms; Cell Differentiation; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Humans; Myofibroblasts; Nerve Tissue Proteins; Phosphorylation; Receptors, Vitronectin; Signal Transduction; Tenascin; Transforming Growth Factor beta

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Movement; Collagen; Humans; Lipoma; Podosomes; Src Homology 2 Domain-Containing, Transforming Protein 1; Transforming Growth Factor beta

Breast tumor heterogeneity is a major impediment to oncotherapy. Cancer cells undergo rapid clonal evolution, thereby acquiring significant growth and invasive advantages. The absence of specific markers of these high-risk populations precludes efficient therapeutic and diagnostic management of the disease. Given the critical function of tumor microenvironment in the oncogenic circuitry, we sought to determine the expression profile of the extracellular matrix oncoprotein, extradomain-B fibronectin (EDB-FN) in invasive breast cancer. Analyses of TCGA/GTEx databases and immunostaining of clinical samples found a significant overexpression of EDB-FN in breast tumors, which correlated with poor overall survival. Significant upregulation of EDB-FN was observed in invasive cell populations generated from relatively less invasive MCF7 and MDA-MB-468 cells by long-term TGF-β treatment and acquired chemoresistance. Treatment of the invasive cell populations with an AKT inhibitor (MK2206-HCl) reduced their invasive potential, with a concomitant decrease in their EDB-FN expression, partly through the phosphoAKT-SRp55 pathway. EDB-FN downregulation, with direct RNAi of EDB-FN or indirectly through RNAi of SRp55, also resulted in reduced motility of the invasive cell populations, validating the correlation between EDB-FN expression and invasion of breast cancer cells. These data establish EDB-FN as a promising molecular marker for non-invasive therapeutic surveillance of aggressive breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Phosphoproteins; Proto-Oncogene Proteins c-akt; Serine-Arginine Splicing Factors; Transforming Growth Factor beta; Tumor Microenvironment

Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlled by TGF-β with one of them-Arrdc3-being able to modulate Epithelial to Mesenchymal Transition. This experimental framework allowed us to correlate lncRNA/chromatin interactions with the real outcome of gene expression and to start defining the gene network regulated by EPR as a component of the TGF-β pathway.

Topics: Animals; Arrestins; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; RNA, Long Noncoding; Transcriptome; Transforming Growth Factor beta

Circulating long non-coding RNAs (lncRNAs) have proven to be useful non-invasive tools for diagnosis of various cancers. FAM83H antisense RNA 1 (FAM83H-AS1) and lncRNA activated by TGF β (lncRNA-ATB) are two lncRNAs that have been shown to play an important role in different cancer types including breast cancer. The primary aim of our study was to investigate the potential role of serum FAM83H-AS1 and lncRNA-ATB as diagnostic/prognostic markers for breast cancer patients.. Serum expression levels of FAM83H-AS1 and lncRNA-ATB were analyzed in 90 breast cancer patients and 30 age- and sex-matched healthy controls using RT-qPCR.. We found that FAM83H-AS1 and lncRNA-ATB were significantly overexpressed in sera of breast cancer patients compared to controls (p = 0.000 for both). Analysis of receiver operating characteristic curve demonstrated that lncRNA-ATB had a higher area under curve (AUC) value than the conventional tumor marker cancer antigen 15-3 (CA15-3) (AUC: 0.844, p = 0.000 versus 0.738, p = 0.002) for early diagnosis of breast cancer in patients with stage I-II. On the other hand, FAM83H-AS1 showed a significant correlation with tumor-node metastasis (TNM) stages, large tumor size and lymph node metastasis, suggesting a prognostic rather than diagnostic value.. This is the first study to demonstrate that serum lncRNA-ATB could be used as a non-invasive diagnostic marker for early stages of breast cancer. Furthermore, serum FAM83H-AS1 has a potential ability for monitoring of progression and staging of breast cancer.

Topics: Adult; Aged; Biomarkers; Breast Neoplasms; Early Diagnosis; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Middle Aged; Mucin-1; Predictive Value of Tests; Prognosis; Proteins; RNA, Antisense; RNA, Long Noncoding; ROC Curve; Transforming Growth Factor beta

Angiogenesis is one of the hallmarks of cancer. We hypothesized that intra-tumoral angiogenesis correlates with inflammation and metastasis in breast cancer patients. To test this hypothesis, we generated an angiogenesis pathway score using gene set variation analysis and analyzed the tumor transcriptome of 3999 breast cancer patients from The Cancer Genome Atlas Breast Cancer (TCGA-BRCA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), GSE20194, GSE25066, GSE32646, and GSE2034 cohorts. We found that the score correlated with expression of various angiogenesis-, vascular stability-, and sphingosine-1-phosphate (S1P)-related genes. Surprisingly, the angiogenesis score was not associated with breast cancer subtype, Nottingham pathological grade, clinical stage, response to neoadjuvant chemotherapy, or patient survival. However, a high score was associated with a low fraction of both favorable and unfavorable immune cell infiltrations except for dendritic cell and M2 macrophage, and with Leukocyte Fraction, Tumor Infiltrating Lymphocyte Regional Fraction and Lymphocyte Infiltration Signature scores. High-score tumors had significant enrichment for unfavorable inflammation-related gene sets (interleukin (IL)6, and tumor necrosis factor (TNF)α- and TGFβ-signaling), as well as metastasis-related gene sets (epithelial mesenchymal transition, and Hedgehog-, Notch-, and WNT-signaling). High score was significantly associated with metastatic recurrence particularly to brain and bone. In conclusion, using the angiogenesis pathway score, we found that intra-tumoral angiogenesis is associated with immune reaction, inflammation and metastasis-related pathways, and metastatic recurrence in breast cancer.

Topics: Breast Neoplasms; Dendritic Cells; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Interferon-gamma; Interleukin-6; Leukocytes; Lymphocytes, Tumor-Infiltrating; Lysophospholipids; Macrophages; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Signal Transduction; Sphingosine; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) and maturation of a fibrillar tumor microenvironment play important roles in breast cancer progression. A better understanding of how these events promote cancer cell migration and invasion could help identify new strategies to curb metastasis. The nucleus and Golgi affect migration in a microenvironment-dependent manner. Nucleus size and mechanics influence the ability of a cell to squeeze through confined tumor microenvironments. Golgi positioning determines front-rear polarity necessary for migration. While the roles of individual attributes of nucleus and Golgi in migration are being clarified, how their manifold features are inter-related and work together remains to be understood at a systems level. Here, to elucidate relationships among nucleus and Golgi properties, we quantified twelve morphological and positional properties of these organelles during fibrillar migration of human mammary epithelial cells. Principal component analysis (PCA) reduced the twelve-dimensional space of measured properties to three principal components that capture 75% of the variations in organelle features. Unexpectedly, nucleus and Golgi properties that co-varied in a PCA model built with data from untreated cells were largely similar to co-variations identified using data from TGFβ-treated cells. Thus, while TGFβ-mediated EMT significantly alters gene expression and motile phenotype, it did not significantly affect the relationships among nucleus size, aspect ratio and orientation with migration direction and among Golgi size and nucleus-Golgi separation distance. Indeed, in a combined PCA model incorporating data from untreated and TGFβ-treated cells, scores of individual cells occupy overlapping regions in principal component space, indicating that TGFβ-mediated EMT does not promote a unique "Golgi-nucleus phenotype" during fibrillar migration. These results suggest that migration along spatially-confined fiber-like tracks employs a conserved nucleus-Golgi arrangement that is independent of EMT state.

Topics: Breast Neoplasms; Cell Line; Cell Movement; Cell Nucleus; Cytoskeleton; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Golgi Apparatus; Humans; Transforming Growth Factor beta; Tumor Microenvironment

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (

Topics: Aged; Breast Neoplasms; Circulating MicroRNA; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Hippo Signaling Pathway; Humans; MicroRNAs; Middle Aged; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs) can exist in pro- and anti-inflammatory states. Anti-inflammatory TAMs (also referred to as M2-polarized) generally suppress antitumor immune responses and enhance the metastatic progression of cancer. To explore the mechanisms behind this phenomenon, we isolated macrophages from mice and humans, polarized them ex vivo, and examined their functional interaction with breast cancer cells in culture and in mice. We found that anti-inflammatory TAMs promoted a metabolic state in breast cancer cells that supported various protumorigenic phenotypes. Anti-inflammatory TAMs secreted the cytokine TGF-β that, upon engagement of its receptors in breast cancer cells, suppressed the abundance of the transcription factor STAT1 and, consequently, decreased that of the metabolic enzyme succinate dehydrogenase (SDH) in the tumor cells. The decrease in SDH levels in tumor cells resulted in an accumulation of succinate, which enhanced the stability of the transcription factor HIF1α and reprogrammed cell metabolism to a glycolytic state. TAM depletion-repletion experiments in a 4T1 mouse model additionally revealed that anti-inflammatory macrophages promoted HIF-associated vascularization and expression of the immunosuppressive protein PD-L1 in tumors. The findings suggest that anti-inflammatory TAMs promote tumor-associated angiogenesis and immunosuppression by altering metabolism in breast cancer cells.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; RNA Interference; Signal Transduction; Succinate Dehydrogenase; Transforming Growth Factor beta

An altered consistency of tumor microenvironment facilitates the progression of the tumor towards metastasis. Here we combine data from secretome and proteome analysis using mass spectrometry with microarray data from mesenchymal transformed breast cancer cells (MCF-7-EMT) to elucidate the drivers of epithelial-mesenchymal transition (EMT) and cell invasion. Suppression of connective tissue growth factor (CTGF) reduced invasion in 2D and 3D invasion assays and expression of transforming growth factor-beta-induced protein ig-h3 (TGFBI), Zinc finger E-box-binding homeobox 1 (ZEB1) and lysyl oxidase (LOX), while the adhesion of cell-extracellular matrix (ECM) in mesenchymal transformed breast cancer cells is increased. In contrast, an enhanced expression of CTGF leads to an increased 3D invasion, expression of fibronectin 1 (FN1), secreted protein acidic and cysteine rich (SPARC) and CD44 and a reduced cell ECM adhesion. Gonadotropin-releasing hormone (GnRH) agonist Triptorelin reduces CTGF expression in a Ras homolog family member A (RhoA)-dependent manner. Our results suggest that CTGF drives breast cancer cell invasion in vitro and therefore could be an attractive therapeutic target for drug development to prevent the spread of breast cancer.

Topics: Breast Neoplasms; Connective Tissue Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibronectins; Gene Expression; Humans; Hyaluronan Receptors; MCF-7 Cells; Neoplasm Invasiveness; Osteonectin; Protein-Lysine 6-Oxidase; rhoA GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta; Triptorelin Pamoate; Zinc Finger E-box-Binding Homeobox 1

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Death; Cell Hypoxia; Cell Line, Tumor; Female; HEK293 Cells; Humans; Immunotherapy; Interleukin-4; Lymph Nodes; Male; Mice; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Growth factors and cytokines mediate complex interactions between cells within the breast tumour microenvironment. In advanced cancer, an excess of regulatory T (T. We used 3D breast tumour models to determine morphological alterations, and the levels of secreted transforming growth factor-β (TGFβ) and induced cytokines. 3D luminal phenotype models and basal phenotype models were generated by culturing NK cells and CD4. We identified that an interleukin-6 (IL6)-chemokine axis associated with TGFβ is primarily responsible for differences detected between breast cancer models, with luminal and basal phenotype tumours responding differentially to immune mediation. Identified cytokines are implicated in facilitating tumour cell subversion of immune cell function to promote an invasive phenotype. Moreover, the disruption of the extracellular matrix and failure to form well-differentiated tumour masses/networks is indicative of enhanced malignancy. Tumour cells are implicated in promoting a pro-inflammatory microenvironment to attenuate NK cell function and in inducing a pro-tumorigenic profile that is facilitated by T

Topics: Breast Neoplasms; Cell Culture Techniques; Cell Shape; Female; Humans; Inflammation; Inflammation Mediators; MCF-7 Cells; Models, Biological; Principal Component Analysis; Transforming Growth Factor beta

Treatment of human estrogen receptor (ER)‑positive breast cancer (ER+ BC) using conventional chemotherapy remains a challenge and is often ineffective as a result of tumor metastasis. The present study aimed to investigate the ability of narasin, an ionophore antibiotic, to potentially inhibit tumor metastasis and growth in human ER+ BC. Narasin was found to have significant inhibitory abilities on cell proliferation, migration and invasion in ER+ BC cell lines MCF‑7 and T47D compared with the triple‑negative BC cell MDA‑MB‑231. For the in vivo studies, narasin effectively decreased the number of tumor metastasis nodules, tumor volume and weight without apparent toxicity in human MCF‑7 nude mouse left ventricle injection tumor metastasis and xenograft models. Mechanistically, it demonstrated that exposure to TGF‑β or IL‑6 induced the expression of epithelial‑mesenchymal transition (EMT) markers in ER+ BC cell lines. On the contrary, narasin dose‑dependently reversed EMT by increasing the expression of E‑cadherin and decreasing the expression of N‑cadherin, vimentin, β‑catenin and zinc finger E‑box‑binding homeobox 1 at the protein and gene expression levels. Gene microarray, molecular docking and western blotting were performed to demonstrate that those protein and gene expression levels are regulated by the inactivation of the TGF‑β/phosphorylated (p)‑SMAD3 and IL‑6/p‑STAT3 signaling pathways. Taken together, these findings indicated that narasin may be a promising candidate that can be further optimized for the treatment of human ER+ BC.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Docking Simulation; Neoplasm Metastasis; Pyrans; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-β signaling can lead to breast cancer. TGF-β elicits its effects on proliferation and differentiation via specific cell surface TGF-β type I and type II receptors (i.e., TβRI and TβRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-β-induced heteromeric complex formation, activated TβRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-β acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-β signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-β-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-β-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-β receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-β-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-β-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

Topics: Animals; Benzamides; Breast Neoplasms; Dioxoles; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Phosphorylation; Pyrazoles; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-β (TGF-β). TGF-β is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3β which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-β-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-β/SMAD signaling pathway by stabilizing SMAD4.

Topics: Acetylglucosamine; Breast Neoplasms; Female; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Protein Processing, Post-Translational; Proteolysis; Serine; Signal Transduction; Smad4 Protein; Threonine; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin

Evidence shows that the anticancer effects of microtubule targeting agents are not due solely to their antimitotic activities but also their ability to impair microtubule-dependent oncogenic signalling.. The effects of microtubule targeting agents on regulators of TGF-β-induced epithelial-to-mesenchymal transition (EMT) were evaluated in breast cancer cell lines using high content imaging, gene and protein expression, siRNA-mediated knockdown and chromatin immunoprecipitation.. Microtubule targeting agents rapidly and differentially alter the expression of Snail and Slug, key EMT-promoting transcription factors in breast cancer. Eribulin, vinorelbine and in some cases, ixabepalone, but not paclitaxel, inhibited TGF-β-mediated Snail expression by impairing the microtubule-dependent nuclear localisation of Smad2/3. In contrast, eribulin and vinorelbine promoted a TGF-β-independent increase in Slug in cells with low Smad4. Mechanistically, microtubule depolymerisation induces c-Jun, which consequently increases Slug expression in cells with low Smad4.. These results identify a mechanism by which eribulin-mediated microtubule disruption could reverse EMT in preclinical models and in patients. Furthermore, high Smad4 levels could serve as a biomarker of this response. This study highlights that microtubule targeting drugs can exert distinct effects on the expression of EMT-regulating transcription factors and that identifying differences among these drugs could lead to their more rational use.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Chromatin Immunoprecipitation; Epithelial-Mesenchymal Transition; Epothilones; Female; Furans; Gene Expression; Genes, jun; Humans; Ketones; Microtubules; Paclitaxel; Smad2 Protein; Smad3 Protein; Smad4 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta; Tubulin Modulators; Vinorelbine

Topics: Animals; Antigens, CD; Breast Neoplasms; Cadherins; Carcinoma, Ductal, Breast; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

GATA3 is a basic and essential transcription factor that regulates many pathophysiological processes and is required for the development of mammary luminal epithelial cells. Loss-of-function GATA3 alterations in breast cancer are associated with poor prognosis. Here, we sought to understand the tumor-suppressive functions GATA3 normally performs. We discovered a role for GATA3 in suppressing epithelial-to-mesenchymal transition (EMT) in breast cancer by activating miR-455-3p expression. Enforced expression of miR-455-3p alone partially prevented EMT induced by transforming growth factor β (TGF-β) both in cells and tumor xenografts by directly inhibiting key components of TGF-β signaling. Pathway and biochemical analyses showed that one miRNA-455-3p target, the TGF-β-induced protein ZEB1, recruits the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex to the promotor region of miR-455 to strictly repress the GATA3-induced transcription of this microRNA. Considering that ZEB1 enhances TGF-β signaling, we delineated a double-feedback interaction between ZEB1 and miR-455-3p, in addition to the repressive effect of miR-455-3p on TGF-β signaling. Our study revealed that a feedback loop between these two axes, specifically GATA3-induced miR-455-3p expression, could repress ZEB1 and its recruitment of NuRD (MTA1) to suppress miR-455, which ultimately regulates TGF-β signaling. In conclusion, we identified that miR-455-3p plays a pivotal role in inhibiting the EMT and TGF-β signaling pathway and maintaining cell differentiation. This forms the basis of that miR-455-3p might be a promising therapeutic intervention for breast cancer.

Topics: Animals; Base Sequence; Breast; Breast Neoplasms; Cell Proliferation; Epithelial Cells; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Female; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, SCID; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer.. In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models.. Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor β (TGFβ) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models.. Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

Topics: Animals; Bone Morphogenetic Proteins; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression; Humans; Intercellular Signaling Peptides and Proteins; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Neoplastic Stem Cells; Phosphorylation; Prognosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Zebrafish

Alternatively-activated pathways have been observed in biological experiments in cancer studies, but the concept had not been fully explored in computational cancer system biology. Therefore, an alternatively-activated pathway identification method was proposed and applied to primary breast cancer and breast cancer liver metastasis research using microarray data. Interestingly, the results show that

Topics: Breast Neoplasms; Calcium Signaling; Cytokines; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Metabolic Networks and Pathways; Oligonucleotide Array Sequence Analysis; Transforming Growth Factor beta

Metastasis is the leading cause of mortality in patients with solid tumors. In this regard, we previously reported that Pseudopodium-Enriched Atypical Kinase One (PEAK1) is necessary for non-canonical Transforming Growth Factor β (TGFβ) signaling and TGFβ/fibronectin-induced metastasis. Here, we demonstrate that inhibition of DHPS-dependent eIF5A1/2 hypusination blocks PEAK1 and E-Cadherin expression, breast cancer cell viability and TGFβ/fibronectin-induced PEAK1-dependent breast cancer metastasis. Interestingly, TGFβ stimulation of high-grade metastatic breast cancer cells increases and sustains eIF5A1/2 hypusination. We used a suite of bioinformatics platforms to search biochemical/functional interactions and clinical databases for additional control points in eIF5A1/2 and PEAK1-Epithelial to Mesenchymal Transition (EPE) pathways. This effort revealed that interacting EPE genes were enriched for TP53 transcriptional targets and were commonly co-amplified in breast cancer patients harboring inactivating TP53 mutations. Taken together, these results suggest that combinatorial therapies targeting DHPS and protein activities elevated in TP53-mutant breast cancers may reduce systemic tumor burden and improve patient outcomes.

Topics: Breast Neoplasms; Cadherins; Eukaryotic Translation Initiation Factor 5A; Female; Fibronectins; Guanine; Humans; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; Prognosis; Protein-Tyrosine Kinases; RNA-Binding Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Our search for signaling pathways regulated by breast tumor kinase (BRK), a nonreceptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to find that BRK competitively binds and phosphorylates SMAD4 and regulates transforming growth factor-β/SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F) phosphorylates SMAD4, resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. Activated BRK-mediated degradation of SMAD4 is associated with the repression of tumor suppressor gene

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Neoplasm Proteins; Phosphorylation; Protein-Tyrosine Kinases; Smad4 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta; Tyrosine; Ubiquitination

Transforming growth factor-β (TGFβ) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional effectors of TGFβ. Smads form complexes with other DNA-binding transcription factors to elicit cell-type-dependent responses. Previously, we found that the collagen invasion and migration of pre-malignant breast cancer cells in response to TGFβ and epidermal growth factor (EGF) critically depend on multiple Jun and Fos components of the activator protein (AP)-1 transcription factor complex. Here we report that the same process is negatively regulated by Jun N-terminal kinase (JNK)-dependent cJun phosphorylation. This was demonstrated by analysis of phospho-deficient, phospho-mimicking, and dimer-specific cJun mutants, and experiments employing a mutant version of the phosphatase MKP1 that specifically inhibits JNK. Hyper-phosphorylation of cJun by JNK strongly inhibited its ability to induce several Jun/Fos-regulated genes and to promote migration and invasion. These results show that MEK-AP-1 and JNK-phospho-cJun exhibit distinct pro- and anti-invasive functions, respectively, through differential regulation of Smad- and AP-1-dependent TGFβ target genes. Our findings are of importance for personalized cancer therapy, such as for patients suffering from specific types of breast tumors with activated EGF receptor-Ras or inactivated JNK pathways.

Topics: Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Genes, jun; HeLa Cells; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-jun; Recombinant Proteins; Signal Transduction; Transcription Factor AP-1; Transforming Growth Factor beta

Breast cancer is one of the major malignancies with a mounting mortality rate in the world. Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) has been identified to regulate the initiation and progression of multiple tumorous diseases according to previous studies. However, its biological role has been rarely reported in breast cancer. In the present study, lncRNA CASC2 was found to be significantly downregulated in breast cancer tissues and cell lines using real-time quantitative PCR. Furthermore, gain-of-function assays demonstrated that overexpression of lncRNA CASC2 significantly repressed breast cancer cell proliferation and metastasis. Moreover, CASC2 induced cell cycle arrest and much more early apoptosis of breast cancer. Additionally, based on the above research, we illustrated that inactivation of the TGF-β signaling pathway was involved in the function of lncRNA CASC2. Collectively, lncRNA CASC2 was a key factor in the tumorigenesis and malignancy of breast cancer, suggesting it may possibly be a potential therapy target for the treatment of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Up-Regulation

To examine the outcomes of sexual function in postmenopausal women and women with a history of breast cancer treated with endocrine therapy who were experiencing the symptoms of GSM for which they were treated with fractional microablative CO. From July 2015 to October 2016, a retrospective chart review of women who underwent fractional microablative CO. There was a statistically significant improvement in every domain of FSFI, WBFS, and FSDS-R when comparing baseline symptom scores to after treatment three symptom scores for all patients. The secondary outcome was to evaluate the differences, if any, in outcomes of sexual function between postmenopausal women and women with a history of breast cancer treated with endocrine therapy. Both groups had statistically significant improvements in many domains studied.. Fractional microablative CO

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cancer Survivors; Female; Female Urogenital Diseases; HSP47 Heat-Shock Proteins; Humans; Laser Therapy; Lasers, Gas; Middle Aged; Mucous Membrane; Postmenopause; Retrospective Studies; Sexual Dysfunction, Physiological; Surveys and Questionnaires; Transforming Growth Factor beta; Treatment Outcome; Vagina

The glycosaminoglycan hyaluronan has a crucial role in tissue organization and cell signaling. Hyaluronan accumulates in conjunction with rapid tissue remodeling during embryogenesis, as well as in inflammatory conditions and cancer. We report a negative correlation between the expression of genes encoding hyaluronan synthase HAS2, its natural antisense transcript HAS2-AS, the chromatin modulating factor HMGA2 and transforming growth factor-β (TGFβ), and survival of patients with invasive breast carcinomas. In mouse mammary epithelial cells, TGFβ activates Smad and non-Smad signaling pathways, resulting in the transcriptional induction of Has2, Has2as (the mouse ortholog of HAS2-AS) and Hmga2, as well as epithelial-mesenchymal transition (EMT)-promoting transcription factors, such as Snail. Importantly, Has2as abrogation suppressed the TGFβ induction of EMT markers, including Snai1, Hmga2, Fn1, and suppressed the mesenchymal phenotype. TGFβ induction of Hmga2, Has2as and Has2, and synthesis of hyaluronan were accompanied with activation of Akt and Erk1/2 MAP-kinase signaling and were required for breast cancer cell motility. Importantly, the hyaluronan receptor Cd44, but not Hmmr, was required for TGFβ-mediated EMT phenotype. Interestingly, Has2as was found to contribute to the maintenance of stem cell factors and breast cancer stemness. Our findings show that Has2as has a key role in TGFβ- and HAS2-induced breast cancer EMT, migration and acquisition of stemness.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HMGA2 Protein; Humans; Hyaluronan Synthases; Hyaluronic Acid; Mice; Neoplasm Transplantation; Neoplastic Stem Cells; RNA, Long Noncoding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The epithelial mesenchymal transition (EMT) is one step in the process through which carcinoma cells metastasize by gaining the cellular mobility associated with mesenchymal cells. This work examines the dual influence of the TGF-β pathway and intercellular contact on the activation of EMT in colon (SW480) and breast (MCF7) carcinoma cells. While the SW480 population revealed an intermediate state between the epithelial and mesenchymal states, the MC7 cells exhibited highly adhesive behavior. However, for both cell lines, an exogenous TGF-β signal and a reduction in cellular confluence can push a subgroup of the population towards the mesenchymal phenotype. Together, these results highlight that, while EMT is induced by the synergy of multiple signals, this activation varies across cell types.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Adhesion; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Movement; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Protein Stability; RNA, Small Interfering; Snail Family Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Specific Proteases; Xenograft Model Antitumor Assays

Breast cancer (BCa) bone metastases (BMETs) drive osteolysis via a feed-forward loop involving tumoral secretion of osteolytic factors (e.g., PTHrP) induced by bone-matrix-derived growth factors (e.g., TGFβ). In prior experiments, turmeric-derived curcumin inhibited in vivo BMET progression and in vitro TGFβ/Smad-signaling in a TGFβ-stimulated PTHrP-dependent human xenograft BCa BMET model (MDA-SA cells). However, it is unclear whether curcumin or curcumin-glucuronide mediates in vivo protection since curcumin-glucuronide is the primary circulating metabolite in rodents and in humans. Thus, effects of curcumin vs. curcumin-glucuronide on Smad-dependent TGFβ signaling were compared in a series of BCa cell lines forming TGFβ-dependent BMET in murine models, and tissue-specific metabolism of curcumin in mice was examined by LC-MS. While curcumin inhibited TGFβ-receptor-mediated Smad2/3 phosphorylation in all BCa cells studied (human MDA-SA, MDA-1833, MDA-2287 and murine 4T1 cells), curcumin-glucuronide did not. Similarly, curcumin, but not curcumin-glucuronide, blocked TGFβ-stimulated secretion of PTHrP from MDA-SA and 4T1 cells. Because the predominant serum metabolite, curcumin-glucuronide, lacked bioactivity, we examined tissue-specific metabolism of curcumin in mice. Compared to serum and other organs, free curcumin (both absolute and percentage of total) was significantly increased in bone, which was also a rich source of enzymatic deglucuronidation activity. Thus, curcumin, and not curcumin-glucuronide, appears to inhibit bone-tropic BCa cell TGFβ-signaling and to undergo site-specific activation (deconjugation) within the bone microenvironment. These findings suggest that circulating curcumin-glucuronide may act as a prodrug that preferentially targets bone, a process that may contribute to the bone-protective effects of curcumin and other highly glucuronidated dietary polyphenols.

Topics: Administration, Oral; Animals; Antineoplastic Agents, Phytogenic; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Curcumin; Female; Glucuronides; Humans; Mice; Mice, Inbred C57BL; Parathyroid Hormone-Related Protein; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Metastasis is the process through which the primary cancer cells spread beyond the primary tumor and disseminate to other organs. Most cancer patients die of metastatic disease. EMT is proposed to be the initial event associated with cancer metastasis and how it occurred is still a mystery. CtBP is known as a co-repressor abundantly expressed in many types of cancer and regulates genes involved in cancer initiation, progression, and metastasis. We found that CtBP regulates intracellular cholesterol homeostasis in breast cancer cells by forming a complex with ZEB1 and transcriptionally repressing SREBF2 expression. Importantly, CtBP repression of intracellular cholesterol abundance leads to increased EMT and cell migration. The reason is that cholesterol negatively regulates the stability of TGF-β receptors on the cell membrane. Interestingly, TGF-β is also capable of reducing intracellular cholesterol relying on the increased recruitment of ZEB1 and CtBP complex to SREBF2 promoter. Thus, we propose a feedback loop formed by CtBP, cholesterol, and TGF-β signaling pathway, through which TGF-β triggers the cascade that mobilizes the cancer cells for metastasis. Consistently, the intravenous injection of breast cancer cells with ectopically CtBP expression show increased lung metastasis depending on the reduction of intracellular cholesterol. Finally, we analyzed the public breast cancer datasets and found that CtBP expression negatively correlates with SREBF2 and HMGCR expressions. High expression of CtBP and low expression of SREBF2 and HMGCR significantly correlates with high EMT of the primary tumors.

Topics: Alcohol Oxidoreductases; Breast Neoplasms; Cell Membrane; Cell Movement; Cholesterol; Co-Repressor Proteins; DNA-Binding Proteins; Feedback, Physiological; Homeostasis; Humans; MCF-7 Cells; Neoplasm Metastasis; Nerve Tissue Proteins; Signal Transduction; Sterol Regulatory Element Binding Protein 2; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

The influence and underlying mechanisms of human adipose‑derived stem cells (Hu‑ADSCs) on breast cancer cells in the tumor microenvironment remain unclear. Understanding the association between Hu‑ADSCs and cancer cells may provide targets for breast cancer treatment and reference for the clinical application of stem cells. Therefore, a Hu‑ADSC and breast cancer MCF7 cell coculture system was established to investigate the paracrine effects of Hu‑ADSCs on MCF7 cell migration and invasion, in addition to the potential mechanism of action by reverse transcription‑quantitative polymerase chain reaction and western blotting. Hu‑ADSCs enhanced MCF7 cell migration and invasion by decreasing the expression of epithelial marker E‑cadherin, and increasing the expression of interstitial marker N‑cadherin and epithelial‑mesenchymal transition (EMT) transcription factors in vitro. The EMT effect of cocultured MCF7 cells was inhibited with the addition of anti‑transforming growth factor (TGF)‑β1 or phosphoinositide 3‑kinase (PI3K) inhibitor LY294002, accompanied by a significant decrease in phosphorylated (p)‑mothers against decapentaplegic homolog (Smad) and p‑protein kinase B (AKT) expression. The data suggested that the paracrine effect of Hu‑ADSCs in the tumor microenvironment promoted the EMT of MCF7 cells by cross interacting with the TGF‑β/Smad and PI3K/AKT pathways.

Topics: Adipose Tissue; Adult; Biomarkers, Tumor; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Coculture Techniques; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Mesenchymal Stem Cells; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment; Young Adult

To investigate the role and mechanism of action of miR-153 in the migration, invasion, and epithelial-mesenchymal transition (EMT) of breast cancer cells.. Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-153 and transforming growth factor beta receptor 2 (TGFBR2) in tissue specimens and cells. miR-153 overexpression in breast cancer cells was achieved by miR-153 mimic transfection. Mobility and invasiveness of breast cancer cells were evaluated by transwell assay. EMT was evaluated by Western blot detecting the protein level of E-cadherin and Vimentin. Interaction of miR-153 and 3'-untranslated region (UTR) of TGFBR2 messenger RNA (mRNA) was investigated by luciferase reporter assay.. The expression of miR-153 in breast cancer tissue specimens and MDA-MB-231 cells was significantly lower than that in nonmalignant counterparts, inversely correlating with that of TGFBR2 mRNA. Transfection with miR-153 mimic significantly increased miR-153 level in MDA-MB-231 cells while inhibiting its migration, invasion, and EMT in vitro, which could be mimicked by TGFBR2 knockdown. Luciferase reporter assay confirmed two targets of miR-153 on the 3'-UTR of TGFBR2 mRNA. Restoring TGFBR2 protein level by transient overexpression largely rescued migration, invasion, and EMT of MDA-MB-231 cells that were repressed by miR-153 mimic transfection.. miR-153 inhibits breast cancer cell migration, invasion, and EMT by targeting TGFBR2.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Invasiveness; Receptor, Transforming Growth Factor-beta Type II; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFβ-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGFβ-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGFβ-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chromatin; Epithelial-Mesenchymal Transition; Female; Gene Knockdown Techniques; Histone-Lysine N-Methyltransferase; Humans; Neoplasm Invasiveness; Phosphorylation; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays; Zebrafish

Advanced breast cancer frequently metastasizes to the bone, resulting in patient morbidity and mortality. The interaction of tumor cells with osteoclasts and osteoblasts seriously affects the occurrence and development of bone metastasis in breast cancer. The signaling crosstalk among the Jagged1/Notch, TGF-β and IL-6 signaling pathways plays a significant role in the context of bone metastasis.. Although Wenshen Zhuanggu (WSZG) formula efficiently decreased the risk of bone metastases in tumor-bearing mice, it remains unclear how WSZG formula regulates the interaction of cancer cells with osteoclasts and osteoblasts in bone metastasis of breast cancer.. In this study, we investigated the role of WSZG formula in the progress of bone metastasis in breast cancer and focused on the cell-cell interactions of tumor cells with osteoclasts and osteoblasts. Western blotting and quantitative real-time PCR were utilized to evaluate the inhibitory activities of WSZG formula on Jagged1 expression both in vivo and in vitro. Osteoblast co-culture and osteoclastogenesis co-culture were applied to analyze the effects of WSZG formula on the interaction of tumor cells with osteoclasts and osteoblasts. A breast cancer xenograft model was also used to test the inhibitory effects of WSZG formula on bone metastasis in breast cancer.. WSZG formula decreased Jagged1 expression in osteolytic lesions in the breast cancer xenograft model. Additionally, WSZG formula decreased Jagged1 expression in tumor cell culture alone or co-culture with pre-osteoclasts and osteoblasts. In addition, WSZG formula decreased Jagged1 expression in Jagged1-overexpressing tumor cells.. The results of this study suggest that WSZG formula mitigates breast cancer bone metastasis through the Jagged1/Notch signaling pathway mediated by TGF-β and IL-6.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Cell Line; Coculture Techniques; Female; Humans; Interleukin-6; Jagged-1 Protein; Male; Mice, Inbred BALB C; Mice, Nude; Osteoblasts; Osteoclasts; Osteogenesis; Plant Extracts; Rats, Sprague-Dawley; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

A major obstacle for effective cancer treatment by radiation therapy is the development of radio-resistance and identification of underlying mechanisms and activated pathways will lead to better combination therapies.. Irradiated MCF-7 and MDA-MB-231 breast cancer cell lines were characterised following different recovery periods. Proliferation was assessed by MTT, BrdU and clonogenic assays and apoptosis by Annexin V/ propidium iodide staining and flow cytometry. Gene expression was monitored by real time PCR/ELISA/antibody labelling and migration using transwell inserts.. Breast cancer cell lines exposed to 6 Gy followed by recovery period for 7 days (D7-6 G) had increased ability for proliferation as well as apoptosis. D7-6 G from both cell lines had increased expression of transforming growth factor isoforms (TGF)-β1, β2 and β3, their receptors TGF-βR1 and TGF-βR2 which are known for such dual effects. The expression of downstream transcription factors Snail, Zeb-1 and HMGA2 also showed a differential pattern in D7-6 G cells with upregulation of at least two of these transcription factors. D7-6 G cells from both cell lines displayed hybrid epithelial-mesenchymal (E/M) phenotype with increased expression of E/M markers and migration. D7-6 G cells had increased expression of cancer stem cells markers Oct4, Sox2, and Nanog; aldehyde dehydrogenase expression and activity; proportion of CD44. Blocking of TGF-β signalling may therefore be an effective strategy for overcoming radio resistance induced by radiation exposure.

Topics: Breast Neoplasms; Cell Survival; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Neoplastic Stem Cells; Phenotype; Radiation Dosage; Radiation Tolerance; Signal Transduction; Transforming Growth Factor beta

Cancer cell plasticity facilitates the development of therapy resistance and malignant progression. De-differentiation processes, such as an epithelial-mesenchymal transition (EMT), are known to enhance cellular plasticity. Here, we demonstrate that cancer cell plasticity can be exploited therapeutically by forcing the trans-differentiation of EMT-derived breast cancer cells into post-mitotic and functional adipocytes. Delineation of the molecular pathways underlying such trans-differentiation has motivated a combination therapy with MEK inhibitors and the anti-diabetic drug Rosiglitazone in various mouse models of murine and human breast cancer in vivo. This combination therapy provokes the conversion of invasive and disseminating cancer cells into post-mitotic adipocytes leading to the repression of primary tumor invasion and metastasis formation.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Female; Flavonoids; Humans; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Proto-Oncogene Proteins c-met; Rosiglitazone; Signal Transduction; Transforming Growth Factor beta

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Self Renewal; Cytokines; DNA-Binding Proteins; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Leukemia Inhibitory Factor Receptor alpha Subunit; Mammary Neoplasms, Experimental; Mice, Inbred NOD; Neoplasm Proteins; RNA-Binding Proteins; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from "normal" (<150 Pascals) to "malignant" (>3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit "memory" of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor β (TGFβ), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Hydrogels; Mechanotransduction, Cellular; Paracrine Communication; Phosphoproteins; Signal Transduction; Spheroids, Cellular; Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1; YAP-Signaling Proteins

Breast cancer is the most common cancer among women worldwide, and approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. ERα has been identified to promote the growth of primary breast cancer, however, it can also antagonize signaling pathways that lead to epithelial-mesenchymal transition (EMT), including transforming growth factor-β (TGF-β) signaling. miRNA alteration or dysfunction is involved in cancer development and progression. Although miR-1271 has identified as a tumor suppressor in various cancers, the role of miR-1271 in breast cancer is still limited.. The effect of miR-1271 on breast cancer progression was investigated both in vitro and in vivo. The EMT-related protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Chromatin immunoprecipitation and dual-luciferase reporter assays were used to validate the regulation of ERα-miR-1271-SNAI2 feedback loop.. miR-1271 suppresses breast cancer progression and EMT phenotype both in vitro and in vivo by targeting SNAI2. Estrogen reverses TGF-β-induced EMT in a miR-1271 dependent manner. Furthermore, ERα transactivates the miR-1271 expression and is also transcriptionally repressed by SNAI2.. Our data uncover the ERα-miR-1271-SNAI2 feedback loop and provide a mechanism to explain the TGF-β network in breast cancer progression.

Topics: Animals; Breast Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Snail Family Transcription Factors; Transforming Growth Factor beta

Family with sequence similarity three member C (FAM3C) (interleukin-like EMT inducer [ILEI]), heat shock factor 1 (HSF1) and Ying-Yang 1 (YY1) have been independently reported to be involved in the pathogenesis of various cancers. However, whether they are coordinated to trigger the development of cancer remains unknown. This study determined the role and mechanism of YY1 and HSF1 in FAM3C-induced proliferation and migration of breast cancer cells. In human MDA-MB-231 breast cancer cell line, transforming growth factor-β (TGFβ) up-regulated FAM3C, HSF1 and YY1 expressions. FAM3C overexpression promoted the proliferation and migration of MDA-MB-231 cells with YY1 and HSF1 up-regulation, whereas FAM3C silencing exerted the opposite effects. FAM3C inhibition repressed TGFβ-induced HSF1 activation, and proliferation and migration of breast cancer cells. YY1 was shown to directly activate HSF1 transcription to promote the proliferation and migration of breast cancer cells. YY1 silencing blunted FAM3C- and TGFβ-triggered activation of HSF1-Akt-Cyclin D1 pathway, and proliferation and migration of breast cancer cells. Inhibition of HSF1 blocked TGFβ-, FAM3C- and YY1-induced proliferation and migration of breast cancer cells. YY1 and HSF1 had little effect on FAM3C expression. Similarly, inhibition of HSF1 also blunted FAM3C- and TGFβ-promoted proliferation and migration of human breast cancer BT-549 cells. In human breast cancer tissues, FAM3C, YY1 and HSF1 protein expressions were increased. In conclusion, FAM3C activated YY1-HSF1 signalling axis to promote the proliferation and migration of breast cancer cells. Furthermore, novel FAM3C-YY1-HSF1 pathway plays an important role in TGFβ-triggered proliferation and migration of human breast cancer MDA-MB-231 cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytokines; Female; Gene Silencing; Heat Shock Transcription Factors; Humans; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; YY1 Transcription Factor

Ruyiping (RYP), a Chinese herbal formula, can remove toxin and clear nodular, showing ability of preventing postoperative recurrence of breast cancer. In this study, network was performed to predict possible targets, genes and pathways associated with RYP and breast cancer. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to quantitatively study RYP formula and its single herbs. MTT methods, Luciferase reporter systems, zebrafish model and western blotting were respectively adopted to verify network prediction. Results showed that the quality of RYP could be controlled and icariin could be selected as mark ingredient; RYP expressed anti-breast tumor effects, which could be associated with inhibiting expression of Transforming Growth Factor β (TGFβ), promoting cells apoptosis and anti-angiogenesis. Parts of these results were consistent with network predictions in some degree, but not all. Network can help us narrow areas, focus on crucial factors, save money as well as time, but the results predicted by network should be confirmed by further experiments.

Topics: Animals; Apoptosis; Breast Neoplasms; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Drugs, Chinese Herbal; Female; Humans; MCF-7 Cells; Neoplasm Recurrence, Local; Transforming Growth Factor beta; Zebrafish

Breast cancer as a molecularly heterogeneous malignancy is associated with dysregulation of several signaling pathways, including transforming growth factor-β (TGF-β) signaling. On the other hand, several recent studies have demonstrated the role of microRNAs (miRNAs) in breast cancer pathogenesis. In the current study, we performed a computerized search to find miR-206 target genes that are functionally linked to the TGF-β signaling pathway. We selected LEF1, Smad2, and Snail2 genes to assess their expression in 65 breast cancer samples and their adjacent noncancerous tissues (ANCTs) in correlation with expression levels of miR-206 as well as clinicopathological characteristics of patients. miR-206 was significantly downregulated in (Estrogen receptor) ER-positive breast cancer samples compared with their corresponding ANCTs. Association analysis between expression levels of genes and demographic features of patients showed significant association between expressions of SMAD2 and LEF1 genes and body mass index ( P values of 0.03 and 0.02, respectively). miR-206 low-expression levels were associated with TNM stage, mitotic rate, and lymph node involvement ( P values of 0.02, 0.01, and 0.01 respectively). In addition, SMAD2 high-expression levels were associated with HER2 status ( P = 0.02). Consequently, our data highlight the role of TGF-β signaling dysregulation in the pathogenesis of breast cancer and warrant further evaluation of miRNAs and messenger RNA coding genes in this pathway to facilitate detection of cancer biomarkers and therapeutic targets.

Topics: Biomarkers, Tumor; Body Mass Index; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphoid Enhancer-Binding Factor 1; MicroRNAs; Middle Aged; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

FUT3 gene is responsible for encode an homonymous α1,3/4-fucosyltransferase involved in the synthesis of sialyl-Lewis antigens. FUT3-fucosylated glycoconjugates play key roles in pathways involved in tumor biology and metastasis, such as cellular ligation to E-selectins, TGF-β-induced epithelial-mesenchymal transition, NK cell-mediated tumor cytotoxicity and apoptosis. Tumor-associated FUT3 promoter polymorphism rs2306969 (-6951 C> T, position related to the gene's translation start site) has been linked to breast, ovarian and intestinal gastric cancer. Although non-coding polymorphisms accounts for the majority of variations founded in breast cancer, their functional roles are still poorly understood. This study aimed to investigate the impact of different alleles for this variation in FUT3 expression of invasive breast tumors. A luciferase reporter assay was performed using two breast tumor cell lines to evaluate respectively the impact of FUT3 rs2306969 (-6951 CC) and (-6951 TT) on protein expression. Gene and protein expressions were also measured in twenty-nine fresh biopsies of invasive breast tumors. Rs2306969 did not significantly influence FUT3 expression in both used systems. However, this study is defiant since the biological role of this polymorphism in breast cancer and other tumor types could be linked to cis/trans modulation of other genes, respond to different environmental stimuli or impact gene expression only in association with other variations. Rs2306969 did not modulate FUT3 expression in breast tumors under non-stimulated conditions. Nevertheless, our study contributes to the notably challenging task that is to understand how non-coding polymorphisms can drive the overall risk in cancer development.

Topics: Alleles; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Gene Frequency; Genetic Predisposition to Disease; Humans; Polymorphism, Single Nucleotide; Risk Factors; Transforming Growth Factor beta

Ductal carcinoma in situ (DCIS) is well-known precursor of invasive ductal carcinoma (IDC). Parts of patients show recurrence as DCIS or IDC after local treatment, but there are no established markers predicting relapse. We analyzed changes in miRNA and oncogene expression during DCIS progression/evolution to identify potential markers predicting recurrence.. Forty archival tissues diagnosed as primary or recurrent DCIS and DCIS adjacent to IDC were analyzed. MiRNA hierarchical clustering showed up-regulation of miR-17-5p and miR-106b-5p in recurrent DCIS and DCIS adjacent to IDC. Target genes were predicted based on pre-formed miRNA databases and PanCancer Pathway panel. MiRNAs were transfected into MCF-10A and MCF-7 cells; western blot analysis was performed with MCF-7 cell line to evaluate the effects on TGF-β downstream pathway.. miRNA hierarchical clustering showed 17 dysregulated miRNAs, including miR-17-5p and miR-106b-5p. Based on miRNA database and nCounter Pancancer pathway analysis, TGFβRII was selected as target of miR-106b-5p and miR-17-5p. MiR-106b-5p- and miR-17-5p-transfected MCF-7 cells showed decreased expression of TGFβRII, especially in cells transfected with both miRNAs.. miR-106b-5p and miR-17-5p might have a role in breast cancer recurrence and progression by suppressing TGF-β activity, leading to early breast cancer carcinogenesis.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Disease Progression; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Middle Aged; Neoplasm Recurrence, Local; RNA Interference; Signal Transduction; Transcriptome; Transforming Growth Factor beta

TGF-β induces epithelial-mesenchymal transition (EMT), which is involved in tumour progression. This study aims to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cancer. We treated the human mammary epithelial cell line MCF10A with TGF-β and observed TGF-β-dependent upregulation of FBN1, involving demethylation of CpG sites, in MCF10A cells undergoing EMT. The biological importance of fibrillin-1, encoded by FBN1, was evaluated through immunohistochemistry on 225 breast cancer specimens of various subtypes. Fibrillin-1 expression was observed only in metaplastic carcinoma of the breast (MCB) (51.7%), and the expression was observed in spindle sarcomatous metaplasia (SSM), but not in other metaplasia, including matrix-producing, pleomorphic, and squamous metaplasia, and carcinomatous components of both MCB and non-MCB. Fibrillin-1 expression was also restricted to the SSM of non-mammary carcinosarcomas of various organs. Overall, fibrillin-1 expression was enriched in MCB and non-mammary carcinosarcoma with SSM (93.7% and 93.3%, respectively), but not in MCBs and non-mammary carcinosarcoma without SSM. FBN1 knockdown in MDA-MB-231 cells with high FBN1 expression did not compromise migration, invasion, and tumourigenesis, and did not alter the expression of other EMT-related markers. In conclusion, fibrillin-1 is a novel TGF-β-induced marker. Fibrillin-1 expression in SSM, but not in other metaplasia and carcinomatous components, in both MCBs and non-mammary carcinosarcomas, together with the inability of FBN1-knockdown to compromise migration and invasion, indicates that fibrillin-1 is a marker induced solely in spindle metaplasia during EMT and does not induce EMT nor lead to tumour aggressiveness.

Topics: Breast; Breast Neoplasms; Carcinosarcoma; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Fibrillin-1; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Metaplasia; Sarcoma; Transforming Growth Factor beta; Up-Regulation

Epithelial-mesenchymal transition (EMT) is the initial event required by cancer cells. Thus, inhibition of the EMT process could have potential benefits for preventing the spread of cancers. The phytochemicals have been reported to have inhibitory activity against the EMT process in breast cancers, but the mechanism behind this effect has not been fully elucidated. 3,3'-Diindolylmethane (DIM) is a major indole derived from bioactive compounds in cruciferous vegetables. In this study, we examined the effects of DIM cotreatment together with TNF-α/TGF-β on the EMT process as well as the mechanisms underlying its effects on human breast cancer cells. DIM significantly enhanced the mRNA and protein expression of E-cadherin and occludin in MCF-7 cells. The protein expression levels of E-cadherin and occludin in MCF-7 cells were significantly decreased after TNF-α/TGF-β treatment alone, but these effects were reversed by the DIM co-treatment. Furthermore, DIM with TNF-α/TGF-β co-treatment attenuated the phosphorylation of Smad2/3 and ERK1/2 proteins. DIM significantly inhibited the TNF-α/TGF-β-induced migration of breast cancer cells. Taken together, the results indicated that DIM effectively suppressed EMT processes through the inhibition of TNF-α/TGF-β-associated signaling pathways in breast cancer cells. Thus, DIM may be a novel preventive and/or therapeutic approach for the treatment of breast cancers.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Drug Interactions; Epithelial-Mesenchymal Transition; Female; Humans; Indoles; MCF-7 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Myofibroblasts are a population of highly contractile fibroblasts that express and require the activity of the transcription factor Snail1. Cancer-associated fibroblasts (CAFs) correlate with low survival of cancer patients when present in the stroma of primary tumors. Remarkably, the presence of myofibroblastic CAFs (which express Snail1) creates mechanical properties in the tumor microenvironment that support metastasis. However, therapeutic blockage of fibroblast activity in patients with cancer is a double-edged sword, as normal fibroblast activities often restrict tumor cell invasion. We used fibroblasts depleted of Snail1 or protein arginine methyltransferases 1 and 4 (PRMT1/-4) to identify specific epigenetic modifications induced by TGFβ/Snail1. Furthermore, we analyzed the in vivo efficiency of methyltransferase inhibitors using mouse models of wound healing and metastasis, as well as fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF). Mechanistically, TGFβ-induced Snail1 promotes the epigenetic mark of asymmetrically dimethylated arginine. Critically, we found that inhibitors of methyltransferases prevent myofibroblast activity (but not regular fibroblast activity) in the extracellular matrix, both in cell culture and in vivo. In a mouse breast cancer model, the inhibitor sinefungin reduces both the myofibroblast activity in the tumor stroma and the metastatic burden in the lung. Two distinct inhibitors effectively blocked the exacerbated myofibroblast activity of patient-derived IPF fibroblasts. Our data reveal epigenetic regulation of myofibroblast transdifferentiation in both wound healing and in disease (fibrosis and breast cancer). Thus, methyltransferase inhibitors are good candidates as therapeutic reagents for these diseases.

Topics: Adenosine; Animals; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Transdifferentiation; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Deletion; Humans; Idiopathic Pulmonary Fibrosis; Lung Neoplasms; Methyltransferases; Mice; Myofibroblasts; Snail Family Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH

Topics: Animals; Biomarkers; Breast; Breast Neoplasms; Disease Susceptibility; Epithelial Cells; Female; Gene Expression; Humans; Mice; Neoplastic Stem Cells; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Precancerous Conditions; Receptor, ErbB-2; Transforming Growth Factor beta

Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.

Topics: Activin Receptors, Type I; Activins; Animals; Apoptosis; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Female; Heterografts; Homeostasis; Humans; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Mice, SCID; Neoplasm Metastasis; Neoplasms; Pancreatic Neoplasms; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Tumor Microenvironment

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-β-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-β-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

Topics: Actins; Breast Neoplasms; Cell Line, Tumor; Endosomal Sorting Complexes Required for Transport; Endosomes; Extracellular Matrix; Female; GTPase-Activating Proteins; HEK293 Cells; HeLa Cells; Humans; Matrix Metalloproteinase 14; Podosomes; rab4 GTP-Binding Proteins; rhoB GTP-Binding Protein; Sorting Nexins; Transforming Growth Factor beta

This study was conducted to explore the function of microRNA-141-3p/cyclin-dependent kinase 8 (miR-141-3p/CDK8) in regulating trastuzumab resistance of breast cancer cells.. Microarray analysis was performed to screen microRNAs that are differentially expressed in wild type and trastuzumab-resistant (TR) breast cancer cell lines. TargetScan helped predict the target gene of miR-141-3p. The regulatory relationship was confirmed through a luciferase reporter assay, quantitative reverse transcriptase polymerase chain reaction, and Western blot analysis. The MTT assay, transwell invasion assay, and wound scratch assay were performed to measure the proliferative, invasive, and migratory ability of breast cancer cells, respectively. Tumor cell xenografts in nude mice were conducted to observe the effect of miR-141-3p on trastuzumab resistance in breast cancer cells in vivo. The enzyme-linked immunosorbent assay was used to detect protein secretion.. miR-141-3p was downregulated in the drug-resistant cell lines. CDK8 was proved to be a target gene of miR-141-3p. Transfection of miR-141-3p or CDK8 small interfering RNA (siRNA) reversed the resistance to trastuzumab in TR cell lines and suppressed cell invasion and migration. Dysregulation of transforming growth factor beta (TGF-β) was detected when the expression of CDK8 was silenced by CDK8 siRNA, and downregulation of TGF-β had a notable effect on reducing the phosphorylation of SMAD2/SMAD3.. miR-141-3p could restore the sensitivity to trastuzumab in breast cancer cells by repressing CDK8, which might regulate the phosphorylation levels of SMAD2/SMAD3 via TGF-β.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 8; Down-Regulation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta; Trastuzumab

Human breast carcinoma-associated fibroblasts (CAFs) increasingly acquire both transforming growth factor-β (TGF-β) and stromal cell-derived factor-1 (SDF-1) signaling in an autocrine fashion during tumor progression. Such signaling mediates activated myofibroblastic and tumor-promoting properties in these fibroblasts. CD26/dipeptidyl peptidase-4 is a serine protease that cleaves various chemokines including SDF-1. Stromal CD26 expression is reportedly undetectable in human skin squamous cell carcinomas. However, whether stromal CD26 expression is also downregulated in human breast cancers and which stromal cells potentially lack CD26 expression remain elusive. To answer these questions, sections prepared from 239 human breast carcinomas were stained with antibodies against CD26 and α-smooth muscle actin (α-SMA), a marker for activated myofibroblasts. We found that tumor-associated stroma involving α-SMA-positive myofibroblasts stained negative or negligible for CD26 in 118 out of 193 (61.1%) tumors, whereas noncancerous stromal regions of the breast showed considerable staining for CD26. This decreased stromal CD26 staining in tumors also tends to be associated with poor outcomes for breast cancer patients. Moreover, we demonstrated that CD26 staining is attenuated on stromal myofibroblasts in human breast cancers. Consistently, CD26 expression is significantly downregulated in cultured CAF myofibroblasts extracted from human breast carcinomas as compared to control human mammary fibroblasts. Inhibition of TGF-β or SDF-1 signaling in CAFs by shRNA clearly upregulated the CD26 expression. Taken together, these findings indicate that CD26 expression is attenuated by TGF-β- and SDF-1-autocrine signaling on stromal myofibroblasts in human mammary carcinomas, and that decreased stromal CD26 expression has potential as a prognostic marker.

Topics: Adult; Aged; Autocrine Communication; Biomarkers; Breast Neoplasms; Cancer-Associated Fibroblasts; Chemokine CXCL12; Dipeptidyl Peptidase 4; Female; Gene Expression; Humans; Immunohistochemistry; Middle Aged; Models, Biological; Myofibroblasts; Signal Transduction; Transforming Growth Factor beta

Theacrine, a purine alkaloid structurally similar to caffeine, has recently become of interest as a potential therapeutic compound. Here, we investigated the antimetastatic potential of theacrine on human breast cancer MDA-MB-231 cells. We observed that theacrine can reverse epithelial-to-mesenchymal transition (EMT), which resulted in a decrease in the levels of mesenchymal markers (Fibronectin, Vimentin, N-cadherin, Twist, and Snail) and an increase in the levels of epithelial markers (Occludin and E-cadherin) in the cells. Additionally, theacrine attenuates TGF-β-induced EMT, cell adhesion, migration, and invasion in MDA-MB-231 cells. Overall, our results suggest that theacrine may inhibit the breast cancer cell metastasis by reversing the EMT process.

Topics: Antineoplastic Agents; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Fibronectins; Humans; Nuclear Proteins; Snail Family Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1; Uric Acid; Vimentin

Epithelial mesenchymal plasticity (EMP) is deemed vital in breast cancer progression, metastasis, stemness and resistance to therapy. Therefore, characterizing molecular mechanisms contributing to EMP are in need enabling the development of more advanced therapeutics against breast cancer. While kinesin superfamily proteins (KIFs) are well known for their role in intracellular cargo movement, our knowledge of their function in breast tumorigenesis is still limited.. Various breast cancer cell lines representing different molecular subtypes were used to determine the role of kinesine-1 subunits KIF5B/KLC1 in regulation of EMP.. In breast cancer, we show that kinesin family member 5B (KIF5B) and its partner protein kinesin light chain 1 (KLC1), subunits of kinesin-1, to play differential roles in regulating EMP and tumorigenesis. Indeed, we found KIF5B to be expressed in triple negative (TN)-basal-like/claudin low breast cancer subtype and to be an inducer of epithelial-mesenchymal transition (EMT), stemness, invasiveness, tumor formation and metastatic colonization. Whereas, we found KLC1 to be expressed in epithelial/luminal breast cancer subtypes and to be a suppressor of EMT, invasion, metastasis and stem cell markers expression as well as to be an inducer of epithelial/luminal phenotype. Interestingly, in TN-basal-like/claudin low cells we found a novel nuclear accumulation of KIF5B and its interaction with the EMT transcriptional regulator Snail1 independent of KLC1. In addition, TGF-β mediated pro-invasive activity was found to be dependent on KIF5B expression. In contrast, the epithelial differentiation factor and EMT suppressor prolactin (PRL) was found to repress KIF5B gene expression and KIF5B-Snail1 nuclear accumulation, but enhanced KLC1 gene expression and KIF5B-KLC1 interaction.. Together, these results highlight a new paradigm for kinesin-1 function in breast tumorigenesis by regulating EMP programing and aggressiveness. FUND: This work was supported by the Canadian Institutes of Health Research (operating grants #233437 and 233438) granted to Suhad Ali.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Kinesins; Mice; Microtubule-Associated Proteins; Prolactin; Transforming Growth Factor beta

Topics: Animals; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Humans; Immunophenotyping; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Transforming Growth Factor beta

MicroRNAs are dysregulated in breast cancer. Heterogeneous Nuclear Ribonucleoprotein A2/B1 (HNRNPA2/B1) is a reader of the N(6)-methyladenosine (m6A) mark in primary-miRNAs (pri-miRNAs) and promotes DROSHA processing to precursor-miRNAs (pre-miRNAs). We examined the expression of writers, readers, and erasers of m6A and report that HNRNPA2/B1 expression is higher in tamoxifen-resistant LCC9 breast cancer cells as compared to parental, tamoxifen-sensitive MCF-7 cells. To examine how increased expression of HNRNPA2/B1 affects miRNA expression, HNRNPA2/B1 was transiently overexpressed (~5.4-fold) in MCF-7 cells for whole genome miRNA profiling (miRNA-seq). 148 and 88 miRNAs were up- and down-regulated, respectively, 48 h after transfection and 177 and 172 up- and down-regulated, respectively, 72 h after transfection. MetaCore Enrichment analysis identified progesterone receptor action and transforming growth factor β (TGFβ) signaling via miRNA in breast cancer as pathways downstream of the upregulated miRNAs and TGFβ signaling via SMADs and Notch signaling as pathways of the downregulated miRNAs. GO biological processes for mRNA targets of HNRNPA2/B1-regulated miRNAs included response to estradiol and cell-substrate adhesion. qPCR confirmed HNRNPA2B1 downregulation of miR-29a-3p, miR-29b-3p, and miR-222 and upregulation of miR-1266-5p, miR-1268a, miR-671-3p. Transient overexpression of HNRNPA2/B1 reduced MCF-7 sensitivity to 4-hydroxytamoxifen and fulvestrant, suggesting a role for HNRNPA2/B1 in endocrine-resistance.

Topics: Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Female; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; MCF-7 Cells; MicroRNAs; Signal Transduction; Tamoxifen; Transcriptome; Transforming Growth Factor beta; Up-Regulation

Epithelial-to-mesenchymal transition (EMT), a fundamental transdifferentiation process in development, produces diverse phenotypes in different physiological or pathological conditions. Many genes involved in EMT have been identified to date, but mechanisms contributing to the phenotypic diversity and those governing the coupling between the dynamics of epithelial (E) genes and that of the mesenchymal (M) genes are unclear. In this study, we employed combinatorial perturbations to mammary epithelial cells to induce a series of EMT phenotypes by manipulating two essential EMT-inducing elements, namely TGF-β and ZEB1. By measuring transcriptional changes in more than 700 E-genes and M-genes, we discovered that the M-genes exhibit a significant diversity in their dependency to these regulatory elements and identified three groups of M-genes that are controlled by different regulatory circuits. Notably, functional differences were detected among the M-gene clusters in motility regulation and in survival of breast cancer patients. We computationally predicted and experimentally confirmed that the reciprocity and reversibility of EMT are jointly regulated by ZEB1. Our integrative analysis reveals the key roles of ZEB1 in coordinating the dynamics of a large number of genes during EMT, and it provides new insights into the mechanisms for the diversity of EMT phenotypes.

Topics: Breast Neoplasms; Cell Line, Tumor; Computational Biology; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mesenchymal Stem Cells; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients.. We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing.. Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients.. Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; CpG Islands; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Extracellular Matrix Proteins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Neoplasm Grading; Neoplasm Staging; Promoter Regions, Genetic; Receptor, ErbB-2; Sequence Analysis, DNA; Transforming Growth Factor beta; Trastuzumab

Galunisertib (Gal) is a transforming growth factor (TGF-β) blockade which is being investigated as a potential tumor immunotherapy candidate drug in clinical trials. However, primary or acquired resistance is often found in the recruited cancer patients, which limits its clinical application. Tumor immune microenvironment can be regulated by intestinal microbiota, leading to different therapeutic outcomes. It is hypothesized that manipulation of cancer patients' intestinal microbiome in the early stage of therapy may be a promising strategy to improve the therapeutic efficacy of Gal.

Topics: Animals; Animals, Genetically Modified; Breast Neoplasms; Cell Line, Tumor; Female; Flow Cytometry; Gastrointestinal Microbiome; Humans; Immunotherapy; Intestinal Mucosa; Liver Neoplasms; Mice; Probiotics; RNA, Ribosomal, 16S; Transforming Growth Factor beta; Tumor Microenvironment

Radiotherapy techniques associated with breast-conserving surgery have evolved in early breast cancer thanks to a better knowledge of tumor radiobiology, highlighting intraoperative radiotherapy (IORT). However, complications have been documented with this procedure, mainly fibrosis. Transforming growth factor beta (TGF-β) is a cytokine with an active role in radiation-induced fibrosis, which could be used as an early biomarker for the development of fibrosis.. Multicentric prospective analysis of 60 patients with breast cancer who underwent breast-conserving surgery, 30 of whom had received additional IORT. TGF-β values were evaluated in serum pre-surgery and in serum collected 24h after surgery. In addition, we evaluated surgical wound fluids collected 6h and 24h following surgery.. Serum and surgical wound fluids TGF-β values collected over 24h following surgery were significantly higher in patients who received additional IORT (P<.0001). Notably, 8 of these patients showed values above 1,000pg/ml. There were no differences between the samples (serum or surgical wound fluids) (P=.5881).. Although further investigation is needed, higher TGF-β values in IORT during breast-conserving surgery can be used as an early biomarker for the development of fibrosis.

Topics: Abnormalities, Radiation-Induced; Aged; Breast; Breast Neoplasms; Female; Fibrosis; Humans; Intraoperative Care; Mastectomy, Segmental; Middle Aged; Prospective Studies; Radiotherapy; Transforming Growth Factor beta

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Humans; Hyaluronan Receptors; Hyaluronic Acid; Molecular Weight; Neoplasm Invasiveness; Organoids; Proteoglycans; Recombinant Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

ITIH5 has been proposed being a novel tumor suppressor in various tumor entities including breast cancer. Recently, ITIH5 was furthermore identified as metastasis suppressor gene in pancreatic carcinoma. In this study we aimed to specify the impact of ITIH5 on metastasis in breast cancer. Therefore, DNA methylation of ITIH5 promoter regions was assessed in breast cancer metastases using the TCGA portal and methylation-specific PCR (MSP). We reveal that the ITIH5 upstream promoter region is particularly responsible for ITIH5 gene inactivation predicting shorter survival of patients. Notably, methylation of this upstream ITIH5 promoter region was associated with disease progression, for example, abundantly found in distant metastases. In vitro, stably ITIH5-overexpressing MDA-MB-231 breast cancer clones were used to analyze cell invasion and to identify novel ITIH5-downstream targets. Indeed, ITIH5 re-expression suppresses invasive growth of MDA-MB-231 breast cancer cells while modulating expression of genes involved in metastasis including Endoglin (ENG), an accessory TGF-β receptor, which was furthermore co-expressed with ITIH5 in primary breast tumors. By performing in vitro stimulation of TGF-β signaling using TGF-β1 and BMP-2 we show that ITIH5 triggered a TGF-β superfamily signaling switch contributing to downregulation of targets like Id1, known to endorse metastasis. Moreover, ITIH5 predicts longer overall survival (OS) only in those breast tumors that feature high ENG expression or inversely regulated ID1 suggesting a clinical and functional impact of an ITIH5-ENG axis for breast cancer progression. Hence, we provide evidence that ITIH5 may represent a novel modulator of TGF-β superfamily signaling involved in suppressing breast cancer metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Down-Regulation; Endoglin; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Neoplasm Invasiveness; Promoter Regions, Genetic; Proteinase Inhibitory Proteins, Secretory; Risk; Signal Transduction; Transforming Growth Factor beta

We investigated expressions of -CC chemokine ligand 2 (CCL2) and CCL5 in tumor samples from 147 breast cancer (BCa) patients and correlated with transforming growth factor-β (TGF-β) expression. We observed an inverse correlation of TGF-β expression with CCL2, CCL5 expression in early stages of BCa. On contrary, in late stages, CCL2, not CCL5, expression was found to be directly proportional with TGF-β expression. TGF-β stimulated MDA-MB-231 cells to express CCL2, however, downregulated both CCL2 and CCL5 in MCF-7. Interestingly, a significant swing of Th1-Th2 ratio towards Th2 is seen within the primary tumors expressing moderate/high-CCL2-low/negative-CCL5. We observed that CCL2-CCR2 interaction induces monocytes/macrophages to secrete Th2-attracting chemokine CCL22 in vitro. Therefore, CCL2 secreted from the tumor microenvironment may attract and interact with monocytes/macrophages, and favor Th2 accumulation by inducing CCL22 secretion. Study in 4T1-BALB/c BCa mouse model demonstrated significant (p<0.05) decrease in CCL2, CCL5 and CCL22 levels and reduction in lung metastatic nodule numbers upon administering TGF-β inhibitor. These findings collectively indicate that TGF-β regulates CCL2 and CCL5 expression in a stage-dependent manner during BCa progression, which in turn, determines Th1-Th2 balance within the tumor microenvironment.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Chemokine CCL2; Chemokine CCL22; Chemokine CCL5; Disease Models, Animal; Disease Progression; Female; Humans; Lung Neoplasms; Macrophages; MCF-7 Cells; Mice; Mice, Inbred BALB C; Th1-Th2 Balance; Th2 Cells; Transforming Growth Factor beta

Chromosome region 3p12-14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down-regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation-specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9-transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E-cadherin, VIM, SNAIL, VEGFA, NFκB-p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR- and TGFβ1/TβR(I/II)-activated AKT signaling.

Topics: ADAMTS9 Protein; Adult; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosomes, Human, Pair 3; CpG Islands; DNA Methylation; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta; Tumor Stem Cell Assay

The tumor microenvironment plays an important role in tumor initiation and progression. It is well documented that nicotine participates in cigarette smoking-related malignancies. Previous studies focused on the effects of nicotine on tumor cells; however, the role of the microenvironment in nicotine-mediated tumorigenesis is poorly understood. Herein, we investigated the effect and molecular mechanism of nicotine on fibroblasts and its contribution to breast cancer. We found that nicotine induced the epithelial-mesenchymal transition (EMT) of breast cancer cells and promoted activation of fibroblasts. Interestingly, conditioned medium from nicotine-activated fibroblasts (Nic-CM) had a greater impact on promoting the EMT and migratory capability toward cancer cells than did treatment with nicotine alone. Production of connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β by nicotine-treated fibroblasts was demonstrated to be crucial for promoting the EMT and cancer cell migration, and blocking of CTGF and TGF-β in Nic-CM-suppressed tumor motility. Moreover, nicotine induced expressions of CTGF, and TGF-β in fibroblasts as identified through α7 nicotinic acetylcholine receptor (nAChR)-dependent activation of the AKT/TAZ signaling mechanism. Together, our data showed for the first time that activation of fibroblasts is largely responsible for accelerating smoking-mediated breast cancer progression.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Breast Neoplasms; Cancer-Associated Fibroblasts; Carcinogens; Cell Movement; Connective Tissue Growth Factor; Culture Media, Conditioned; Epithelial-Mesenchymal Transition; Female; Humans; Intracellular Signaling Peptides and Proteins; MCF-7 Cells; Neoplasm Invasiveness; Nicotine; Paracrine Communication; Proto-Oncogene Proteins c-akt; Signal Transduction; Smoking; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor-beta (TGF-β) functions as a potent proliferation inhibitor and apoptosis inducer in the early stages of breast cancer, yet promotes cancer aggressiveness in the advanced stages. The dual effect of TGF-β on cancer development is known as TGF-β paradox, and the remarkable functional conversion of TGF-β is a pivotal and controversial phenomenon that has been widely investigated for decades. This phenomenon may be attributed to the cross talk between TGF-β signaling and other pathways, including EGF receptor (EGFR) signaling during cancer progression. However, the underlying mechanism by which TGF-β shifts its role from a tumor suppressor to a cancer promoter remains elusive. In this study, TGF-β is positively correlated with EGFR expression in breast cancer tissues, and a functional linkage is observed between TGF-β signaling and EGFR transactivation in breast cancer cell lines. TGF-β promotes the migration and invasion abilities of breast cancer cells, along with the increase in EGFR expression. EGFR is also essential for TGF-β-induced enhancement of these abilities of breast cancer cells. Canonical Smad3 signaling and ERK/Sp1 signaling pathways mediate TGF-β-induced EGFR upregulation. Hence, our study provided insights into a novel mechanism by which TGF-β supports breast cancer progression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; ErbB Receptors; Female; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Smad3 Protein; Sp1 Transcription Factor; Transforming Growth Factor beta

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor β. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

Topics: Animals; Breast Neoplasms; Female; Fibroblasts; Humans; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Proteins; Neoplastic Stem Cells; Stromal Cells; Transforming Growth Factor beta

We show that Cyclooxygenase-2 over-expression induces an oncogenic microRNA miR655 in human breast cancer cells by activation of EP4. MiR655 expression positively correlated with COX-2 in genetically disparate breast cancer cell lines and increased in all cell lines when grown as spheroids, implicating its link with stem-like cells (SLCs). Ectopic miR655 over-expression in MCF7 and SKBR3 cells resulted in increased proliferation, migration, invasion, spheroid formation and Epithelial to Masenchymal transition (EMT). Conversely, knocking down miR655 in aggressive MCF7-COX2 and SKBR3-COX2 cells reverted these phenotypes. MCF7-miR655 cells displayed upregulated NOTCH/WNT genes; both pathway inhibitors abrogated miR655-induced spheroid formation, linking miR655 with SLC-related pathways. MiR655 expression was dependent on EP4 activity and EP4 downstream signaling pathways PI3K/AKT, ERK and NF-kB and led to TGFβ resistance for Smad3 phosphorylation. Tail vein injection of MCF7-miR655 and SKBR3-miR655 cells in NOD/SCID/GUSB-null mice revealed increased lung colony growth and micrometastases to liver and spleen. MiR655 expression was significantly high in human breast tumors (n = 105) compared to non-tumor tissues (n = 20) and associated with reduced patient survival. Thus miR655 could serve as a prognostic breast cancer biomarker.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; MCF-7 Cells; Mice; MicroRNAs; Neoplastic Stem Cells; NF-kappa B; Phenotype; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; Receptors, Prostaglandin E, EP4 Subtype; RNA Interference; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Studies have shown that aspirin and metformin play important roles in chemoprevention and repression of breast cancers, even though the exact mechanism remains unclear. Aspirin is capable of stimulating apoptosis through prostaglandin-dependent or prostaglandin-independent pathways. Metformin inhibits cell growth by enhancing the tumor suppressive function of transforming growth factor (TGF-β). In the present study, we report a new link between aspirin, metformin, TGF-β1 and murine breast cancer inhibition. Specifically, we showed that aspirin and metformin enhanced 4T1 cell apoptosis by inducing secretion of TGF-β1, whereas estradiol weakened the effect.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Aspirin; Breast Neoplasms; Cell Proliferation; Female; Humans; Hypoglycemic Agents; Metformin; Mice; Mice, Inbred BALB C; Mice, Nude; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Animals; Bone Morphogenetic Protein 2; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Hyaluronan Receptors; MCF-7 Cells; Mice; Models, Biological; Neoplastic Stem Cells; Recombinant Proteins; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and forkhead box P3 (Foxp3) have important roles in breast cancer development. Previous studies confirmed a correlation between these immune molecules and tumor characteristics, but their association with nutritional status in breast cancer is largely unknown. We aimed to investigate the association between body mass index (BMI), hemoglobin, total protein, albumin, globulin (GLB), albumin/GLB ratio (AGR), pre-albumin, prognostic nutritional index, and TGF-β, IL-10, and Foxp3 mRNA expression in patients with breast cancer. Quantitative real-time PCR was used to detect the mRNA expression of TGF-β, IL-10, and Foxp3 in the peripheral blood of 107 patients with breast cancer and 21 healthy controls. We found that TGF-β mRNA levels were 2.6-fold, 3.2-fold, and 2.3-fold higher in patients with low BMI (<23), low AGR, and high GLB, respectively, than in their counterparts (P < 0.05). In addition, IL-10 mRNA expression levels in patients with normal BMI (<23) were 2.8-fold and 3.5-fold higher than in those who were overweight (23≤ BMI <25) and obese (BMI ≥ 25), respectively (P < 0.05). In addition, TGF-β, IL-10, and Foxp3 mRNA levels were significantly higher in patients with breast cancer than in healthy controls (P < 0.05). In summary, our results suggest that nutritional status, especially BMI, may strongly affect systematic immune function in patients with breast cancer. © 2018 IUBMB Life, 70(3):237-245, 2018.

Topics: Adult; Body Mass Index; Breast Neoplasms; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Middle Aged; Obesity; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Antibodies; B7-H1 Antigen; Breast Neoplasms; Cohort Studies; CTLA-4 Antigen; Female; Forkhead Transcription Factors; Humans; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Melanoma; Mice; Mice, Inbred NOD; Receptors, Transforming Growth Factor beta; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Transforming Growth Factor beta

A critical mechanism that has been proposed for transcription regulation by estrogen receptor α (ER) is the tethering of ER to DNA via other transcription factors, such as AP-1. However, genome-wide assessment of the overlap in chromatin binding repertoires of these two transcription factors has not been reported. Here, we show that the AP-1 transcription factor c-Jun interacts with ER and that c-Jun chromatin binding shows extensive overlap with ER binding at the global level. Further, we show that c-Jun overexpression reprograms ER chromatin binding and modulates ER-mediated gene regulation. Our data are consistent with a mechanism where estrogen/ER-dependent crosstalk with AP-1 at the transcriptional level is mediated through the tethering of ER to DNA bound AP-1. Additionally, in our system c-Jun overexpression causes reduced sensitivity to tamoxifen in ER+ breast cancer cells. Integrated cistrome, transcriptome, and clinical data reveal TGFBI as a candidate gene which may confer tamoxifen resistance by ER and AP-1 crosstalk. Further, we show that TGFBI expression is elevated in breast cancer compared to normal breast. Together, our data provide a novel genome-wide footprint of ER and AP-1 crosstalk and suggest AP-1 and TGFBI signaling as potential therapeutic targets in AP-1-overexpressing ER-positive breast tumors.

Topics: Antineoplastic Agents, Hormonal; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-fos; Signal Transduction; Tamoxifen; Transforming Growth Factor beta; Up-Regulation

Breast cancer is the most common cancer among women worldwide and metastasis is the leading cause of death among patients with breast cancer. The transforming growth factor-β (TGF-β) pathway plays critical roles during breast cancer epithelial-mesenchymal transition (EMT) and metastasis. SMAD2, a positive regulator of TGF-β signaling, promotes breast cancer metastasis through induction of EMT.. The expression of miR-190 and SMAD2 in breast cancer tissues, adjacent normal breast tissues and cell lines were determined by RT-qPCR. The protein expression levels and localization were analyzed by western blotting and immunofluorescence. ChIP and dual-luciferase report assays were used to validate the regulation of ZEB1-miR-190-SMAD2 axis. The effect of miR-190 on breast cancer progression was investigated both in vitro and in vivo.. miR-190 down-regulation is required for TGF-β-induced EMT. miR-190 suppresses breast cancer metastasis both in vitro and in vivo by targeting SMAD2. miR-190 expression is down-regulated and inversely correlates with SMAD2 in breast cancer samples, and its expression level was associated with outcome in patients with breast cancer. Furthermore, miR-190 is transcriptionally regulated by ZEB1.. Our data uncover the ZEB1-miR-190-SMAD2 axis and provide a mechanism to explain the TGF-β network in breast cancer metastasis.

Topics: 3' Untranslated Regions; Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Models, Biological; Neoplasm Metastasis; Neoplasm Staging; Nucleotide Motifs; Promoter Regions, Genetic; RNA Interference; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The multifunctional cytokine TGF-β crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-β and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-β in late stages compared to early stages of the disease. In vitro studies showed that TGF-β elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-β-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-β, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-β signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients.

Topics: Adult; Aged; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Computational Biology; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Staging; Octamer Transcription Factor-3; Promoter Regions, Genetic; Protein Multimerization; Signal Transduction; Smad3 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta; Young Adult

Breast cancer (BC) in the Asia Pacific regions is enriched in younger patients and rapidly rising in incidence yet its molecular bases remain poorly characterized. Here we analyze the whole exomes and transcriptomes of 187 primary tumors from a Korean BC cohort (SMC) enriched in pre-menopausal patients and perform systematic comparison with a primarily Caucasian and post-menopausal BC cohort (TCGA). SMC harbors higher proportions of HER2+ and Luminal B subtypes, lower proportion of Luminal A with decreased ESR1 expression compared to TCGA. We also observe increased mutation prevalence affecting BRCA1, BRCA2, and TP53 in SMC with an enrichment of a mutation signature linked to homologous recombination repair deficiency in TNBC. Finally, virtual microdissection and multivariate analyses reveal that Korean BC status is independently associated with increased TIL and decreased TGF-β signaling expression signatures, suggesting that younger Asian BCs harbor more immune-active microenvironment than western BCs.

Topics: Adult; Asian People; Biomarkers, Tumor; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Carcinoma, Ductal; Carcinoma, Lobular; Cohort Studies; Estrogen Receptor alpha; Exome Sequencing; Female; Humans; Middle Aged; Neoplasm Staging; Postmenopause; Premenopause; Receptor, ErbB-2; Transcriptome; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Suppressor Protein p53; White People

An epithelial to mesenchymal transition (EMT) has been correlated to malignant tumor progression and metastasis by promoting cancer cell migration and invasion and chemoresistance. Hence, finding druggable EMT effectors is critical to efficiently interfere with metastasis formation and to overcome therapy resistance. We have employed a high-content microscopy screen in combination with a kinome and phosphatome-wide siRNA library to identify signaling pathways underlying an EMT of murine mammary epithelial cells and breast cancer cells. This screen identified the MEK5-ERK5 axis as a critical player in TGFβ-mediated EMT. Suppression of MEK5-ERK5 signaling completely prevented the morphological and molecular changes occurring during a TGFβ-induced EMT and, conversely, forced highly metastatic breast cancer cells into a differentiated epithelial state. Inhibition of MEK5-ERK5 signaling also repressed breast cancer cell migration and invasion and substantially reduced lung metastasis without affecting primary tumor growth. The results suggest that the MEK5-ERK5 signaling axis via activation of MEF2B and other transcription factors plays an important role in the induction and maintenance of breast cancer cell migration and invasion and thus represents an exploitable target for the pharmacological inhibition of cancer cell metastasis.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 5; Mice; Mitogen-Activated Protein Kinase 7; Neoplasm Metastasis; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a conserved cellular plasticity program that is reactivated in carcinoma cells and drives metastasis. Although EMT is well studied its regulatory mechanisms remain unclear. Therefore, to identify novel regulators of EMT, a data mining approach was taken using published microarray data and a group of deubiquitinases (DUB) were found to be upregulated in cells that have undergone EMT. Here, it is demonstrated that one DUB, ubiquitin-specific peptidase 11 (USP11), enhances TGFβ-induced EMT and self-renewal in immortalized human mammary epithelial cells. Furthermore, modulating USP11 expression in human breast cancer cells altered the migratory capacity

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Plasticity; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Thiolester Hydrolases; Transforming Growth Factor beta

TGF-β plays a central role in mediating epithelial-mesenchymal transition (EMT) by activating the Smad pathway. In addition, accumulating evidence suggests that TGF-β-induced EMT is NF-κB-dependent in various cancer types. However, it is largely unclear if NF-κB mediates TGF-β-induced EMT in breast cancer, and if this mediation occurs, the regulatory mechanisms are unknown. In our study, we found that TGF-β activates the NF-κB pathway. Inhibition of NF-κB signaling markedly abrogates TGF-β-induced EMT. By studying the regulatory mechanism of TGF-β-induced NF-κB signaling, we found that lncRNA NKILA was upregulated by TGF-β and was essential for the negative feedback regulation of the NF-κB pathway. Accordingly, overexpression of NKILA significantly reduced TGF-β-induced tumor metastasis in vivo. Consistent with the results from mice, the expression of NKILA was negatively correlated with EMT phenotypes in clinical breast cancer samples. Collectively, our study indicated that the NKILA-mediated negative feedback affects TGF-β-induced NF-κB activation and that NKILA may be a therapeutic molecule in breast cancer metastasis via inhibition of EMT.

Topics: Animals; Apoptosis; Breast Neoplasms; Cadherins; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Mice, Inbred NOD; Mice, SCID; NF-kappa B; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β) has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) during cancer progression, mainly by activating a set of pleiotropic transcription factors including SNAI2/Slug. However, the involvement of long non-coding RNAs (lncRNAs) in TGF-β-induced Slug activation and EMT remains largely unknown.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; RNA, Long Noncoding; Snail Family Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Deposition of type I collage in ECM is an important property of various fibrotic diseases including breast cancer. The excessive expression of type I collagen contributes to the rigidity of cancer tissue and increases the mechanical stresses which facilitate metastasis and proliferation of cancer cells via the activation of TGF-β signaling pathway. The increased mechanical stresses also cause the compression of blood vessels and result in hypoperfusion and impaired drug delivery in cancer tissue. Additionally, type I collage functions as the ligand of α2β1-integrin and DDR1/2 receptors on the membrane of cancer cells to initiate signal transduction leading to metastasis. The expression of type I collage in cancer cells is previously shown to be inducible by TGF-β however the detailed mechanism by which the synthesis of type I collagen is regulated in breast cancer cells remains unclear. Herein, we report that MRTF-A, a co-activator of SRF, is important for the regulation of type I collagen gene COL1A1 in breast cancer cells. MRTF-A physically interacted with the promoter of COL1A1 to facilitate histone acetylation and RNA polymerase II recruitment. The RhoC-ROCK signaling pathway which controls the nuclear localization of MRTF-A regulated the transcription of COL1A1 in human breast cancer cells. TGF-β and Wnt signaling increased the expression of both MRTF-A and COL1A1. Furthermore, depletion of MRTF-A abolished the upregulation of COL1A1 in response to the TGF-β or Wnt signaling, indicating the importance of MRTF-A in the synthesis of type I collagen in breast cancer. Given the crucial roles of type I collagen in the formation of metastasis-prone and hypoperfusion microenvironment, MRTF-A would be a potential target for the development of anti-breast cancer activities.

Topics: Breast Neoplasms; Cell Nucleus; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Humans; MCF-7 Cells; Neoplasm Metastasis; Promoter Regions, Genetic; Signal Transduction; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation

The breast cancer microenvironment promotes tumor vascularization through the complex interactions involving tumor-associated fibroblasts (TAFs). Emerging data indicate that TAFs increase production and signaling by TGF-β cytokines, while the role of TGF-β signaling in the regulation of tumor blood vessels is not fully understood. The current study presents evidence that TAFs enhance the organization of tumor blood capillaries, and TGF-β signaling plays an important role in this response.. Tumor vascularization was studied in xenograft models of breast carcinoma cells, alone and in combination with fibroblasts. TGF-β signaling in breast cancer cells was modulated by expression of kinase-inactive TGFBR1-K232R (dnTGFBR1) or constitutive-active TGFBR1-T204D (caTGFBR1) receptor mutants. The architecture of tumor blood capillaries was assessed by immune-histochemical analysis of endothelium and pericytes. The role of TGF-β-Smad signaling in fibronectin expression was examined using adenoviral transduction of signaling components.. Our studies revealed that TAFs significantly increase the lumen size of blood microvessels. Inactivation of TGF-β signaling in tumor cells by dnTGFBR1 reduced the microvessel density and lumen sizes, decreasing tumor growth. In contrast, caTGFBR1-tumors exhibited greater vessel density and lumen sizes. Tumors with inactive dnTGFBR1 showed lower amounts of TAFs, while caTGFBR1 increased amounts of TAFs compared to the control. Inspection of pericytes and endothelial cells in tumor vasculature revealed that TAFs enhanced vessel coverage by pericytes, vascular cells supporting capillaries. This effect was impaired in dnTGFBR1-tumors, whereas active caTGFBR1 enhanced the association of pericytes with endothelium. Accordingly, dnTGFBR1-tumors exhibited the presence of hemorrhages, a sign of fragile blood vessels. Biochemical analysis showed that TGFBR1-SMAD signaling up-regulates fibronectin, a prominent regulator of endothelium-pericyte interactions.. The current study indicates that tumor-fibroblast crosstalk enhances tumor vascularization by increasing the pericyte-endothelium association via a mechanism involving the TGFβ-fibronectin axis. The tumor-fibroblast model represents a useful system for dissecting the complex interactions governing tumor angiogenesis and developing new approaches to therapeutic targeting tumor vasculature.

Topics: Animals; Breast Neoplasms; Cancer-Associated Fibroblasts; Endothelium, Vascular; Female; Heterografts; Humans; Mice; Mice, SCID; Neovascularization, Pathologic; Pericytes; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial-mesenchymal transition (EMT) is a key event associated with metastasis and dissemination in breast tumor pathogenesis. Promyelocytic leukemia (PML) gene produces several isoforms due to alternative splicing; however, the biological function of each specific isoform has yet to be identified. In this study, we report a previously unknown role for PMLIV, the most intensely studied nuclear isoform, in transforming growth factor-β (TGF-β) signaling-associated EMT and migration in breast cancer. This study demonstrates that PMLIV overexpression promotes a more aggressive mesenchymal phenotype and increases the migration of MCF-7 cancer cells. This event is associated with activation of the TGF-β canonical signaling pathway through the induction of Smad2/3 phosphorylation and the translocation of phospho-Smad2/3 to the nucleus. In this study, we report a previously unknown role for PMLIV in TGF-β signaling-induced regulation of breast cancer-associated EMT and migration. Targeting this pathway may be therapeutically beneficial.

Topics: Breast Neoplasms; Cell Movement; Cell Nucleus; Epithelial-Mesenchymal Transition; Female; HEK293 Cells; Humans; MCF-7 Cells; Models, Biological; Phosphorylation; Promyelocytic Leukemia Protein; Protein Domains; Protein Isoforms; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

With improved screening methods, the numbers of abnormal breast lesions diagnosed in women have been increasing over time. However, it remains unclear whether these breast lesions will develop into invasive cancers. To more effectively predict the outcome of breast lesions and determine a more appropriate course of treatment, it is important to understand the underlying mechanisms that regulate progression of non-invasive lesions to invasive breast cancers. A hallmark of invasive breast cancers is the accumulation of fibroblasts. Fibroblast proliferation and activation in the mammary gland is in part regulated by the Transforming Growth Factor beta1 pathway (TGF-β). In animal models, TGF-β suppression of CCL2 and CXCL1 chemokine expression is associated with metastatic progression of mammary carcinomas. Here, we show that transgenic overexpression of the Polyoma middle T viral antigen in the mouse mammary gland of C57BL/6 mice results in slow growing non-invasive lesions that progress to invasive carcinomas in a stage dependent manner. Invasive carcinomas are associated with accumulation of fibroblasts that show decreased TGF-β expression and high levels of CXCL1, but not CCL2. Using co-transplant models, we show that decreased TGF-β signaling in fibroblasts contribute to mammary carcinoma progression through enhancement of CXCL1/CXCR2 dependent mechanisms. Using cell culture models, we show that CXCL1 mediated mammary carcinoma cell invasion through NF-κB, AKT, Stat3 and p42/44MAPK dependent mechanisms. These studies provide novel mechanistic insight into the progression of pre-invasive lesions and identify new stromal biomarkers, with important prognostic implications.

Topics: Animals; Breast; Breast Neoplasms; Cell Proliferation; Chemokine CXCL1; Disease Progression; Female; Fibroblasts; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred C57BL; RAW 264.7 Cells; Receptors, Interleukin-8B; Signal Transduction; Transforming Growth Factor beta

An epithelial-mesenchymal transition (EMT) has been implicated in cancer metastasis, drug resistance, and in conferring stem cell-like traits to cancer cells. Most studies investigating EMT in cancer have either utilized immortalized or cancer cell lines that are already primed to undergo an EMT and do not adequately represent a fully differentiated epithelial state in the absence of an EMT induction. Hence, model systems are required which recapitulate all stages of EMT in cancer cells. Here, we report the derivation and characterization of epithelial PyMT-1099 cancer cells from the MMTV-PyMT mouse model of breast cancer. We demonstrate that PyMT-1099 cells undergo an EMT upon TGFβ treatment, while upon TGFβ withdrawal they go through a mesenchymal-epithelial transition (MET), as assessed by changes in cell morphology and marker expression and comparable to normal murine mammary gland NMuMG cells. However, in contrast to NMuMG cells, PyMT-1099 cells show an increase in cell migration and are highly tumorigenic and metastatic when transplanted into immunocompromised mice. Finally, we report cancer cell-specific changes in gene expression during EMT of PyMT-1099 cells not found in non-transformed NMuMG cells. Thus, PyMT-1099 cells are a versatile tool to study breast cancer-associated EMT and MET in vitro and in vivo.

Topics: Animals; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mice; Transforming Growth Factor beta

Dormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells.. We used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H. Exogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture.. Dormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

Topics: Bone Marrow Cells; Breast Neoplasms; Cell Proliferation; Cellular Senescence; Estrogens; Humans; Interleukin-6; Interleukin-8; MCF-7 Cells; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Expression of the transcription factor SOX4 is often elevated in human cancers, where it generally correlates with tumor-progression and poor-disease outcome. Reduction of SOX4 expression results in both diminished tumor-incidence and metastasis. In breast cancer, TGF-β-mediated induction of SOX4 has been shown to contribute to epithelial-to-mesenchymal transition (EMT), which controls pro-metastatic events. Here, we identify SMAD3 as a novel, functionally relevant SOX4 interaction partner. Genome-wide analysis showed that SOX4 and SMAD3 co-occupy a large number of genomic loci in a cell-type specific manner. Moreover, SOX4 expression was required for TGF-β-mediated induction of a subset of SMAD3/SOX4-co-bound genes regulating migration and extracellular matrix-associated processes, and correlating with poor-prognosis. These findings identify SOX4 as an important SMAD3 co-factor controlling transcription of pro-metastatic genes and context-dependent shaping of the cellular response to TGF-β. Targeted disruption of the interaction between these factors may have the potential to disrupt pro-oncogenic TGF-β signaling, thereby impairing tumorigenesis.

Topics: Breast Neoplasms; Carcinogenesis; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Prognosis; Signal Transduction; Smad3 Protein; SOXC Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

The most life-threatening step during malignant tumor progression is reached when cancer cells leave the primary tumor mass and seed metastasis in distant organs. To infiltrate the surrounding tissue and disseminate throughout the body, single motile tumor cells leave the tumor mass by breaking down cell-cell contacts in a process called epithelial to mesenchymal transition (EMT). An EMT is a complex molecular and cellular program enabling epithelial cells to abandon their differentiated phenotype, including cell-cell adhesion and cell polarity, and to acquire mesenchymal features and invasive properties.. We employed gene expression profiling and functional experiments to study transcriptional control of transforming growth factor (TGF)β-induced EMT in normal murine mammary gland epithelial (NMuMG) cells.. We identified that expression of the transcription factor forkhead box protein F2 (Foxf2) is upregulated during the EMT process. Although it is not required to gain mesenchymal markers, Foxf2 is essential for the disruption of cell junctions and the downregulation of epithelial markers in NMuMG cells treated with TGFβ. Foxf2 is critical for the downregulation of E-cadherin by promoting the expression of the transcriptional repressors of E-cadherin, Zeb1 and Zeb2, while repressing expression of the epithelial maintenance factor Id2 and miRNA 200 family members. Moreover, Foxf2 is required for TGFβ-mediated apoptosis during EMT by the transcriptional activation of the proapoptotic BH3-only protein Noxa and by the negative regulation of epidermal growth factor receptor (EGFR)-mediated survival signaling through direct repression of its ligands betacellulin and amphiregulin. The dual function of Foxf2 during EMT is underscored by the finding that high Foxf2 expression correlates with good prognosis in patients with early noninvasive stages of breast cancer, but with poor prognosis in advanced breast cancer.. Our data identify the transcription factor Foxf2 as one of the important regulators of EMT, displaying a dual function in promoting tumor cell apoptosis as well as tumor cell migration.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Intercellular Junctions; MCF-7 Cells; Mice; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) are activated fibroblasts associated with cancer. They have an important role in tumor growth and metastasis. Artemisinin (ART) is a sesquiterpene lactone extracted from Chinese herb qinghao, and artemether (ARM), artesunate (ARS) and dihydroartemisinin (DHA) were synthesized derivatives of artemisinin, which also have anti-malarial and anti-cancer effects such as artemisinin.. In this study, we investigated the in-vitro and in-vivo effects of artemisinin derivatives on inactivating cancer-associated fibroblasts and uncovered its underlying mechanism.. We demonstrated that ARS and DHA could revert L-929-CAFs and CAFs from activated to inactivated state in vitro. Mechanically, ARS and DHA could suppress TGF-β signaling to inhibit activation of L-929-CAFs and CAFs, and decreased interaction between tumor and tumor microenvironment. The results showed that ARS and DHA could suppress CAFs-induced breast cancer growth and metastasis in the orthotopic model. Conformably, ARS and DHA suppressed TGF-β signaling to inactivate cancer-associated fibroblasts and inhibit cancer metastasis in vivo.. Artemisinin derivatives are potential therapeutic agents for the treatment of breast cancer.

Topics: Animals; Antimalarials; Artemisinins; Breast Neoplasms; Cancer-Associated Fibroblasts; Disease Models, Animal; Female; Humans; Mice; Mice, Nude; Transforming Growth Factor beta; Tumor Microenvironment

In the era of immunotherapy and personalized medicine, there is an urgent need for advancing the knowledge of immune evasion in different cancer types and identifying reliable biomarkers that guide both therapy selection and patient inclusion in clinical trials. Given the differential immune responses and evasion mechanisms in breast cancer, we expect to identify different breast cancer groups based on their expression of immune-related genes. For that, we used the sequential biclustering method on The Cancer Genome Atlas RNA-seq breast cancer data and identified 7 clusters. We found that 77.4% of the clustered tumor specimens evade through transforming growth factor-beta (TGF-β) immunosuppression, 57.7% through decoy receptor 3 (DcR3) counterattack, 48.0% through cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and 34.3% through programmed cell death-1 (PD-1). TGF-β and DcR3 are potential novel drug targets for breast cancer immunotherapy. Targeting TGF-β and DcR3 may provide a powerful approach for treating breast cancer because 57.7% of patients overexpressed these two molecules. Furthermore, triple-negative breast cancer (TNBC) patients clustered equally into two subgroups: one with impaired antigen presentation and another with high leukocyte recruitment but four different evasion mechanisms. Thus, different TNBC patients may be treated with different immunotherapy approaches. We identified biomarkers to cluster patients into subgroups based on immune evasion mechanisms and guide the choice of immunotherapy. These findings provide a better understanding of patients' response to immunotherapies and shed light on the rational design of novel combination therapies.

Topics: B7-H1 Antigen; Biomarkers, Tumor; Breast Neoplasms; CTLA-4 Antigen; Female; Gene Expression; Humans; Immune Evasion; Immunotherapy; Programmed Cell Death 1 Receptor; Receptors, Tumor Necrosis Factor, Member 6b; Sequence Analysis, RNA; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

Recent studies indicate that the long noncoding RNA ATB (lncATB) can induce the epithelial-mesenchymal transition (EMT) in cancer cells, but the specific cellular targets of lncATB require further investigation. In the present study, the upregulation of lncATB in breast cancer cells was validated in a TGF-β-induced EMT model. Gain- and loss-of-function studies demonstrated that lncATB enhanced cell migration, invasion and clonogenicity in vitro and in vivo. LncATB promoted the EMT by acting as a sponge for the miR-200 family and restoring Twist1 expression. Subsequently, the clinical significance of lncATB was investigated in a cohort of breast cancer patients (N = 131). Higher lncATB expression was correlated with increased nodal metastasis (P = 0.036) and advanced clinical stage (P = 0.011) as well as shorter disease-free survival (P = 0.043) and overall survival (P = 0.046). These findings define Twist1 as a major target of lncATB in the induction of the EMT and highlight lncATB as a biomarker in breast cancer patients.

Topics: Adult; Animals; Breast Neoplasms; Cell Movement; Cell Proliferation; Cohort Studies; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; MCF-7 Cells; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Nuclear Proteins; Prognosis; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Tumor Burden; Twist-Related Protein 1; Xenograft Model Antitumor Assays

The aim of this study is to investigate the mechanisms of interactions between TGF-β and Wnt/β-catenin pathways that induce and regulate EMT and promote breast cancer cells to become resistant to treatment.. The effect of TGF-β on Wnt/β-catenin signaling pathway was examined by using a human Wnt/β-catenin-regulated cDNA plate array and western blot analysis. The interaction of Twist at promoter of Wnt3 was examined by chromatin immunoprecipitation (ChIP) assay. Secreted Wnt3 level was determined by ELISA assay.. HER2-overexpressing breast cancer cells treated with TGF-β have a reduced response to trastuzumab and exhibited EMT-like phenotype. The TGF-β-induced EMT in HER2-cells was concordant with upregulation of Wnt3 and β-catenin pathways. The TGF-β-induced induction of Wnt3 during EMT was found to be Smad3-dependent. ChIP analysis identified occupancy of Twist at promoter region of Wnt3. Knock-down of Twist by shRNA confirmed the significance of Twist in response to TGF-β regulating Wnt3 during EMT. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, MMP26, Vascular endothelial growth factors (VEGF), and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment. TGF-βR1 ALK5 kinase inhibitor, A83-01 can effectively prevent the TGF-β-induced Twist and Wnt3. Co-treating A83-01 and trastuzumab inhibited TGF-β-induced cell invasion significantly in both trastuzumab responsive and resistant cells.. Our data demonstrated an important interdependence between TGF-β and Wnt/β-catenin pathways inducing EMT in HER2-overexpressing breast cancer cells. Twist served as a linkage between the two pathways during TGF-β-induced EMT. A83-01 could inhibit the TGF-β-initiated pathway interactions and enhance HER2-cells response to trastuzumab treatment.

Topics: beta Catenin; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Protein Serine-Threonine Kinases; Pyrazoles; Receptor, ErbB-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Thiosemicarbazones; Transforming Growth Factor beta; Trastuzumab; Twist-Related Protein 1; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway; Wnt3 Protein

A high incidence of tumor recurrence and metastasis has been reported in breast cancer patients; nevertheless, the underlying molecular mechanisms are largely unknown. Epithelial-mesenchymal transition (EMT), which is induced by transforming growth factor-β (TGF-β), has been implicated in tumorigenesis and breast cancer metastasis. EMT events are now directly associated with tumor metastasis, and this progress is dependent on the inflammatory microenvironment. Cytosolic phospholipase A2α (cPLA2α) has been shown to participate in a series of biological processes including inflammation and cancer development. However, the role and molecular mechanism of cPLA2α in breast cancer EMT and metastasis remain enigmatic. In this study, we found that cPLA2α was commonly overexpressed in most human breast cancer tissues and significantly correlated with a poor prognosis for human breast cancer. Functional studies demonstrated that cPLA2α overexpression was significantly associated with elevated migration and invasion in MDA-MB-231 and T47D cells. Conversely, reduced cPLA2α expression strongly attenuated metastasis and the EMT program of MDA-MB-231 cells. Further study found that knockdown of cPLA2α in MDA-MB-231 cells inhibited TGF-β-induced EMT through the PI3K/Akt signaling pathway. Animal experiments revealed that cPLA2α downregulation in MDA-MB-231 cells markedly restrained tumorigenesis and metastasis in vivo. This study indicates the potential role of cPLA2α in breast cancer metastasis and indicates that this molecule is a promising therapeutic target for breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Knockdown Techniques; Group IV Phospholipases A2; Humans; Mice; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Delta-aminolevulinate dehydratase (ALAD) catalyzes the second step in the biosynthesis of heme and is also an endogenous inhibitor of the 26S proteasome. The role of ALAD in breast cancer progression is still unclear. In this study, we found that the expression of ALAD was downregulated in breast cancer tissues compared with adjacent normal breast tissues. Enhanced ALAD expression was associated with a favorable outcome in patients with breast cancer. Overexpression of ALAD suppresses breast cancer cell proliferation and invasion and inhibits the epithelial-mesenchymal transition phenotype. Furthermore, we found that ALAD regulates transforming growth factor-β-mediated breast cancer progression. This finding suggests that ALAD might be a potential biomarker for breast cancer that suppresses breast cancer progression by regulating transforming growth factor-β-mediated epithelial-mesenchymal transition.

Topics: Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Microscopy, Fluorescence; Porphobilinogen Synthase; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sensitivity and Specificity; Transforming Growth Factor beta

Topics: Breast Neoplasms; Core Binding Factor Alpha 2 Subunit; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Phenotype; Transforming Growth Factor beta

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the βsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor β induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.

Topics: Biomechanical Phenomena; Breast Neoplasms; Carcinogenesis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Size; Cell Transformation, Neoplastic; Collagen Type I; Contractile Proteins; Female; Humans; Receptor, ErbB-2; Transforming Growth Factor beta

Great progress has been achieved in the study of the role of TGF-β signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-β signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-β signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-β signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-β signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.

Topics: Animals; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Kaplan-Meier Estimate; Mice; Neoplasm Metastasis; Prognosis; Protein Binding; Signal Transduction; Smad4 Protein; Smad7 Protein; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

Sodium salicylate (NaS) is a derivate of acetylsalicylic acid or aspirin, used as a nonsteroidal anti-inflammatory drug for centuries, for its analgesic and anti-inflammatory effects. It was found to modulate different signaling pathways, in a cell-specific way. Here, we explore the effect of NaS on cell growth and urokinase activity in MDA MB-231 breast cancer cells.. We analyzed the effect of NaS treatment on cell growth by flow cytometry and viability test. The transwell migration assay was used to study the migratory response of the cells. The gene expression was analyzed by qRT-PCR on RNA level and by Western blot analysis on protein level. Urokinase activity was assessed by caseinolysis.. Sublethal concentrations of NaS decreased cell growth and inhibited urokinase activity. The latter was a consequence of decrease in urokinase expression and increase in expression of its inhibitors. Analysis of signaling molecules revealed activation of transforming growth factor-β signaling, increase in master transcription factors for epithelial-mesenchymal transition and changes in integrin expression.. We propose that NaS causes partial cellular reprogramming through transforming growth factor-β signaling which, together with direct NaS influence, causes changes in expression in a set of genes involved in extracellular proteolysis. These data could be beneficial for the development of new therapeutic approaches in invasive breast cancer treatment.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclooxygenase Inhibitors; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Gene Expression Profiling; Humans; Integrins; Real-Time Polymerase Chain Reaction; Signal Transduction; Sodium Salicylate; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

Activin A belongs to the superfamily of transforming growth factor beta (TGFβ) and is a critical regulatory cytokine in breast cancer and inflammation. However, the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here, a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast, activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally, activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively, our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers.

Topics: Activins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Humans; Inflammation; Neutrophils; Transforming Growth Factor beta

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Topics: Breast Neoplasms; Calcium; Epithelial-Mesenchymal Transition; Humans; Neoplasm Proteins; Stromal Interaction Molecule 1; Stromal Interaction Molecule 2; Transforming Growth Factor beta; Tumor Cells, Cultured

Metastasis is the ultimate cause of breast cancer related mortality. Epithelial-mesenchymal transition (EMT) is thought to play a crucial role in the metastatic potential of breast cancer. Growing evidence has implicated the SUMO E3 ligase PIAS1 in the regulation of EMT in mammary epithelial cells and breast cancer metastasis. However, the relevance of PIAS1 in human cancer and mechanisms by which PIAS1 might regulate breast cancer metastasis remain to be elucidated. Using tissue-microarray analysis (TMA), we report that the protein abundance and subcellular localization of PIAS1 correlate with disease specific overall survival of a cohort of breast cancer patients. In mechanistic studies, we find that PIAS1 acts via sumoylation of the transcriptional regulator SnoN to suppress invasive growth of MDA-MB-231 human breast cancer cell-derived organoids. Our studies thus identify the SUMO E3 ligase PIAS1 as a prognostic biomarker in breast cancer, and suggest a potential role for the PIAS1-SnoN sumoylation pathway in controlling breast cancer metastasis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cohort Studies; Female; HEK293 Cells; Humans; Middle Aged; Neoplasm Invasiveness; Organoids; Protein Inhibitors of Activated STAT; Protein Stability; Protein Transport; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Survival Analysis; Tissue Array Analysis; Transforming Growth Factor beta

A long noncoding RNA (lncRNA) activated by transforming growth factor (TGF)-β (lncRNA-ATB) has been recently shown to promote the invasion-metastasis cascade in various types of cancers via upregulation of some targets including ZEB1.. The aim of the present study was to elucidate the expression of lncRNA-ATB and ZEB in breast cancer patients.. The expression of these genes was evaluated by real-time reverse transcription polymerase chain reaction in tumor samples form 50 newly diagnosed breast cancer patients as well as their corresponding adjacent non-cancerous tissues (ANCTs). Patients were divided into subsequent groups according to the median lncRNA-ATB expression.. LncRNA-ATB has been shown to be downregulated in about two third of tumor samples compared with their ANCTs.A significant association has been found between ZEB1 expression and Ki-67 status. In addition, we demonstrated a correlation between expression of lncRNA-ATB and ZEB1 in tumor samples and not in ANCTs.. Collectively, out data show downregulation of lncRNA-ATB in a significant number of breast tumor tissues compared with ANCTs and imply that lncRNA-ATB might have distinct roles in the pathogenesis of different cancers or even different subtypes of a certain cancer which should be evaluated in future studies.

Topics: Adult; Aged; Aged, 80 and over; Breast; Breast Neoplasms; Cadherins; Carcinoma, Ductal, Breast; Down-Regulation; Female; Gene Expression; Humans; Ki-67 Antigen; Middle Aged; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; RNA, Long Noncoding; Transforming Growth Factor beta; Young Adult; Zinc Finger E-box-Binding Homeobox 1

Epithelial‑mesenchymal transition (EMT) is a key mechanism underlying metastatic breast cancer. Reactive oxygen species (ROS) play an important role in EMT. Heme oxygenase‑1 (HMOX‑1) can reduce oxidative stress. However, the effect of HMOX‑1 on the EMT process in breast cancer cells is unknown. We treated the MCF‑7 breast cancer cell line with the HMOX‑1 inducer hemin and observed that hemin induced HMOX‑1 expression and inhibited migration, invasion and ROS generation in transforming growth factor‑β (TGF‑β)‑treated MCF‑7 cells using quantitative RT‑qPCR, western blotting, wound‑healing and cell invasion assays as well as fluorescent probe DCFDA. Hemin inhibited TGF‑β‑induced EMT in the MCF‑7 cells, whereas HMOX‑1 siRNA attenuated the suppressive effect of hemin as determined by the expression and cellular distribution of selected EMT markers. In summary, our results revealed that hemin treatment increased HMOX‑1 expression and inhibited TGF‑β‑induced EMT in MCF‑7 cells.

Topics: Breast Neoplasms; Cell Movement; Epithelial-Mesenchymal Transition; Female; Heme Oxygenase-1; Humans; MCF-7 Cells; Neoplasm Invasiveness; Reactive Oxygen Species; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that has a critical role in promoting carcinoma's metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, as their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell-cell junctions. Additionally, miR-143 appears to have a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of a number of junction proteins and increased motility, correlating with enhanced basal and TGF-β-induced SMAD-mediated transcription. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role of miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Co-Repressor Proteins; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Transgenic; MicroRNAs; Neoplasm Invasiveness; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) pathway plays a vital role in driving cancer cell epithelial-mesenchymal transition (EMT). Zonula occludens-1 (ZO-1), which is downregulated in response to TGF-β, is able to control endothelial cell-cell tension, cell migration, and barrier formation. However, the molecular mechanism of how TGF-β regulates ZO-1 expression remains unclear.. Breast cancer cells were treated with TGF-β to induce an EMT progress. Chromatin immunoprecipitation and dual-luciferase reporter assay were performed to investigate direct relationship between Snail and RNA binding motif protein 38 (RBM38). The RNA immunoprecipitation combined with RNA electrophoretic mobility shift assay and dual-luciferase reporter assay were conducted to testify direct relationship between RBM38 and ZO-1. The ZO-1 siRNA was transfected to breast cancer cells that overexpress RBM38 and the control, followed by transwell and Matrigel invasion assays to examine cell migratory and invasive ability.. Transforming growth factor-β induced a remarkable downregulation of RBM38 in breast cancer that was directly regulated by transcription repressor Snail targeting the E-box elements in promoter region of RBM38 gene. Additionally, RBM38 positively regulated ZO-1 transcript via directly binding to AU/U-rich elements in its mRNA 3'-UTR. Moreover, by magnifying RBM38 expression, cell migration and invasion mediated by knockdown of ZO-1 in breast cancer were reversed.. All the results clarified a linear regulation relationship among Snail, RBM38, and ZO-1, implicating RBM38 as a pivotal mediator in TGF-β-induced EMT in breast cancer.

Topics: 3' Untranslated Regions; Benzamides; Breast Neoplasms; Cell Movement; Dioxoles; Down-Regulation; E-Box Elements; Epithelial-Mesenchymal Transition; Female; Gene Expression; Humans; MCF-7 Cells; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; Transfection; Transforming Growth Factor beta; Zonula Occludens-1 Protein

Secreted frizzled-related protein 1 (SFRP1) expression is down-regulated in a multitude of cancers, including breast cancer. Loss of Sfrp1 also exacerbates weight gain as well as inflammation. Additionally, loss of SFRP1 enhances TGF-β signaling and the downstream MAPK pathway. TGF-β has been shown to increase the expression of Early Growth Response 2 (EGR2), a transcription factor implicated in immune function in a wide variety of cell types. The work described here was initiated to determine whether SFRP1 modulation affects TGF-β mediated EGR2 expression in mammary tissues as well as macrophage polarization.. Knockdown of SFRP1 expression increases the expression of EGR2 mRNA in human mammary epithelial cells and addition of rSFRP1 decreases the expression of EGR2 when added to explant mammary gland tissues. Chemical inhibition of both TGF-β and MAPK signaling in Sfrp1. Loss of SFRP1 likely contributes to tumor progression by altering the expression of a critical transcription factor in both the epithelium and the immune system.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Early Growth Response Protein 2; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Macrophages; Mammary Glands, Animal; MAP Kinase Signaling System; Mice; Mice, Knockout; Proteins; Transforming Growth Factor beta

Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor β (TGFβ) is a cytokine involved in the acquisition of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGFβ and its downstream SMAD pathway on resistance to anti-HER2 drugs.. HER2-positive breast cancer cell lines were stimulated with TGFβ for 14 days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored.. We found that continuous activation of the TGFβ-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGFβ required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells.. This study indicates that the TGFβ-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Immunohistochemistry; Neoadjuvant Therapy; Neoplasm Metastasis; Neoplasm Staging; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Receptor, ErbB-2; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; Female; Gene Expression; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Metastasis; Phenotype; Phosphorylation; Protein Binding; rac1 GTP-Binding Protein; Transforming Growth Factor beta

Few long noncoding RNAs (lncRNAs) that act as oncogenic genes in breast cancer have been identified.. Oncogenic lncRNAs associated with tumourigenesis and worse survival outcomes were examined and validated in Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA), respectively. Then, the potential biological functions and expression regulation of these lncRNAs were studied via bioinformatics and genome data analysis. Moreover, progressive breast cancer subtype-specific lncRNAs were investigated via high-throughput sequencing in our cohort and TCGA validation. To elucidate the mechanisms of the regulation of these lncRNAs, genomic alterations from the TCGA, Broad, Sanger and BCCRC data, as well as epigenetic modifications from GEO data, were then applied and examined to meet this objective. Finally, cell proliferation assays, flow cytometry analyses and TUNEL assays were applied to validate the oncogenic roles of these lncRNAs in vitro.. A cluster of oncogenic lncRNAs that was upregulated in breast cancer tissue and was associated with worse survival outcomes was identified. These oncogenic lncRNAs are involved in regulating immune system activation and the TGF-beta and Jak-STAT signalling pathways. Moreover, TINCR, LINC00511, and PPP1R26-AS1 were identified as subtype-specific lncRNAs associated with HER-2, triple-negative and luminal B subtypes of breast cancer, respectively. The up-regulation of these oncogenic lncRNAs is mainly caused by gene amplification in the genome in breast cancer and other solid tumours. Finally, the knockdown of TINCR, DSCAM-AS1 or HOTAIR inhibited breast cancer cell proliferation, increased apoptosis and inhibited cell cycle progression in vitro.. These findings enhance the landscape of known oncogenic lncRNAs in breast cancer and provide insights into their roles. This understanding may potentially aid in the comprehensive management of breast cancer.

Topics: Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Computational Biology; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Oncogenes; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation

Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1β and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFβ-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1β and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1β and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Chemokine CCL2; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1beta; Macrophages; Mice; Mice, Transgenic; Paracrine Communication; Receptor, Notch1; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Distant metastasis is the main cause of breast cancer-related death; however, effective therapeutic strategies targeting metastasis are still scarce. This is largely attributable to the spatiotemporal intratumor heterogeneity during metastasis. Here we show that protein deacetylase SIRT7 is significantly downregulated in breast cancer lung metastases in human and mice, and predicts metastasis-free survival. SIRT7 deficiency promotes breast cancer cell metastasis, while temporal expression of Sirt7 inhibits metastasis in polyomavirus middle T antigen breast cancer model. Mechanistically, SIRT7 deacetylates and promotes SMAD4 degradation mediated by β-TrCP1, and SIRT7 deficiency activates transforming growth factor-β signaling and enhances epithelial-to-mesenchymal transition. Significantly, resveratrol activates SIRT7 deacetylase activity, inhibits breast cancer lung metastases, and increases survival. Our data highlight SIRT7 as a modulator of transforming growth factor-β signaling and suppressor of breast cancer metastasis, meanwhile providing an effective anti-metastatic therapeutic strategy.Metastatic disease is the major reason for breast cancer-related deaths; therefore, a better understanding of this process and its players is needed. Here the authors report the role of SIRT7 in inhibiting SMAD4-mediated breast cancer metastasis providing a possible therapeutic avenue.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mice, Transgenic; RNA Interference; Signal Transduction; Sirtuins; Smad4 Protein; Transforming Growth Factor beta; Transplantation, Heterologous

Breast cancer (BrC) is a major public health problem worldwide. The intra-tumoral heterogeneity and tumor cell plasticity importantly contribute to disease progression and treatment failure. However, the dynamic interactions between different tumor clones, as well as their contribution to tumor aggressiveness are still poorly understood. In this study, we provide evidence of a lateral transmission of aggressive features between aggressive and non-aggressive tumor cells, consisting of gain of expression of cancer stem cell markers, increased expression of CXCL12 receptors CXCR4 and CXCR7 and increased invasiveness in response to CXCL12, which correlated with high levels of secretion of pro-inflammatory mediators G-CSF, GM-CSF, MCP-1, IL-8 and metalloproteinases 1 and 2 by the aggressive cells. Noteworthy, we found no evidence of a TGF-β participation in the inducible-invasive phenotype. Altogether, our results provide evidence of communication between tumor cells with different potentials for aggressiveness, which could influence intra-tumoral population dynamics promoting the emergence of clones with novel functions. Understanding these interactions will provide better targets for diagnosis, prognosis and therapeutic strategies.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Lineage; Cell Movement; Cell Proliferation; Chemokine CXCL12; Clonal Evolution; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Humans; Neoplasm Invasiveness; Receptors, CXCR; Receptors, CXCR4; Signal Transduction; Transforming Growth Factor beta

Some microRNAs (miRNAs) are known to suppress breast cancer. However, whether the expressions of these tumor suppressive miRNAs translate to patient survival were not investigated in large cohort. Nine miRNAs (miR-30a, miR-30c, miR-31, miR-126, miR-140, miR-146b, miR-200c, miR-206, and miR-335) known to be tumor suppressive miRNAs in breast cancer were investigated in Genomic Data Common data portal miRNA-Seq dataset and The Cancer Genome Atlas (TCGA) (n = 1052). Of the 9 miRNAs, miR-30a, miR-30c, miR-126, miR-140, miR-206, and miR-335 were found to have significantly lower expression in breast cancer tissues compared to paired normal breast tissue. High expression of miR-30a or miR-200c was associated with significantly better overall survival (OS). Gene Set Enrichment Analysis (GSEA) demonstrated that low expression levels of miR-30a had the tendency to associate with gene enrichment of EMT, while miR-200c did not, in TCGA cohort, and our findings support the need of validation using large cohort to use miRNA as prognostic biomarker for patients with breast cancer.

Topics: Breast Neoplasms; Disease-Free Survival; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kaplan-Meier Estimate; MicroRNAs; Neoplasm Staging; Prognosis; Signal Transduction; Transforming Growth Factor beta

Topics: Anoikis; Breast Neoplasms; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Extracellular Matrix; Female; Humans; Integrin alpha6; MAP Kinase Signaling System; MCF-7 Cells; Placenta; Pregnancy; Pregnancy Complications, Neoplastic; Pregnancy Trimester, First; Signal Transduction; Transforming Growth Factor beta

The purpose of this study was to clarify the alterations of major immune regulators in peripheral blood mononuclear cells (PBMCs) of cancer patients and to analyze the association with the disease progression in breast cancer patients.. The study included 6 healthy volunteers (HVs), 12 primary breast cancer (PBC) patients, and 30 metastatic breast cancer (MBC) patients. The expression of immune regulators such as, CCR6, CD4, CD8, CD14, CD40, CD56, CD80, CTLA4, CXCR4, FOXP3, IDO-1, IDO-2, NKG2D, NRP-1, PD-1, and PD-L1 mRNA in PBMCs was measured by quantitative RT-PCR. Analysis of variance with contrasts was performed to find expression patterns of the three groups (HVs, PBC, MBC).. We clarified the alterations of mRNA of major immune regulators PD-L1, FOXP3, CD80, CD40, and CD14 in PBMCs of cancer patients and the association of these alternations with disease progression. Furthermore, PD-L1 expression was correlated with serum interferon-γ production.. Our data suggested that mRNA expressions of PD-L1, FOXP3, CD80, CD40 and CD14 in PBMCs are affected by disease progression. Understanding the roles of these various interactions will be of importance to future studies aiming to uncover biomarkers for predicting response to immune therapy.

Topics: Adult; Aged; Antigens, CD; B7-H1 Antigen; Biomarkers, Tumor; Breast Neoplasms; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Leukocytes, Mononuclear; Middle Aged; Programmed Cell Death 1 Receptor; Prospective Studies; Transforming Growth Factor beta

Protein arginine methyltransferase 5 (PRMT5) complexed with MEP50/WDR77 catalyzes arginine methylation on histones and other proteins. PRMT5-MEP50 activity is elevated in cancer cells and its expression is highly correlated with poor prognosis in many human tumors. We demonstrate that PRMT5-MEP50 is essential for transcriptional regulation promoting cancer cell invasive phenotypes in lung adenocarcinoma, lung squamous cell carcinoma and breast carcinoma cancer cells. RNA-Seq transcriptome analysis demonstrated that PRMT5 and MEP50 are required to maintain expression of metastasis and Epithelial-to-mesenchymal transition (EMT) markers and to potentiate an epigenetic mechanism of the TGFβ response. We show that PRMT5-MEP50 activity both positively and negatively regulates expression of a wide range of genes. Exogenous TGFβ promotes EMT in a unique pathway of PRMT5-MEP50 catalyzed histone mono- and dimethylation of chromatin at key metastasis suppressor and EMT genes, defining a new mechanism regulating cancer invasivity. PRMT5 methylation of histone H3R2me1 induced transcriptional activation by recruitment of WDR5 and concomitant H3K4 methylation at targeted genes. In parallel, PRMT5 methylation of histone H4R3me2s suppressed transcription at distinct genomic loci. Our decoding of histone methylarginine at key genes supports a critical role for complementary PRMT5-MEP50 transcriptional activation and repression in cancer invasion pathways and in response to TGFβ stimulation and therefore orients future chemotherapeutic opportunities.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Arginine; Breast Neoplasms; Carcinoma, Squamous Cell; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histones; Humans; Lung Neoplasms; MCF-7 Cells; Methylation; Neoplasm Invasiveness; Neoplasms; Prognosis; Protein-Arginine N-Methyltransferases; Sequence Analysis, RNA; Transcription, Genetic; Transforming Growth Factor beta

Histone demethylase PHF8 is upregulated and plays oncogenic roles in various cancers; however, the mechanisms underlying its dysregulation and functions in carcinogenesis remain obscure. Here, we report the novel functions of PHF8 in EMT (epithelial to mesenchymal transition) and breast cancer development. Genome-wide gene expression analysis revealed that PHF8 overexpression induces an EMT-like process, including the upregulation of SNAI1 and ZEB1. PHF8 demethylates H3K9me1, H3K9me2 and sustains H3K4me3 to prime the transcriptional activation of SNAI1 by TGF-β signaling. We show that PHF8 is upregulated and positively correlated with MYC at protein levels in breast cancer. MYC post-transcriptionally regulates the expression of PHF8 via the repression of microRNAs. Specifically, miR-22 directly targets and inhibits PHF8 expression, and mediates the regulation of PHF8 by MYC and TGF-β signaling. This novel MYC/microRNAs/PHF8 regulatory axis thus places PHF8 as an important downstream effector of MYC. Indeed, PHF8 contributes to MYC-induced cell proliferation and the expression of EMT-related genes. We also report that PHF8 plays important roles in breast cancer cell migration and tumor growth. These oncogenic functions of PHF8 in breast cancer confer its candidacy as a promising therapeutic target for this disease.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Heterografts; Histone Demethylases; Histones; Humans; Mice; MicroRNAs; Proto-Oncogene Proteins c-myc; Signal Transduction; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

CQ is an anti-malaria drug, which has been used for years. However, there are published articles about its activity in anti-cancers. The aim of this approach was to look at possibility and related mechanisms of anti-breast cancer (mouse breast cancer cell line 4T1) by CQ alone. The studies of anti 4T1 in vitro and in vivo by CQ were performed. The growth of 4T1 in vitro and in vivo, survival of mice post treatment with CQ, changes of immune parameters and microenvironment in mice were evaluated. Our results demonstrate that CQ could markedly inhibit growth of 4T1 in vitro through inducing apoptosis of cells, inhibiting secretion of TGF-β and prolong the mice survival in vivo through boosting immune system by upregulating CD8+ T cell, and through down-regulating tumor associated macrophages (TAM), myeloid derived suppressing cells (MDSC) and Tregs, in microenvironment of mice bearing tumor. This provides a new mode of action for CQ and it is therefore concluded that CQ could be with potential in breast cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cellular Microenvironment; Chloroquine; Female; Humans; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Neoplasm Transplantation; Transforming Growth Factor beta; Tumor Burden

While we previously demonstrated that CITED2 expression in primary breast tumor tissues is elevated relative to normal mammary epithelium and inversely correlated with patient survival, its functional impact on primary tumor development and progression remained unknown. To address this issue, we examined the effect of CITED2 silencing on the growth of human breast cancer cell lines MDA-MB-231 and MDA-MB-468 following orthotopic administration in vivo. Here, we show that CITED2 silencing significantly attenuated MDA-MB-231 primary tumor growth concordant with reduced tumor vascularization, while MDA-MB-468 primary tumor growth and tumor vascularization remained unaffected. Correspondingly, expression of VEGFA was significantly reduced in shCITED2-expressing MDA-MB-231, but not MDA-MB-468 tumors. Consistent with the observed pattern of vascularization and VEGFA expression, we found that TGF-β stimulation induced expression of VEGFA and enhanced CITED2 recruitment to the VEGFA promoter in MDA-MA-231 cells, while failing to induce VEGFA expression in MDA-MB-468 cells. Further supporting its involvement in TGF-β-induced expression of VEGFA, CITED2 silencing prevented TGF-β induction of VEGFA expression in MDA-MB-231 cells. Collectively, these data indicate that CITED2 regulates primary breast tumor growth, likely by influencing tumor vasculature via TGF-β-dependent regulation of VEGFA.

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; Neovascularization, Pathologic; Promoter Regions, Genetic; Repressor Proteins; RNA Interference; Signal Transduction; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Burden; Vascular Endothelial Growth Factor A

Histological inspection of visually normal tissue adjacent to neoplastic lesions often reveals multiple foci of cellular abnormalities. This suggests the presence of a regional carcinogenic signal that spreads oncogenic transformation and field cancerization. We observed an abundance of mutagenic reactive oxygen species in the stroma of cryosectioned patient tumor biopsies, indicative of extratumoral oxidative stress. Diffusible hydrogen peroxide (H

Topics: Breast Neoplasms; Cancer-Associated Fibroblasts; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Oxidative Stress; PTEN Phosphohydrolase; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4-USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3-mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and metastasis in overweight/obese patient breast cancer specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGFβ signaling and dyslipidemia-induced aberrant TGFβ activation during breast cancer metastasis.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Cell Proliferation; Fatty Acids, Nonesterified; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Obesity; Phosphorylation; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin Thiolesterase; Ubiquitination

Long non-coding RNA (LncRNA) CCAT2 plays an important role in tumorigenesis, tumor growth and metastasis. In this study, we reported that long noncoding RNA CCAT2 (LncRNA CCAT2) could regulate TGF-β signaling pathway in breast cancer.. The relative mRNA expression level of CCAT2 in adjacent non-cancerous and breast cancer tissues without or with metastasis were analyzed by quantitative Real-time polymerase chain reaction (qRT-PCR). The mRNA expression levels of CCAT2 in breast cancer cell lines were analyzed by qRT-PCR. Proliferation, invasion and migration of breast cancer cells were detected after infected with si-NC or si-CCAT2. Flow cytometry analysis was used to detect the cell cycle distribution and apoptosis rate in breast cancer cells after infected with si-NC or si-CCAT2. The relative protein expression level of TGF-β, Smad2 and α-SMA in breast cancer cells after infected with si-NC or si-CCAT2 were analyzed by Western blot.. The relative mRNA expression level of CCAT2 in breast cancer tissues was significantly increased compared with adjacent non-cancerous tissues. The expression level of CCAT2 in breast cancer without metastasis was decreased compared with breast cancer metastasis. Meanwhile, down-regulation of CCAT2 inhibited the proliferation, invasion and migration in breast cancer cells. Furthermore, down-regulation of CCAT2 caused breast cancer cells cycle arrested in G0/G1 phase and promoted cell apoptosis. Down-regulation of CCAT2 significantly down-regulated the protein expression levels of TGF-β, Smad2 and α-SMA in breast cancer cells.. CCAT2 was highly expressed in breast cancer. Down-regulation of CCAT2 inhibited the proliferation, invasion and migration and promoted cell apoptosis in breast cancer cells by regulating TGF-β signaling pathway.

Topics: Actins; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; RNA, Long Noncoding; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

The miR-200 family promotes the epithelial state by suppressing the Zeb1/Zeb2 epithelial gene transcriptional repressors. To identify other miR-200-regulated genes, we isolated mRNAs bound to transfected biotinylated miR-200c in mouse breast cancer cells. In all, 520 mRNAs were significantly enriched in miR-200c binding at least twofold. Putative miR-200-regulated genes included Zeb2, enriched 3.5-fold in the pull down. However, Zeb2 knockdown does not fully recapitulate miR-200c overexpression, suggesting that regulating other miR-200 targets contributes to miR-200's enhancement of epithelial gene expression. Candidate genes were highly enriched for miR-200c seed pairing in their 3'UTR and coding sequence and for genes that were downregulated by miR-200c overexpression. Epidermal growth factor receptor and downstream MAPK signaling pathways were the most enriched pathways. Genes whose products mediate transforming growth factor (TGF)-β signaling were also significantly overrepresented, and miR-200 counteracted the suppressive effects of TGF-β and bone morphogenic protein 2 (BMP-2) on epithelial gene expression. miR-200c regulated the 3'UTRs of 12 of 14 putative miR-200c-binding mRNAs tested. The extent of mRNA binding to miR-200c strongly correlated with gene suppression. Twelve targets of miR-200c (Crtap, Fhod1, Smad2, Map3k1, Tob1, Ywhag/14-3-3γ, Ywhab/14-3-3β, Smad5, Zfp36, Xbp1, Mapk12, Snail1) were experimentally validated by identifying their 3'UTR miR-200 recognition elements. Smad2 and Smad5 form a complex with Zeb2 and Ywhab/14-3-3β and Ywhag/14-3-3γ form a complex with Snail1. These complexes that repress transcription assemble on epithelial gene promoters. miR-200 overexpression induced RNA polymerase II localization and reduced Zeb2 and Snail1 binding to epithelial gene promoters. Expression of miR-200-resistant Smad5 modestly, but significantly, reduced epithelial gene induction by miR-200. miR-200 expression and Zeb2 knockdown are known to inhibit cell invasion in in vitro assays. Knockdown of each of three novel miR-200 target genes identified here, Smad5, Ywhag and Crtap, also profoundly suppressed cell invasion. Thus, miR-200 suppresses TGF-β/BMP signaling, promotes epithelial gene expression and suppresses cell invasion by regulating a network of genes.

Topics: 3' Untranslated Regions; Animals; Bone Morphogenetic Protein 2; Breast Neoplasms; Cadherins; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Homeodomain Proteins; Mice; MicroRNAs; NIH 3T3 Cells; Repressor Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box Binding Homeobox 2

Breast cancer (BCa) bone metastases cause osteolytic bone lesions, which result from the interactions of metastatic BCa cells with osteoclasts and osteoblasts. Osteoclasts differentiate from myeloid lineage cells. To understand the cell-specific role of transforming growth factor beta (TGF-β) in the myeloid lineage, in BCa bone metastases, MDA-MB-231 BCa cells were intra-tibially or intra-cardially injected into LysM(Cre)/Tgfbr2(floxE2/floxE2) knockout (LysM(Cre)/Tgfbr2 KO) or Tgfbr2(floxE2/floxE2) mice. Metastatic bone lesion development was compared by analysis of both lesion number and area. We found that LysM(Cre)/Tgfbr2 knockout significantly decreased MDA-MB-231 bone lesion development in both the cardiac and tibial injection models. LysM(Cre)/Tgfbr2 knockout inhibited the tumor cell proliferation, angiogenesis and osteoclastogenesis of the metastatic bones. Cytokine array analysis showed that basic fibroblast growth factor (bFGF) was downregulated in MDA-MB-231-injected tibiae from the LysM(Cre)/Tgfbr2 KO group, and intravenous injection of the recombinant bFGF to LysM(Cre)/Tgfbr2 KO mice rescued the inhibited metastatic bone lesion development. The mechanism by which bFGF rescued the bone lesion development was by promotion of tumor cell proliferation through the downstream mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)-cFos pathway after binding to the FGF receptor 1 (FGFR1). Consistent with animal studies, we found that in human BCa bone metastatic tissues, TGF-β type II receptor (TβRII) and p-Smad2 were expressed in osteoclasts and tumor cells, and were correlated with the expression of FGFR1. Our studies suggest that myeloid-specific TGF-β signaling-mediated bFGF in the bone promotes BCa bone metastasis.

Topics: Animals; Antibodies, Neoplasm; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Humans; Mice; Myeloid Cells; Osteoclasts; Protein Serine-Threonine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The biological heterogeneity of breast cancer leads to the need for finding new approaches to understand the mechanisms implicated in breast cancer progression. The tumor stroma appears as a key in the progression of solid tumors towards a malignant phenotype. Cancer associated fibroblasts (CAFs) may orchestrate a functional "corrupted" stroma which in turn helps metastatic spread. In this study, we investigated by real-time PCR, the expression of 19 factors by normal breast-associated fibroblasts (NAFs) and CAFs, which were implicated in several actions promoting tumor growth, such as extracellular matrix remodeling, inflammation and invasion. Also, we explored the influence of inflammatory cells phenotypes (MMP11 status) and breast cancer cell lines (MCF-7 and MDA-MB-231) on the molecular profile of CAFs. If we consider that one of the major sources of CAFs are resident NAFs, the transition of NAFs into CAFs is associated with molecular changes involving the overexpression of some molecular factors of biological importance in tumor progression. In addition, the characterization of the tumor stroma regarding to the MMP11 status by MICs reflects a type of fibroblasts which contribute even more to tumor progression. Moreover, different patterns in the induction of the expression of factors by CAFs were observed, depending on the tumor cell line which they were co-cultured with. Furthermore, CAFs influence TGFβ expression in both cancer cell lines. Therefore, this study can help to a better characterization of tumor stroma in order to improve the prognostic evaluation, as well as to define the different populations of CAFs as potential therapeutic targets in breast cancer. © 2015 Wiley Periodicals, Inc.

Topics: Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Coculture Techniques; Disease Progression; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 11; MCF-7 Cells; Phenotype; Prospective Studies; Transforming Growth Factor beta; Tumor Microenvironment

In mouse mammary epithelial cells, cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the apical localization of ZO-1 mRNA, which encodes a critical tight junction component. In mice lacking CPEB1 and in cultured cells from which CPEB has been depleted, randomly distributed ZO-1 mRNA leads to the loss of cell polarity. We have investigated whether this diminution of polarity results in an epithelial-to-mesenchyme (EMT) transition and possible increased metastatic potential. Here, we show that CPEB1-depleted mammary epithelial cells alter their gene expression profile in a manner consistent with an EMT and also become motile, which are made particularly robust when cells are treated with transforming growth factor-β, an enhancer of EMT. CPEB1-depleted mammary cells become metastatic to the lung following injection into mouse fat pads while ectopically expressed CPEB1 prevents metastasis. Surprisingly, CPEB1 depletion causes some EMT/metastasis-related mRNAs to have shorter poly(A) tails while other mRNAs to have longer poly(A) tails. Matrix metalloproteinase 9 (MMP9) mRNA, which encodes a metastasis-promoting factor, undergoes poly(A) lengthening and enhanced translation upon CPEB reduction. Moreover, in human breast cancer cells that become progressively more metastatic, CPEB1 is reduced while MMP9 becomes more abundant. These data suggest that at least in part, CPEB1 regulation of MMP9 mRNA expression mediates metastasis of breast cancer cells.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Glands, Animal; Matrix Metalloproteinase 9; Mice; mRNA Cleavage and Polyadenylation Factors; Neoplasm Metastasis; Poly A; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta

MicroRNA-21 (miR-21) upregulation, smad family member 7 (smad7) downregulation, epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) actions contribute to breast cancer cell aggressiveness. However, their correlation and the relevant molecular mechanisms involved remain to be elucidated. The present study was undertaken to determine the association of miR-21, smad7, EGF and TGF-β with breast cancer cell invasion and migration and to identify the molecular mechanisms involved using immunohistochemistry and western blot analysis. In the present study, the plasma miR-21 levels were significantly increased in patients with breast cancer, as compared to the controls. Smad7 was confirmed to be a direct target of miR-21, by luciferase reporter and western blot assays. The downregulation of smad7 by miR-21 or sismad7 enhanced EGF-dependent invasion and migration, as well as TGF-β-dependent invasion and migration. The actions of miR-21 were abrogated by expressing a modified smad7 cDNA resistant to miR-21. Moreover, miR-21, EGF and TGF-β combined to markedly increase cancer cell invasion and migration, and this effect was blocked by the combination of erlotinib (an EGF receptor kinase inhibitor) and SB505124 (a type I TGF-β receptor inhibitor). A lower smad7 expression was identified in poorly differentiated breast cancers, as compared to well- to moderately differentiated breast cancers. Notably, antagonism of miR-21 decreased breast cancer cell proliferation and tumor growth in mouse models. In conclusion, our results demonstrated that plasma miR-21 levels may serve as a diagnostic marker in breast cancers, whereas miR-21 promotes breast cancer cell proliferation and invasion by suppressing smad7, which enhances EGF and TGF-β pathways.

Topics: 3' Untranslated Regions; Animals; Breast Neoplasms; Cell Movement; Epidermal Growth Factor; Female; Humans; MCF-7 Cells; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Transplantation; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

The multifunctional cytokine transforming growth factor-β (TGFβ) is produced by several types of cancers, including prostate cancer, and promote tumour progression in autocrine and paracrine manners. In response to ligand binding, the TGFβ type I receptor (TβRI) activates Smad and non-Smad signalling pathways. The ubiquitin-ligase tumour necrosis factor receptor-associated factor 6 (TRAF6) was recently linked to regulate intramembrane proteolytic cleavage of the TβRI in cancer cells. Subsequently, the intracellular domain (ICD) of TβRI enters in an unknown manner into the nucleus, where it promotes the transcription of pro-invasive genes, such as MMP2 and MMP9. Here we show that the endocytic adaptor molecules APPL1 and APPL2 are required for TGFβ-induced nuclear translocation of TβRI-ICD and for cancer cell invasiveness of human prostate and breast cancer cell lines. Moreover, APPL proteins were found to be expressed at high levels in aggressive prostate cancer tissues, and to be associated with TβRI in a TRAF6-dependent manner. Our results suggest that the APPL-TβRI complex promotes prostate tumour progression, and may serve as a prognostic marker.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Male; Microscopy, Confocal; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; TNF Receptor-Associated Factor 6; Transforming Growth Factor beta

Tumor initiating cells (TICs) serve as the root of tumor growth. After identifying TICs in spontaneous breast tumors of the MMTV-Wnt1 mouse model, we confirmed the specific expression and activation of Yes-associated protein 1 (Yap1) within TICs. To investigate the role of Yap1 in the self-renewal of breast TICs and the underlying mechanism, we sorted CD49fhighEpCAMlow cells as breast TICs. Active Yap1 with ectopic expression in breast TICs promoted their colony formation in vitro (p< 0.01) and self-renewal in vivo (p< 0.01), and led to a 4-fold increase in TIC frequency (p< 0.05).A conditional knock-out mouse was reconstructed to generate Yap1 knock-out breast tumors. The loss of Yap1 led to a dramatic growth disadvantage of breast TICs in vitro (p< 0.01) and in vivo (p< 0.01), and it also led to an over 200-fold decrease in TIC frequency (p< 0.01). The expression of active Yap1 was negatively correlated with that of phosphorylated Smad3 (p-Smad3).Transforming growth factor β (TGF-β) served as a strong enhancer of Smad3 and an inhibitor of clonogenesis of TICs. The presence of SIS3, a specific inhibitor of Smad3, could rescue the TGF-β -induced growth inhibition and reverse the Smad3 inhibition by Yap1. Analysis of a database containing 2,072 human breast cancer samples showed that higher expressions of Yap1 correlated with a poorer outcome of a 15-year survival rate and median overall survival (mOS)in patients, especially in those with basal breast tumors without estrogen receptor 1 (ER) expression. The findings indicate that active Yap1 promotes the self-renewal of breast TICs by inhibiting Smad3 signaling.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Cycle Proteins; Estrogen Receptor alpha; Female; Humans; Isoquinolines; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Knockout; Neoplastic Stem Cells; Phosphoproteins; Phosphorylation; Pyridines; Pyrroles; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; Wnt1 Protein; YAP-Signaling Proteins

The prostate transmembrane protein, androgen-induced 1 (PMEPA1) has been previously shown to promote solid malignancies in a variety of cancers, but the role and mechanisms of PMEPA1 in breast cancer has not been fully addressed. Here, we found that PMEPA1 was upregulated in breast cancer cell lines as well as in a set of clinical invasive breast ductal carcinomas. Interestingly, depletion of PMEPA1 decreased breast cancer stem cell (CSC)-enriched populations, while ectopic overexpression of PMEPA1 increased breast CSC-enriched populations. Furthermore, transforming growth factor-β (TGF-β) treatment was also found to upregulate PMEPA1 expression and the CSC-enriched populations in triple-negative breast cancer cell lines. TGF-β-induced PMEPA1 expression partially contributed to TGF-β-induced breast CSC maintenance. These findings suggest that TGF-β-PMEPA1 axis might provide new diagnosis and therapeutic targets for breast cancer treatment.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Gene Knockdown Techniques; Humans; Membrane Proteins; Neoplastic Stem Cells; Transforming Growth Factor beta; Up-Regulation

Compelling efficacy on intervention of tumorigenesis by nonsteroidal anti-inflammatory drugs (NSAIDs) has been documented intensively. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for human cancer chemoprevention. A novel derivative of the NSAID sulindac sulfide (SS), referred as sulindac sulfide amide (SSA), was recently developed, which lacks COX inhibitory activity, yet shows greater suppressive effect than SS on growth of various cancer cells. In this study, we focus on the inhibitory activity of SSA on breast tumor cell motility, which has not been studied previously. Our results show that SSA treatment at non-cytotoxic concentrations can specifically reduce breast tumor cell motility without influencing tumor cell growth, and the mechanism of action involves the suppression of TGFβ signaling by directly blocking Smad2/3 phosphorylation. Moreover, miR-21, a well-documented oncogenic miRNA for promoting tumor cell metastasis, was also found to be involved in inhibitory activity of SSA in breast tumor cell motility through the modulation of TGFβ pathway. In conclusion, we demonstrate that a non-COX inhibitory derivative of sulindac can inhibit breast tumor metastasis by a mechanism involving the TGFβ/miR-21 signaling axis.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; MicroRNAs; Sulindac; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

Cerberus is a key regulator of vertebrate embryogenesis. Its biological function has been studied extensively in frog and mouse embryos. Its ability to bind and antagonize the transforming growth factor-β (TGF-β) family ligand Nodal is well established. Strikingly, the molecular function of Cerberus remains poorly understood. The underlying reason is that Cerberus is a complex, multifunctional protein: It binds and inhibits multiple TGF-β family ligands, it may bind and inhibit some Wnt family members, and two different forms with distinct activities have been described. In addition, sequence homology between frog and mammalian Cerberus is low, suggesting that previous studies, which analyzed frog Cerberus function, may not accurately describe the function of mammalian Cerberus. We therefore undertook to determine the molecular activities of human Cerberus in TGF-β family signaling. Using purified proteins, surface plasmon resonance, and reporter gene assays, we discovered that human Cerberus bound and inhibited the TGF-β family ligands Activin B, BMP-6, and BMP-7, but not the frog Cerberus ligand BMP-2. Notably, full-length Cerberus successfully blocked ligand binding to type II receptors, but the short form was less effective. In addition, full-length Cerberus suppressed breast cancer cell migration but the short form did not. Thus, our findings expand the roles of Cerberus as TGF-β family signaling inhibitor, provide a molecular rationale for the function of the N-terminal region, and support the idea that Cerberus could have regulatory activities beyond direct inhibition of TGF-β family signaling.

Topics: Activins; Amino Acid Sequence; Animals; Bone Morphogenetic Proteins; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cytokines; Female; Humans; Molecular Sequence Data; Proteolysis; Sequence Alignment; Signal Transduction; Transforming Growth Factor beta

TGF-β and cancer progression share a multifaceted relationship. Despite the knowledge of TGF-β biology in the development of cancer, several factors that mediate the cancer-promoting role of TGF-β continue to be identified. This study aimed to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cells. We treated the human mammary epithelial cell line MCF10A with TGF-β and identified TGF-β-dependent upregulation of PRRX2, the gene encoding paired-related homeobox 2 transcription factor. Overexpression of PRRX2 enhanced migration, invasion and anchorage-independent growth of MCF10A cells and induced partial epithelial mesenchymal transition (EMT), as determined by partial fibroblastoid morphology of cells, upregulation of EMT markers and partially disrupted acinar structure in a three-dimensional culture. We further identified PLAT, the gene encoding tissue-type plasminogen activator (tPA), as the highest differentially expressed gene in PRRX2-overexpressing MCF10A cells, and demonstrated direct binding and transactivation of the PLAT promoter by PRRX2. Furthermore, PLAT knockdown inhibited PRRX2-mediated enhanced migration and invasion, suggesting that tPA may mediate PRRX2-induced migration and invasion. Finally, the significant correlation of PRRX2 expression with poor survival in 118 primary breast tumour samples (P = 0.027) and the increased PRRX2 expression in metaplastic breast carcinoma samples, which is pathogenetically related to EMT, validated the biological importance of PRRX2-enhanced migration and invasion and PRRX2-induced EMT. Thus, our data suggest that upregulation of PRRX2 may be a mechanism contributing to TGF-β-induced invasion and EMT in breast cancer. © 2016 Wiley Periodicals, Inc.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Neoplasm Invasiveness; Prognosis; Tissue Plasminogen Activator; Transforming Growth Factor beta

Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity.

Topics: Amphiregulin; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation; Humans; Ligands; Mammary Glands, Human; MAP Kinase Signaling System; Phenotype; Phosphorylation; Signal Transduction; Transforming Growth Factor beta

Store-operated calcium entry (SOCE) signaling is involved in cancer progression. Stromal interaction molecule 1 (STIM1) triggers store-operated calcium channels to induce SOCE. Transforming growth factor-β (TGF-β) influences a wide range of cellular behaviors, including cell proliferation. However, little is known about the relationship between calcium signaling and TGF-β signaling in cancer cell proliferation. Here, we found that TGF-β induced cell cycle arrest at the G0/G1 phase and suppressed cell proliferation in MDA-MB-231 and MCF-7 breast cancer cells. These effects were impaired by extracellular Ca2+ chelator EGTA or SOCE specific inhibitor SKF96365 in MDA-MB-231 cells. Treating MDA-MB-231 cells with TGF-β for 24 and 48 h markedly decreased STIM1 expression and thapsigargin-induced SOCE. A transcriptional inhibitor of STIM1, Wilm's tumor suppressor 1 (WT1), was upregulated in TGF-β-treated MDA-MB-231 cells, and knockdown of WT1 expression partially restored the TGF-β-induced downregulation of STIM1. Stably overexpressing STIM1 in MDA-MB-231 cells restored the TGF-β-induced effects. The p21 mRNA level increased in SKF96365- or TGF-β-treated MDA-MB-231 cells, whereas that for cyclin E1 decreased. Our findings demonstrate for the first time that STIM1 and SOCE are involved in the TGF-β-induced suppression of cell proliferation. Furthermore, our studies also provide a new approach to inhibit breast cancer cell proliferation with small molecules targeting STIM1 and SOCE.

Topics: Breast Neoplasms; Calcium Signaling; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Stromal Interaction Molecule 1; Transforming Growth Factor beta

Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Indoles; Integrin beta1; Integrin beta3; Mice, Inbred BALB C; Nuclear Receptor Subfamily 4, Group A, Member 1; Phenols; Phenylacetates; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Seventy-five percent of breast cancers are estrogen receptor α positive (ER⁺). Research on these tumors is hampered by lack of adequate in vivo models; cell line xenografts require non-physiological hormone supplements, and patient-derived xenografts (PDXs) are hard to establish. We show that the traditional grafting of ER⁺ tumor cells into mammary fat pads induces TGFβ/SLUG signaling and basal differentiation when they require low SLUG levels to grow in vivo. Grafting into the milk ducts suppresses SLUG; ER⁺ tumor cells develop, like their clinical counterparts, in the presence of physiological hormone levels. Intraductal ER⁺ PDXs are retransplantable, predictive, and appear genomically stable. The model provides opportunities for translational research and the study of physiologically relevant hormone action in breast carcinogenesis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Estrogen Receptor alpha; Female; Humans; Mammary Glands, Human; MCF-7 Cells; Mice; Mice, Inbred NOD; Mice, SCID; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interacting RNAs and play key roles in the biogenesis and functions of PIWI-interacting RNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms and that they are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cells derived from breast cancer. Reducing PIWIL4 expression drastically impairs the migration ability of MDA-MB-231 cells, significantly increases their apoptosis, and mildly affects their proliferation. Our transcriptome and proteome analysis reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-β and FGF signaling pathways and MHC class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer.

Topics: Apoptosis; Argonaute Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Fibroblast Growth Factors; Gene Knockdown Techniques; Humans; RNA-Binding Proteins; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling.

Topics: Bone Morphogenetic Protein 4; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Culture Media, Conditioned; Eye Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Nerve Tissue Proteins; Prognosis; Transforming Growth Factor beta; Up-Regulation

Previous reports have shown a mutual negative feedback loop between microRNA (miR)-206 and estrogen receptor (ER) expression. Furthermore, decreased miR-206 expression in breast cancer (BC) is associated with the advanced clinical stage and lymph node metastasis. However, its role and the mechanism underlying the migration and invasion of ER positive BC remain unclear.. In this study, miR-206 was stably transfected into ER positive cell lines MCF-7 and T47D to investigate the effect of miR-206. The results showed that miR-206 overexpression markedly impaired the migration and invasive abilities of these cells, followed by suppression of the epithelial mesenchymal transition (EMT). Mechanistic analyses showed that miR-206 inhibited the autocrine production of transforming growth factor (TGF)-β as well as the downstream expression of neuropilin-1 (NRP1) and SMAD2, responsible for the decreased migration, invasion, and EMT in these cells.. Our data demonstrate that miR-206 inhibits TGF-β transcription and autocrine production, as well as downstream target genes of EMT. Restoring miR-206 expression may provide an effective therapeutic strategy for ER positive BC.

Topics: 3' Untranslated Regions; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Neuropilin-1; Receptors, Estrogen; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

MicroRNAs act as key regulators in carcinogenesis and progression in various cancers. In present study, we explored the role of miR-340 in the breast cancer progression. Our results showed that overexpression of miR-340 inhibits breast cancer cell proliferation and invasion, whereas depletion of miR-340 promotes breast cancer progression. Molecularly, ZEB1 was identified as a target gene of miR-340 and miR-340 suppressed the expression of ZEB1 by directly binding to the 3'-UTR of ZEB1. Furthermore, ZEB1 transcriptionally suppresses miR-340 expression. The negative feedback loop regulated TGF-β-mediated breast cancer progression. In conclusion, our data suggested that miR-340 acted as a tumor suppressor in breast cancer progression.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Adhesion; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Transforming Growth Factor beta; Tumor Cells, Cultured; Zinc Finger E-box-Binding Homeobox 1

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Topics: Adipogenesis; Adult; Aged; Animals; Breast; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Differentiation; Cell Hypoxia; Collagen; Energy Metabolism; Extracellular Matrix; Female; Gels; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Glycolysis; Humans; Lipid Droplets; Middle Aged; Myofibroblasts; Rats; Tissue Donors; Transforming Growth Factor beta

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Gene Knockdown Techniques; Humans; Membrane Proteins; Models, Biological; Neoplasm Metastasis; Receptor, ErbB-2; Transforming Growth Factor beta; Tumor Microenvironment

Basal subtype breast cancers have a particularly poor prognosis, with high invasiveness and resistance to most targeted therapies. TGFβ and MYC drive central features of basal breast cancer: TGFβ is an autocrine and paracrine signaling factor that drives cell invasion and metastasis, and MYC is a central regulator of cellular proliferation that is upregulated in many cancer types. We show here that genetic or pharmacologic inhibition of MYC in MCF10A basal breast cells results in increased sensitivity to TGFβ-stimulated invasion and metastasis and also show that this signaling loop is dependent on activation of SRC. Analysis of human breast cancer datasets and additional experiments with breast cancer cell lines further suggest the relevance of this signaling loop in basal, but not luminal, breast cancers. Our results imply precaution should be taken when utilizing therapeutic inhibitors of MYC with basal breast cancer patients as this could lead to increased metastasis; however, simultaneous pharmacologic inhibition of SRC and MYC for these patients could facilitate the antiproliferative effects of MYC inhibition while blocking the consequent promotion of metastasis. Cancer Res; 76(12); 3520-30. ©2016 AACR.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Integrin alphaVbeta3; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins c-myc; src-Family Kinases; Transforming Growth Factor beta

Cancer stem-like cells contribute to tumor heterogeneity and have been implicated in disease relapse and drug resistance. Here we show the coexistence of distinct breast cancer stem-like cells (BCSC) as identified by ALDH(+) and CD29(hi)CD61(+) markers, respectively, in murine models of breast cancer. While both BCSC exhibit enhanced tumor-initiating potential, CD29(hi)CD61(+) BCSC displayed increased invasive abilities and higher expression of epithelial-to-mesenchymal transition and mammary stem cell-associated genes, whereas ALDH(+) BCSC were more closely associated with luminal progenitors. Attenuating the autophagy regulator FIP200 diminished the tumor-initiating properties of both ALDH(+) and CD29(hi)CD61(+) BCSC, as achieved by impairing either the Stat3 or TGFβ/Smad pathways, respectively. Furthermore, combining the Stat3 inhibitor Stattic and the Tgfβ-R1 inhibitor LY-2157299 inhibited the formation of both epithelial and mesenchymal BCSC colonies. In vivo, this combination treatment was sufficient to limit tumor growth and reduce BCSC number. Overall, our findings reveal a differential dependence of heterogeneous BCSC populations on divergent signaling pathways, with implications on how to tailor drug combinations to improve therapeutic efficacy. Cancer Res; 76(11); 3397-410. ©2016 AACR.

Topics: Animals; Apoptosis; Autophagy; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Mice; Neoplasm Invasiveness; Neoplastic Stem Cells; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad Proteins; STAT3 Transcription Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFβ signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFβ in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Cancer Res; 76(11); 3376-86. ©2016 AACR.

Topics: Animals; Antigens, Ly; Antigens, Surface; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Proliferation; Disease Progression; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Escape; Xenograft Model Antitumor Assays

We have previously reported that RORγ expression was decreased in ER-ve breast cancer, and increased expression improves clinical outcomes. However, the underlying RORγ dependent mechanisms that repress breast carcinogenesis have not been elucidated. Here we report that RORγ negatively regulates the oncogenic TGF-β/EMT and mammary stem cell (MaSC) pathways, whereas RORγ positively regulates DNA-repair. We demonstrate that RORγ expression is: (i) decreased in basal-like subtype cancers, and (ii) inversely correlated with histological grade and drivers of carcinogenesis in breast cancer cohorts. Furthermore, integration of RNA-seq and ChIP-chip data reveals that RORγ regulates the expression of many genes involved in TGF-β/EMT-signaling, DNA-repair and MaSC pathways (including the non-coding RNA, LINC00511). In accordance, pharmacological studies demonstrate that an RORγ agonist suppresses breast cancer cell viability, migration, the EMT transition (microsphere outgrowth) and mammosphere-growth. In contrast, RNA-seq demonstrates an RORγ inverse agonist induces TGF-β/EMT-signaling. These findings suggest pharmacological modulation of RORγ activity may have utility in breast cancer.

Topics: Benzamides; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; DNA Repair; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplasm Metastasis; Nuclear Receptor Subfamily 1, Group F, Member 3; Piperazines; Propanols; Sequence Analysis, RNA; Signal Transduction; Transforming Growth Factor beta

The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is an attractive target for the development of new targeted therapies. We synthesized several indolo-pyrido-isoquinolin based alkaloids to activate p53 function and examined their therapeutic efficacy using NCI-60 screening. Here, we provide molecular evidence that one of these compounds, 11-methoxy-2,3,4,13-tetrahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ylium-bromide (termed P18 or NSC-768219) inhibits growth and clonogenic potential of cancer cells. P18 treatment results in downregulation of mesenchymal markers and concurrent upregulation of epithelial markers as well as inhibition of migration and invasion. Experimental epithelial-mesenchymal-transition (EMT) induced by exposure to TGFβ/TNFα is also completely reversed by P18. Importantly, P18 also inhibits mammosphere-formation along with a reduction in the expression of stemness factors, Oct4, Nanog and Sox2. We show that P18 induces expression, phosphorylation and accumulation of p53 in cancer cells. P18-mediated induction of p53 leads to increased nuclear localization and elevated expression of p53 target genes. Using isogenic cancer cells differing only in p53 status, we show that p53 plays an important role in P18-mediated alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells. Furthermore, P18 increases miR-34a expression in p53-dependent manner and miR-34a is integral for P18-mediated inhibition of growth, invasion and mammosphere-formation. miR-34a mimics potentiate P18 efficacy while miR-34a antagomirs antagonize P18. Collectively, these data provide evidence that P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of breast cancer and p53-miR-34a axis is important for P18 function.

Topics: Alkaloids; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Clone Cells; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Isoquinolines; MicroRNAs; Neoplasm Invasiveness; Neoplastic Stem Cells; Phosphorylation; Pyridines; Secologanin Tryptamine Alkaloids; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Up-Regulation

Metastasis is the leading lethal factor severely restraining the effectiveness of clinical treatment. TGF-beta is the key regulator for metastasis and influences paradoxically on cancer progression. The known TGF-beta blockers exert little selectivity on its functions, indiscriminately causing the anti-metastatic and pro-growth effects. Under such circumstances, specifically rebalancing the oncological function of TGF-beta provides a crucial oncotarget against metastasis. In our study, we established the screening platform targeting cell motility and identified a potential flavonoid, Chamaejasmenin B (ICJ), extracted from Stellera chamaejasme L..It suppressed the migration and invasion in breast cancer cells in vitro. Moreover, by dynamical quantification of breast cancer progression in small-animal imaging system, ICJ was proved to be a potent inhibitor of metastasis with minimal toxic side effects. Mechanism study further revealed that ICJ efficiently blocked TGF-beta induced EMT, disrupted the interaction between β3 integrin-TβRII complex and, consequently, resulted in the selective inhibition of FAK:Src:p38 pathway. Meanwhile, specific blockage of this pathway largely attenuated the anti-metastatic function of ICJ. Importantly, in contrast with the antagonistic effects on TGF-beta induced metastasis, ICJ obviously sensitized its cytostatic activity, suggesting that it was not a pan-blocker but a rebalancer for the functional output of TGF-beta. Collectively, by targeting TGF-beta Paradox, we experimentally provided a promising candidate for metastatic intervention.

Topics: Animals; Biflavonoids; Breast Neoplasms; Disease Models, Animal; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Transforming Growth Factor beta

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Epithelium; Humans; Mice, SCID; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta

The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-β (TGF-β) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-β. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-β plays a crucial role in this process.

Topics: Breast Neoplasms; Cell Line, Tumor; Cellular Senescence; Female; Fibroblasts; Humans; Mammary Glands, Human; NF-kappa B; Paracrine Communication; Radiation, Ionizing; Signal Transduction; Smad Proteins; Syndecan-1; Transforming Growth Factor beta

The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context-dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal-truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor-β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β-induced or epidermal growth factor (which activates Ras)-induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program.

Topics: Breast Neoplasms; Cell Line, Transformed; Cell Movement; Dual Specificity Phosphatase 6; Female; HEK293 Cells; Humans; Oncogene Protein p21(ras); Signal Transduction; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFβ and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFβ-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFβ-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFβ-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFβ-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

Topics: A549 Cells; Antigens, CD; Breast Neoplasms; Cadherins; CD24 Antigen; Cell Cycle Proteins; Cell Movement; Cell Self Renewal; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Etoposide; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Hep G2 Cells; Humans; Hyaluronan Receptors; Mammary Glands, Human; Neoplastic Stem Cells; Octamer Transcription Factor-3; Phenotype; Poly(ADP-ribose) Polymerases; Protein Glutamine gamma Glutamyltransferase 2; Reactive Oxygen Species; RNA Interference; Signal Transduction; Snail Family Transcription Factors; SOXB1 Transcription Factors; Spheroids, Cellular; Time Factors; Topoisomerase II Inhibitors; Transfection; Transforming Growth Factor beta; Transglutaminases

We previously reported that constitutive c-Abl activity (CST-Abl) abrogates the tumorigenicity of triple-negative breast cancer cells through the combined actions of two cellular events: downregulated matrix metalloproteinase (MMP) and upregulated p21Waf1/Cip1 expression. We now find decreased c-Abl expression to be significantly associated with diminished relapse-fee survival in breast cancer patients, particularly those exhibiting invasive and basal phenotypes. Moreover, CST-Abl expression enabled 4T1 cells to persist innocuously in the mammary glands of mice, doing so by exhausting their supply of cancer stem cells. Restoring MMP-9 expression and activity in CST-Abl-expressing 4T1 cells failed to rescue their malignant phenotypes; however, rendering these same cells deficient in p21 expression not only delayed their acquisition of senescent phenotypes, but also partially restored their tumorigenicity in mice. Although 4T1 cells lacked detectable expression of p53, those engineered to express CST-Abl exhibited robust production and secretion of TGF-β1 that engendered the reactivated expression of p53. Mechanistically, TGF-β-mediated p53 expression transpired through the combined actions of Smad1/5/8 and Smad2, leading to the dramatic upregulation of p21 and its stimulation of TNBC senescence. Collectively, we identified a novel c-Abl:p53:p21 signaling axis that functions as a powerful suppressor of mammary tumorigenesis and metastatic progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Matrix Metalloproteinase 9; Mice; Models, Biological; Proto-Oncogene Proteins c-abl; Smad Proteins, Receptor-Regulated; Transcriptional Activation; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53

In addition to contraction, myoepithelia have diverse paracrine effects, including a tumor suppression effect. However, certain myoepithelial markers have been shown to contribute to tumor progression. Transforming growth factor-β (TGF-β) is involved in the transdifferentiation of fibroblasts to contractile myofibroblasts. We investigated whether TGF-β can upregulate potential myoepithelial markers, which may have functional and clinicopathological significance in breast cancer. We found that TGF-β induced SPOCK1 expression in MCF10A, MCF12A, and M10 breast cells and demonstrated SPOCK1 as a novel myoepithelial marker that was immunolocalized within or beneath myoepithelia lining ductolobular units. A functional study showed that overexpression of SPOCK1 enhanced invasiveness in mammary immortalized and cancer cells. To further determine the biological significance of SPOCK1 in breast cancer, we investigated the expression of SPOCK1 in 478 invasive ductal carcinoma (IDC) cases through immunohistochemistry and correlated the expression with clinicopathological characteristics. SPOCK1 expression was significantly correlated with high pathological tumor size (P = 0.012), high histological grade (P = 0.013), the triple-negative phenotype (P = 0.022), and the basal-like phenotype (P = 0.026) and was correlated with a significantly poorer overall survival on univariate analysis (P = 0.001, log-rank test). Multivariate Cox regression analysis demonstrated that SPOCK1 expression maintained an independent poor prognostic factor of overall survival. Analysis of SPOCK1 expression on various non-IDC carcinoma subtypes showed an enrichment of SPOCK1 expression in metaplastic carcinoma, which is pathogenetically closely related to epithelial-mesenchymal transition (EMT). In conclusion, we identified SPOCK1 as a novel TGF-β-induced myoepithelial marker and further demonstrated that SPOCK1 enhanced invasion in breast cancer cells and correlated with poor prognosis in breast cancer clinical samples. The enrichment of SPOCK1 expression in metaplastic carcinoma and the correlation between SPOCK1 expression and high histological grading and basal-like phenotypes in IDC evidence an association between SPOCK1 and EMT.

Topics: Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Prognosis; Proportional Hazards Models; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Transforming Growth Factor beta

Tumor hypoxia induces the expression of growth arrest and DNA damage-inducible protein (GADD34). However, the role of GADD34 in tumor growth remains unclear.. Gadd34 expression was knocked-down through lentivirus-mediated short hairpin RNA (shRNA) in tumor cells, which were subsequently injected subcutaneously into mice. Tumor volumes and myeloid-derived suppressor cells (MDSCs) were monitored. Isolated MDSCs were incubated with tumor supernatant to investigate the impact of GADD34 on cytokine secretion of MDSCs.. We observed that reduction of GADD34 expression significantly suppressed tumor, and resulted in decreased accumulation of MDSCs and T-cells, and inhibition of GADD34 reduced secretion of vascular epithelial growth factor α and transforming growth factor β by MDSCs.. These findings provide a promising strategy for targeting GADD34 activity in order to inhibit tumor growth.

Topics: Animals; Breast Neoplasms; Carcinoma, Lewis Lung; CD11b Antigen; Culture Media, Conditioned; DNA Damage; Female; Flow Cytometry; Humans; Hypoxia; Mice; Mice, Inbred C57BL; Mice, Nude; Myeloid Cells; Myeloid-Derived Suppressor Cells; Neoplasms; Protein Phosphatase 1; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Chemotherapeutic response is critical for the successful treatment and good prognosis in cancer patients. In this study, we analysed the gene expression profiles of preoperative samples from oestrogen receptor (ER)-negative breast cancer patients with different responses to taxane-anthracycline-based (TA-based) chemotherapy, and identified a group of genes that was predictive. Pregnancy specific beta-1-glycoprotein 1 (PSG1) played a central role within signalling pathways of these genes. Inhibiting PSG1 can effectively reduce chemoresistance via a transforming growth factor-β (TGF-β)-related pathway in ER-negative breast cancer cells. Drug screening then identified dicumarol (DCM) to target the PSG1 and inhibit chemoresistance to TA-based chemotherapy in vitro, in vivo, and in clinical samples. Taken together, this study highlights PSG1 as an important mediator of chemoresistance, whose effect could be diminished by DCM.

Topics: Anticoagulants; Breast Neoplasms; Bridged-Ring Compounds; Cell Line, Tumor; Dicumarol; Drug Resistance, Neoplasm; Female; Humans; Pregnancy-Specific beta 1-Glycoproteins; Taxoids; Transforming Growth Factor beta

Activation of Epithelial-to-Mesenchymal Transition (EMT) is important for tumor metastasis. Although growth factors such as TGFβ and EGF have been shown to induce EMT in breast epithelial cells, the mechanism resulting in migration is not well understood. Herein, we provide evidence that Ca2+ entry into the cell, especially upon store-depletion, plays an important role in TGFβ-induced EMT by promoting cellular migration and potentially leading to metastasis. The increased migration by TGFβ in non-cancerous cells was due to the loss of E-cadherin along with a subsequent increase in N-cadherin levels. Importantly, TGFβ-treatment increases store-mediated Ca2+ entry, which was essential for the activation of calpain leading to the loss of E-cadherin and MMP activation. Inhibition of Ca2+ entry by using Ca2+ channel blocker SKF-96365, significantly decreased Ca2+ entry, decreased TGFβ-induced calpain activation, and suppressed the loss of E-cadherin along with inhibiting cell migration. Furthermore, TRPC1 function as an endogenous Ca2+ entry channel and silencing of either TRPC1 or its activator, STIM1, significantly decreased TGFβ induced Ca2+ entry, inhibited TGFβ-mediated calpain activation and cell migration. In contrast, overexpression of TRPC1 showed increased Ca2+ entry and promoted TGFβ-mediated cell migration. Moreover, increased TRPC1 expression was observed in ductal carcinoma cells. Together these results suggest that disrupting Ca2+ influx via TRPC1/STIM1 mechanism reduces calpain activity, which could restore intercellular junction proteins thereby inhibiting EMT induced motility.

Topics: Antigens, CD; Breast Neoplasms; Cadherins; Calcium Signaling; Calpain; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Matrix Metalloproteinases; Membrane Potentials; Neoplasm Metastasis; Neoplasm Proteins; RNA Interference; Stromal Interaction Molecule 1; Time Factors; Transfection; Transforming Growth Factor beta; TRPC Cation Channels

An emerging characteristic of drug resistance in cancer is the induction of epithelial-mesenchymal transition (EMT). However, the mechanisms of EMT-mediated drug resistance remain poorly defined. Therefore, we conducted long-term treatments of human epidermal growth factor receptor-2 (Her2)-transformed breast cancer cells with either the EGFR/Her2 kinase inhibitor, Lapatinib or TGF-β, a known physiological inducer of EMT. Both of these treatment regimes resulted in robust EMT phenotypes, but upon withdrawal a subpopulation of TGF-β induced cells readily underwent mesenchymal-epithelial transition, where as Lapatinib-induced cells failed to reestablish an epithelial population. The mesenchymal population that remained following TGF-β stimulation and withdrawal was quickly selected for during subsequent Lapatinib treatment, manifesting in inherent drug resistance. The Nanostring cancer progression gene panel revealed a dramatic upregulation of fibroblast growth factor receptor 1 (FGFR1) and its cognate ligand FGF2 in both acquired and inherent resistance. Mechanistically, FGF:Erk1/2 signaling functions to stabilize the EMT transcription factor Twist and thus maintain the mesenchymal and drug resistant phenotype. Finally, Lapatinib resistant cells could be readily eliminated using recently characterized covalent inhibitors of FGFR. Overall our data demonstrate that next-generation targeting of FGFR can be used in combination with Her2-targeted therapies to overcome resistance in this breast cancer subtype.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Fibroblast Growth Factor 2; Humans; Hyaluronan Receptors; Lapatinib; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Proteins; Phenotype; Quinazolines; Receptor, Fibroblast Growth Factor, Type 1; Signal Transduction; Time Factors; Transforming Growth Factor beta; Twist-Related Protein 1; Xenograft Model Antitumor Assays

Transforming growth factor beta (TGFβ) signaling in breast cancer is selectively associated with pulmonary metastasis. However, the underlying mechanisms remain unclear. Here we show that Bcl-3, a member of the IκB family, serves as a critical regulator in TGFβ signaling to modulate breast cancer pulmonary metastasis. Bcl-3 expression was significantly associated with metastasis-free survival in breast cancer patients. Bcl-3 deletion inhibited the migration and invasion of breast cancer cells in vitro, as well as breast cancer lung metastasis in vivo. Bcl-3 was required for the expression of downstream TGFβ signaling genes that are involved in breast cancer lung metastasis. Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFβ treatment. Bcl-3 could bind to Smad3 and prevent the ubiquitination and degradation of Smad3 protein. These results indicate that Bcl-3 serves as a promising target to prevent breast tumor lung metastasis.

Topics: Animals; B-Cell Lymphoma 3 Protein; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Mice; Protein Stability; Proto-Oncogene Proteins; Signal Transduction; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta

GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGFβ, which is expressed naturally by platelets and regulatory T cells (Treg). Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here, we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGFβ in the tumor microenvironment. We found that human breast, lung, and colon cancers expressed GARP aberrantly. In genetic studies in normal mammary gland epithelial and carcinoma cells, GARP expression increased TGFβ bioactivity and promoted malignant transformation in immunodeficient mice. In breast carcinoma-bearing mice that were immunocompetent, GARP overexpression promoted Foxp3

Topics: Animals; Blotting, Western; Breast Neoplasms; Carcinogenesis; Female; Heterografts; Humans; Immunohistochemistry; Immunoprecipitation; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Polymerase Chain Reaction; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Escape

The transforming growth factor β (TGFβ) pathway plays critical roles during cancer cell epithelial-mesenchymal transition (EMT) and metastasis. SMAD7 is both a transcriptional target and a negative regulator of TGFβ signalling, thus mediating a negative feedback loop that may potentially restrain TGFβ responses of cancer cells. Here, however, we show that TGFβ treatment induces SMAD7 transcription but not its protein level in a panel of cancer cells. Mechanistic studies reveal that TGFβ activates the expression of microRNA-182 (miR-182), which suppresses SMAD7 protein. miR-182 silencing leads to SMAD7 upregulation on TGFβ treatment and prevents TGFβ-induced EMT and invasion of cancer cells. Overexpression of miR-182 promotes breast tumour invasion and TGFβ-induced osteoclastogenesis for bone metastasis. Furthermore, miR-182 expression inversely correlates with SMAD7 protein in human tumour samples. Therefore, our data reveal the miR-182-mediated disruption of TGFβ self-restraint and provide a mechanism to explain the unleashed TGFβ responses in metastatic cancer cells.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; RNA Interference; Signal Transduction; Smad7 Protein; Transcriptional Activation; Transforming Growth Factor beta

TGF-β has paradoxical effects on cancer cell proliferation, as it suppresses proliferation of normal epithelial and low-invasive cancer cells, but enhances that of high-invasive cancer cells. However, how cancer cells acquire the ability to evade the tumor-suppressing effects of TGF-β, yet still take advantage of its tumor-promoting effects, remains elusive. Here, we identified miR-106b as a molecular switch to determine TGF-β effects on cell proliferation. TGF-β1 enhances the transcription of miR-106b via a promoter independent of its host gene MCM7 by activating c-jun. In high-invasive breast cancer cells, miR-106b is upregulated by TGF-β1 at a much higher level than that in normal or low-invasive cancer cells. Accumulation of miR-106b counterbalances TGF-β growth-inhibiting effects by eliminating activated retinoblastoma (RB) and results in enhanced proliferation. Furthermore, miR-106b mediates TGF-β effects on tumor growth and metastasis in breast cancer xenografts. In addition, miR-106b expression is elevated in higher stage tumors and correlated with tumor progression in breast cancer patients. These findings suggest that high level of miR-106b induced by TGF-β determines the tumor-promoting effects of TGF-β in breast cancer.

Topics: Adult; Animals; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial Cells; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Transplantation; Promoter Regions, Genetic; Retinoblastoma Protein; Transcription, Genetic; Transforming Growth Factor beta

We have shown previously that withaferin A (WA), a bioactive component of the medicinal plant Withania somnifera, inhibits growth of cultured and xenografted human breast cancer cells and prevents breast cancer development and pulmonary metastasis incidence in a transgenic mouse model. The present study was undertaken to determine if the anticancer effect of WA involved inhibition of epithelial-mesenchymal transition (EMT). Experimental EMT induced by exposure of MCF-10A cells to tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β) was partially reversed by treatment with WA but not by its structural analogs withanone or withanolide A. Combined TNF-α and TGF-β treatments conferred partial protection against MCF-10A cell migration inhibition by WA. Inhibition of TNF-α and TGF-β-induced MCF-10A cell migration by WA exposure was modestly attenuated by knockdown of E-cadherin protein. MCF-7 and MDA-MB-231 cells exposed to WA exhibited sustained (MCF-7) or transient (MDA-MB-231) induction of E-cadherin protein. On the other hand, the level of vimentin protein was increased markedly after 24 h treatment of MDA-MB-231 cells with WA. WA-induced apoptosis was not affected by vimentin protein knockdown in MDA-MB-231 cells. Protein level of vimentin was significantly lower in the MDA-MB-231 xenografts as well as in MMTV-neu tumors from WA-treated mice compared with controls. The major conclusions of the present study are that (a) WA treatment inhibits experimental EMT in MCF-10A cells, and (b) mammary cancer growth inhibition by WA administration is associated with suppression of vimentin protein expression in vivo.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Mice, Nude; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vimentin; Withanolides

Aberrant glycosylation is a hallmark of most human cancers and affects many cellular properties, including cell proliferation, apoptosis, differentiation, transformation, migration, invasion, and immune responses. Here, we report that N-acetylgalactosaminyltransferase14 (GALNT14), which mediates the initial step of mucin-type O-glycosylation and is heterogeneously expressed in most breast cancers, plays a critical role in the invasion and migration of breast cancers by regulating the activity of MMP-2 and expression of some EMT genes. We have modulated the expression of GALNT14 by RNAi and overexpression in MCF-7 cells. Overexpression of GALNT14 significantly enhanced cell migration and invasion and promoted the proliferation of breast cancer cells. Knockdown of GALNT14 reduced clonogenicity and attenuates cell migration and cell invasion. The mRNAs for N-cadherin, vimentin, E-cadherin, MMP-2, VEGF, and TGF-β were determined by RT-qPCR involving GALNT14-overexpressing or knockdown MCF-7 cells. Expression profiling revealed the upregulation of N-cadherin, vimentin, MMP-2, VEGF, TGF-β and the downregulation of E-cadherin in GALNT14 overexpressing cells, with the opposite seen in GALNT14 knockdowns. Gelatin zymography analysis further indicated that overexpression of GALNT14 increased MMP-2 activity in MCF-7 cells. Conversely, downregulation of GALNT14 reduced MMP-2 activity. Promoter analysis revealed that GALNT14 stimulates MMP-2 expression through the AP-1-binding site. Western blot analyses showed that knockdown of GALNT14 significantly reduced the expression of an oncoprotein mucin 1 (MUC1). These findings indicate that GALNT14 contributes to breast cancer invasion by altering the cell proliferation, motility, expression levels of EMT genes, and by stimulating MMP-2 activity, suggesting GALNT14 may be a potential target for breast cancer treatment.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; MCF-7 Cells; Mucin-1; N-Acetylgalactosaminyltransferases; Neoplasm Invasiveness; Polypeptide N-acetylgalactosaminyltransferase; Promoter Regions, Genetic; Transcription Factor AP-1; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vimentin

The epithelial-mesenchymal transition (EMT) is one of the main mechanisms contributing to the onset of cancer metastasis, and has proven to be associated with breast cancer progression. SHON is a novel secreted hominoid-specific protein we have previously identified; it is specifically expressed in all human cancer cell lines tested and is oncogenic for human mammary carcinoma cells. Here, we show that ectopic overexpression of SHON in immortalized human mammary epithelial cells is sufficient for cells to acquire the mesenchymal traits, as well as the enhanced cell migration and invasion, along with the epithelial stem cell properties characterized by increased CD44(high) /CD24(low) subpopulation and mammosphere-forming ability. Moreover, we demonstrate that SHON positively activates the autocrine transforming growth factor-β (TGF-β) pathway to contribute to EMT, while SHON itself is induced by TGF-β in mammary epithelial cells. These data are in favor of a SHON-TGFβ-SHON-positive feedback loop that regulates EMT program in breast cancer progression. Finally, examination of the human clinic breast cancer specimens reveals that tumor cells may extracellularly release SHON protein to promote the cancerization of surrounding cells. Together, our findings define an important function of SHON in regulation of EMT via TGF-β signaling, which is closely associated with the invasive subtypes of human breast cancer.

Topics: Bone Remodeling; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Female; Humans; MAP Kinase Signaling System; Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Patient data suggest that colony-stimulating factor-1 (CSF1) and its receptor (CSF1R) have critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cell lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cell lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight-junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Claudins; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Macrophage Colony-Stimulating Factor; MCF-7 Cells; Mice; Neoplasm Invasiveness; Receptor, Macrophage Colony-Stimulating Factor; Transforming Growth Factor beta

Radiotherapy remains a prime approach to adjuvant therapies in patients with early and advanced breast cancer. In spite of therapeutic success, metastatic progression in patients undergoing therapy, limits its application. However, effective therapeutic strategies to understand the cellular and molecular machinery in inhibiting radiation-induced metastatic progression, which is poorly understood so far, need to be strengthened. Ionizing radiation was known to prompt cancer cell's metastatic ability by eliciting Transforming Growth Factor-beta (TGF-β), a key regulator in epithelial-mesenchymal transdifferentiation and radio-resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invasion reversal abilities in irradiated breast cancer cell lines by assessing their respective attributes. The role of metastasis regulatory molecules like TGF-β, E-cadherin, and integrin αV and its downstream molecules were determined using RT-PCR, western blotting, immunofluorescence, and extracellular TGF-β levels affirmed through ELISA assays. These studies affirmed the TGF-β restoring ability of thymoquinone in radiation-driven migration and invasion. Also, results demonstrated that the epithelial markers E-cadherin and cytokeratin 19 were downregulated whereas mesenchymal markers like integrin αV, MMP9, and MMP2 were upregulated by irradiation treatment; however thymoquinone pre-sensitization has reverted the expression of these proteins back to control proteins expression. Here, paclitaxel was chosen as an apoptosis inducer in TGF-β restored cells and confirmed its cytotoxic effects in radiation alone and thymoquinone sensitized irradiated cells. We conclude that this therapeutic modality is effective in preventing radiation-induced epithelial-mesenchymal transdifferentiation and concomitant induction of apoptosis in breast cancer.

Topics: Apoptosis; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Female; Humans; Paclitaxel; Radiation; Radiotherapy; Transforming Growth Factor beta

Increased expression of Pituitary Tumor Transforming Gene 1 (Pttg1) has been shown in various tumor cells, including breast cancer (BC). However, the precise role of Pttg1 in the tumorigenesis is not clarified yet. Here, we examined BC from the patients and detected significant increases and correlation in Pttg1 and phosphorylated SMAD3 (pSMAD3), a key effector of activated transforming growth factor β (TGFβ) receptor signaling pathway. Pttg1 levels were then modulated by transgene or small hairpin RNA (shRNA) in a human BC cell line, BT474, respectively. We found that Pttg1 overexpression increased the proliferation of BC cells in vitro and in vivo, while Pttg1 inhibition decreased proliferation of BC cells in vitro and in vivo. Moreover, phosphorylation of SMAD3 by TGFβ1 was significantly inhibited by Pttg1 overexpression, suggesting that Pttg1 may promote growth of BC cells by inhibiting pSMAD3-mediated cell-growth inhibition. Thus, Pttg1 appears to be a novel therapeutic target for controlling the tumorigenesis of BC.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Protein Processing, Post-Translational; Securin; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+)  CD25(+)  FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-β (TGF-β) production in tumour cells that essentially blocked TGF-β-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-β-induced Treg cell augmentation.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Case-Control Studies; Chemistry, Pharmaceutical; Coculture Techniques; Curcumin; Dose-Response Relationship, Drug; Female; Forkhead Transcription Factors; Humans; Interleukin-2 Receptor alpha Subunit; MAP Kinase Kinase Kinases; Mice, Inbred BALB C; Nanoparticles; Paracrine Communication; Protein Kinase Inhibitors; Signal Transduction; Smad Proteins; STAT Transcription Factors; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Escape; Tumor Microenvironment

Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Mammary Glands, Human; Mice; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Repressor Proteins; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

The invasion and chemoresistance are crucial causes of morbidity and death for cancer patients. Axl is closely associated with malignant phenotype of breast tumor cells, including invasiveness and metastasis. Both breast cancer cell line and tissue displayed increased expression of Axl, especially in highly metastatic breast cancer. On the contrary, experimental inhibition of Axl or transforming growth factor beta 1 (TGF-β1) by RNAi assay could suppress cell invasion ability and chemoresistance. Moreover, the up-regulation of Axl was induced by TGF-β1, further activated phosphatidylinositol 3-kinase (PI3K)/Akt and PAK1 translocation, and resulted in greater cell motility, invasion, and chemoresistance in vitro and in vivo. After the detection and statistics in human breast cancer specimens, we found that the Axl expression was closely correlated with TGF-β1 level, tumor differentiation, lymph node metastasis, and clinical stage (p < 0.01). Our findings support the possibility that Axl is a significant regulator of invasion and chemosensitivity, and it means by targeting Axl or its related signaling pathways, we can reduce the invasion and chemosensitivity of breast tumor.

Topics: Animals; Axl Receptor Tyrosine Kinase; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; MCF-7 Cells; Mice; Neoplasm Invasiveness; p21-Activated Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

The process of epithelial‑mesenchymal transition (EMT) is essential for the proliferation and metastasis of tumor cells. Our previous study demonstrated that the expression of gangliotetraosylceramide (Gg4) and the transcription of UDP‑Gal: β1,3-galactosyltransferase‑4 (β3GalT4), a gene which controls the expression of Gg4, are reduced during transforming growth factor (TGF)‑β‑induced EMT in normal murine mammary gland (NMuMG) cells. The present study revealed that the transcription level of β3GalT4 in patients with breast cancer was decreased compared with healthy control subjects. However, the molecular basis underlying these effects remains to be elucidated. Analysis of the β3GalT4 promoter sequence revealed a putative Smad‑binding element (SBE) of Smad4, which is a transcriptional factor in TGFβ responses and forms a complex with Smad3. To clarify the association between the Smad3/4 complex and the β3GalT4 gene during EMT in NMuMG cells, an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay were performed. The Smad3/4 complex was found to bind directly to the SBE of the β3GalT4 promoter. Overexpression of Smad3 and Smad4 through stable transfection had no notable effect on cell phenotype, but did alter the expression of various EMT protein markers. Treatment with TGFβ reduced the expression of Gg4 and the mRNA levels of the β3GalT4 gene in Smad3‑ and Smad4‑overexpressing cells compared with vector‑transfected cells. Expression of the epithelial markers E‑cadherin and β‑catenin decreased in parallel with the reduction in Gg4. These findings suggested that the activated Smad3/4 complex downregulated the expression of Gg4 and the β3GalT4 gene through translocation into the nucleus and binding to the β3GalT4 promoter.

Topics: Adult; Breast Neoplasms; Case-Control Studies; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Galactosyltransferases; Gangliosides; Gene Expression Regulation; Humans; Middle Aged; Neoplasm Staging; RNA, Messenger; Smad Proteins; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Burden

The cytotoxic activity and count of natural killer (NK) cells appear to be reduced after surgery; however, it is unknown whether the magnitude of this immune suppression is similar among different types of oncological surgery. In this study, we compared the innate immune function of patients undergoing three different oncological surgeries.. We compared the number and function of NK cells obtained from patients who had undergone mastectomies (n = 17), thoracotomies (n = 21), or liver resections for cancer (n = 22). Cytotoxicity assays were performed to measure the function of NK cells. We also determined the plasma concentrations of interleukins (IL) 2 and 4, interferon-γ, granzyme B, perforin, soluble major histocompatibility complex class I-related chain A, and epinephrine, both before and 24 h after surgery. Differences in immunologic parameters were compared preoperatively and postoperatively and by type of surgery. P values <0.05 were considered statistically significant.. The preoperative NK cell count differed statistically (P < 0.006) among all three types of surgeries; however, within surgery postoperative counts and changes compared with baseline did not. The postoperative function of NK cells was similar among types of surgeries, but was significantly reduced compared with preoperative levels (mastectomy P < 0.0001, thoracotomy P = 0.001, and liver resections P = 0.002). We observed a significant increase in the postoperative plasma concentrations of epinephrine, whereas the concentrations of major histocompatibility class I polypeptide-related sequence A and the IL-2 and/or IL-4 ratio remained unchanged before and after surgery.. The magnitude of innate immune suppression is similar among different oncological procedures. More studies are needed to better understand this complex phenomenon.

Topics: Adult; Aged; Breast Neoplasms; Colorectal Neoplasms; Cytokines; Epinephrine; Female; Humans; Immunity, Innate; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial-mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2'-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion.

Topics: Antigens, CD; Azacitidine; Breast Neoplasms; Cadherins; CCCTC-Binding Factor; Cell Line, Tumor; Cell Movement; Cells, Cultured; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Epithelial-Mesenchymal Transition; Female; Gene Silencing; HMGA2 Protein; Humans; Promoter Regions, Genetic; Repressor Proteins; Transforming Growth Factor beta

Elucidating the mechanisms that underlie metastasis is of paramount importance to understanding tumor progression and to the development of novel therapeutics. Epithelial to Mesenchymal Transition (EMT) plays a vital role in tumor cell dissemination and is regulated by a core cassette of transcription factors. Despite recent advances, the molecular pathways that regulate the EMT program have not yet been fully delineated. We show that Siah ubiquitin ligases regulate Zeb1 protein, a key EMT transcription factor. The induction of EMT in breast cancer cells leads to the down-regulation of Siah, while the loss of Siah induces a mesenchymal phenotype, concurrent with an up-regulation of Zeb1. Overexpression of Siah in vitro mediates Zeb1 degradation, which can be blocked with a Siah peptide inhibitor. Thus, this work demonstrates that Siah is a novel regulator of EMT. This work is the first to identify a mechanism of post-translational regulation of the key Epithelial to Mesenchymal Transition transcription factor Zeb1.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; MCF-7 Cells; Mice; Microscopy, Fluorescence; Models, Genetic; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor alpha; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Zinc Finger E-box-Binding Homeobox 1

A variety of biophysical forces are altered in the tumor microenvironment (TME) and these forces can influence cancer progression. One such force is interstitial fluid flow (IFF)-the movement of fluid through the tissue matrix. IFF was previously shown to induce invasion of cancer cells, but the activated signaling cascades remain poorly understood. Here, it is demonstrated that IFF induces invasion of ERBB2/HER2-expressing breast cancer cells via activation of phosphoinositide-3-kinase (PI3K). In constitutively activate ERBB2-expressing cells that have undergone epithelial-to-mesenchymal transition (EMT), IFF-mediated invasion requires the chemokine receptor CXCR4, a gradient of its ligand CXCL12, and activity of the PI3K catalytic subunits p110α and β. In wild-type ERBB2-expressing cells, IFF-mediated invasion is chemokine receptor-independent and requires only p110α activation. To test whether cells undergoing EMT alter their signaling response to IFF, TGFβ1 was used to induce EMT in wild-type ERBB2-expressing cells, resulting in IFF-induced invasion dependent on CXCR4 and p110β.. This study identifies a novel signaling mechanism for interstitial flow-induced invasion of ERBB2-expressing breast cancer cells, one that depends on EMT and acts through a CXCR4-PI3K pathway. These findings suggest that the response of cancer cells to interstitial flow depends on EMT status and malignancy.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Fluid; Female; Humans; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Receptor, ErbB-2; Receptors, CXCR4; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Topics: Animals; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Mice; Neoplasm Invasiveness; RNA, Long Noncoding; Transcriptome; Transforming Growth Factor beta

The translation initiation factor eIF4E is an oncogene that is commonly overexpressed in primary breast cancers and metastases. In this article, we report that a pharmacologic inhibitor of eIF4E function, ribavirin, safely and potently suppresses breast tumor formation. Ribavirin administration blocked the growth of primary breast tumors in several murine models and reduced the development of lung metastases in an invasive model. Mechanistically, eIF4E silencing or blockade reduced the invasiveness and metastatic capability of breast cancer cells in a manner associated with decreased activity of matrix metalloproteinase (MMP)-3 and MMP-9. Furthermore, eIF4E silencing or ribavirin treatment suppressed features of epithelial-to-mesenchymal transition, a process crucial for metastasis. Our findings offer a preclinical rationale to explore broadening the clinical evaluation of ribavirin, currently being tested in patients with eIF4E-overexpressing leukemia, as a strategy to treat solid tumors such as metastatic breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-4E; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Ribavirin; Transforming Growth Factor beta

Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness.. Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition.. Our results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECs(Mes)) in vitro. The ECs(Mes) acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECs(Mes) were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECs(Mes) were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFβ and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFβ down-regulated Smad1/5 phosphorylation and Jag1(KD) tumor cells were unable to initiate the process.. Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFβ pathways in the initiation of mesenchymal phenotype may propose new stromal targets.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Mesoderm; Mice; Mice, Inbred NOD; Mice, SCID; Phenotype; Receptors, Notch; Signal Transduction; Transcriptome; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays

Cathepsin L, a lysosomal acid cysteine protease, was found to be overexpressed in several types of human carcinomas. However, its functional roles in tumor progression and the underlying mechanisms remain largely unclear. In the present study, we investigated a novel functional aspect of cathepsin L in regulating transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT) in A549 and MCF‑7 cells and examined its possible mechanisms. We found that TGF‑β‑induced cell morphologic changes of EMT were associated with the increased protein level of cathepsin L in A549 and MCF‑7 cells, suggesting that cathepsin L may be involved in the regulation of EMT. Furthermore, we showed that silencing of cathepsin L blocked TGF‑β‑induced cell migration, invasion and actin remodeling and inhibited TGF‑β‑mediated EMT. We also demonstrated that the mechanism of how cathepsin L knockdown regulates EMT may be explained by the suppression of EMT‑inducing molecules, such as Snail, which is associated with the phosphatidylinositol 3‑kinase (PI3K)‑AKT and Wnt signaling pathways. Moreover, we proved that cathepsin L knockdown in A549 cells significantly inhibited xenograft tumor growth and EMT in vivo. The results showed a new mechanism to determine cathepsin L involvement in the regulation of cancer invasion and migration. These results showed that cathepsin L knockdown is important in regulating EMT and suggest that cathepsin L may be utilized as a new target for enhancing the efficacy of chemotherapeutics against epithelial cancer.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cathepsin L; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Heterografts; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Transplantation; RNA, Bacterial; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Breast cancer represents the second leading cause of cancer-related deaths among women worldwide and preventive therapy could reverse or delay the devastating impact of this disease. Ellagic acid (EA), a dietary flavonoid polyphenol which is present in abundance in pomegranate, muscadine grapes, walnuts and strawberries, has been shown to inhibit cancer cells proliferation and induce apoptosis. Here, we investigated the growth inhibitory effects of EA on MCF-7 breast cancer cells. In the present study, we first found that EA inhibits the proliferation of MCF-7 breast cancer cells mainly mediated by arresting cell cycle in the G0/G1 phase. Moreover, gene expression profiling of MCF-7 breast cancer cell line treated with EA for 6, 12 and 24 h was performed using cDNA microarray. A total of 4,738 genes were found with a >2.0-fold change after 24 h of EA treatment. Among these genes, 2,547 were downregulated and 2,191 were upregulated. Furthermore, the changes of 16 genes, which belong to TGF-β/Smads signaling pathway, were confirmed by real-time RT-PCR and/or western blot analysis. TGF-β/Smads signaling pathway was found as the potential molecular mechanism of EA to regulate breast cancer cell cycle arrest in vitro. Therefore, the regulation of TGF-β/Smads pathway in breast cancer cells could be a novel therapeutic approach for the treatment of patients with breast cancer. Further studies with in vitro models, as well as an analysis of additional human samples, are still needed to confirm the molecular mechanisms of EA in inhibition or prevention of breast cancer growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Ellagic Acid; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Oligonucleotide Array Sequence Analysis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) and oncogenic metabolism (including glycolytic switch) are important for tumor development and progression. Here, we show that Dlx-2, one of distal-less (Dlx) homeobox genes, induces EMT and glycolytic switch by activation of Snail. In addition, it was induced by TGF-β and Wnt and regulates TGF-β- and Wnt-induced EMT and glycolytic switch by activating Snail. We also found that TGF-β/Wnt suppressed cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, in a Dlx-2/Snail-dependent manner. TGF-β/Wnt appeared to downregulate the expression of various COX subunits including COXVIc, COXVIIa and COXVIIc; among these COX subunits, COXVIc was a common target of TGF-β, Wnt, Dlx-2 and Snail, indicating that COXVIc downregulation plays an important role(s) in TGF-β/Wnt-induced COX inhibition. Taken together, our results showed that Dlx-2 is involved in TGF-β- and Wnt-induced EMT, glycolytic switch, and mitochondrial repression by Snail activation.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Dogs; Epithelial-Mesenchymal Transition; Female; Glycolysis; HCT116 Cells; Homeodomain Proteins; Humans; Madin Darby Canine Kidney Cells; MCF-7 Cells; Mitochondria; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Wnt Signaling Pathway

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of G1 cell cycle progression. Two key substrates of mTORC1 are ribosomal subunit S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). We reported previously that simultaneous knockdown of S6K and eIF4E causes a transforming growth factor-β (TGF-β)-dependent G1 cell cycle arrest in MDA-MB-231 human breast cancer cells. Rapamycin inhibits the phosphorylation of S6K at nano-molar concentrations in MDA-MB-231 cells; however, micro-molar concentrations of rapamycin are required to inhibit phosphorylation of 4E-BP1 - the phosphorylation of which liberates eIF4E to initiate translation. Micro-molar doses of rapamycin are required for complete G1 cell cycle arrest - indicating that 4E-BP1 is a critical target of mTOR for promoting cell cycle progression. Data are provided demonstrating that G1 cell cycle arrest induced by rapamycin is due to up-regulation of TGF-β signaling and down-regulation of Rb phosphorylation via phosphorylation of the mTORC1 substrates S6K and 4E-BP1 respectively. These findings enhance the current understanding of the cytostatic effects of mTORC1 suppression with therapeutic implications.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phosphorylation; Retinoblastoma Protein; Sirolimus; Transforming Growth Factor beta

TGF-β/SMAD signaling has long been known to exhibit a dual role in cancer, questioning what determines its context-dependent functions. In this issue of Cancer Cell, Xu and colleagues describe a critical role of the adaptor protein 14-3-3ζ in modulating SMAD activities by changing its interaction partners during breast cancer progression.

Topics: 14-3-3 Proteins; Bone Neoplasms; Breast Neoplasms; Female; Humans; Kruppel-Like Transcription Factors; Nuclear Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Zinc Finger Protein Gli2

Transforming growth factor β (TGF-β) functions as a tumor suppressor in premalignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effector Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in premalignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β's tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression.

Topics: 14-3-3 Proteins; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Kruppel-Like Transcription Factors; Molecular Targeted Therapy; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Zinc Finger Protein Gli2

Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects.. Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided.. DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors.. MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.

Topics: Amidines; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Benzylamines; Breast Neoplasms; Cell Line, Tumor; Dose Fractionation, Radiation; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin G; Macrophages; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 14; Mice; Neovascularization, Pathologic; Nitric Oxide Synthase Type II; Phenotype; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Metastasis is a process where only a small subset of cells is capable of successfully migrating to and propagating at secondary sites. TGF-β signalling is widely known for its role in cancer metastasis and is associated with cell migration in whole cell populations.. We extend these findings by investigating the role of TGF-β signalling in promoting migration and motility by imaging the signalling activity in live, individual MDA-MB-231 cancer cells utilizing a novel Smad3 Td-Tomato reporter adenovirus. Here we find that not all MDA-MB-231 cancer cells have similar TGF-β mediated Smad3 transcription activity and display at least two distinct migratory populations. Importantly, Smad3 activity was significantly higher within migratory cells compared to non-migrated cells in wound healing and transwell assays. Furthermore, time-lapse experiments showed that MDA-MB-231 cells displaying Smad3 activity moved faster and a greater distance compared to cells not displaying Smad3 reporter activity. Interestingly, despite being more motile than cells with undetectable levels of Smad3 activity, high Smad3 activity was detrimental to cell motility compared to low and medium level of Smad3 activity.. We have developed a method enabling real-time visualization of TGF-β signalling in single live cells. Breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-β-Smad3 activity. Those sub-populations may be responsible for tumor invasion and metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Signal Transduction; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Kaplan-Meier Estimate; Lung Neoplasms; Mice, Inbred C57BL; Neoplastic Stem Cells; Paracrine Communication; Proto-Oncogene Proteins; Smad2 Protein; Transcriptome; Transforming Growth Factor beta; Wnt Proteins; Wnt-5a Protein

Transforming growth factor-β (TGF-β) is an important regulator of breast cancer progression. However, how the breast cancer microenvironment regulates TGF-β signaling during breast cancer progression remains largely unknown. Here, we identified fibulin-3 as a secreted protein in the breast cancer microenvironment, which efficiently inhibits TGF-β signaling in both breast cancer cells and endothelial cells. Mechanistically, fibulin-3 interacts with the type I TGF-β receptor (TβRI) to block TGF-β induced complex formation of TβRI with the type II TGF-β receptor (TβRII) and subsequent downstream TGF-β signaling. Fibulin-3 expression decreases during breast cancer progression, with low fibulin-3 levels correlating with a poorer prognosis. Functionally, high fibulin-3 levels inhibited TGF-β-induced epithelial-mesenchymal transition (EMT), migration, invasion and endothelial permeability, while loss of fibulin-3 expression/function promoted these TGF-β-mediated effects. Further, restoring fibulin-3 expression in breast cancer cells inhibited TGF-β signaling, breast cancer cell EMT, invasion and metastasis in vivo. These studies provide a novel mechanism for how TGF-β signaling is regulated by the tumor microenvironment, and provide insight into targeting the TGF-β signaling pathway in human breast cancer patients.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The development of breast cancer is linked to the loss of estrogen receptor (ER) during the course of tumor progression, resulting in loss of responsiveness to hormonal treatment. The mechanisms underlying dynamic ERα gene expression change in breast cancer remain unclear. A range of physiological and biological changes, including increased adipose tissue hypoxia, accompanies obesity. Hypoxia in adipocytes can establish a pro-malignancy environment in breast tissues. Epidemiological studies have linked obesity with basal-like breast cancer risk and poor disease outcome, suggesting that obesity may affect the tumor phenotype by skewing the microenvironment toward support of more aggressive tumor phenotypes. In the present study, human SGBS adipocytes were co-cultured with ER-positive MCF7 cells for 24 h. After co-culture, HIF1α, TGF-β, and lectin-type oxidized LDL receptor 1 (LOX1) mRNA levels in the SGBS cells were increased. Expression levels of the epithelial-mesenchymal transition (EMT)-inducing transcription factors FOXC2 and TWIST1 were increased in the co-cultured MCF7 cells. In addition, the E-cadherin mRNA level was decreased, while the N-cadherin mRNA level was increased in the co-cultured MCF7 cells. ERα mRNA levels were significantly repressed in the co-cultured MCF7 cells. ERα gene expression in the MCF7 cells was decreased due to increased HIF1α in the SGBS cells. These results suggest that adipocytes can modify breast cancer cell ER gene expression through hypoxia and also can promote EMT processes in breast cancer cells, supporting an important role of obesity in aggressive breast cancer development.

Topics: Adipocytes; Breast Neoplasms; Cell Hypoxia; Cell Proliferation; Coculture Techniques; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MCF-7 Cells; Nuclear Proteins; Obesity; Scavenger Receptors, Class E; Transforming Growth Factor beta; Tumor Microenvironment; Twist-Related Protein 1

Increased expression of αv integrins is frequently associated with tumor cell adhesion, migration, invasion and metastasis, and correlates with poor prognosis in breast cancer. However, the mechanism by which αv integrins can enhance breast cancer progression is still largely unclear. The effects of therapeutic targeting of αv integrins in breast cancer also have yet to be investigated.. We knocked down αv integrin in MDA-MB-231 and MCF10A-M4 breast cancer cells, or treated these cells with the αv antagonist GLPG0187. The effects of αv integrin depletion on mesenchymal markers, transforming growth factor-β (TGF-β)/Smad signaling and TGF-β-induced target gene expression were analyzed in MDA-MB-231 cells by RNA analysis or Western blotting. The function of αv integrin on breast cancer cell migration was investigated by transwell assay in vitro, and its effect on breast cancer progression was assessed by both zebrafish and mouse xenografts in vivo. In the mouse model, GLPG0187 was administered separately, or in combination with the standard-of-care anti-resorptive agent zoledronate and the chemotherapeutic drug paclitaxel, to study the effects of combinational treatments on breast cancer metastasis.. Genetic interference and pharmacological targeting of αv integrin with GLPG0187 in different breast cancer cell lines inhibited invasion and metastasis in the zebrafish or mouse xenograft model. Depletion of αv integrin in MDA-MB-231 cells inhibited the expression of mesenchymal markers and the TGF-β/Smad response. TGF-β induced αv integrin mRNA expression and αv integrin was required for TGF-β-induced breast cancer cell migration. Moreover, treatment of MDA-MB-231 cells with non-peptide RGD antagonist GLPG0187 decreased TGF-β signaling. In the mouse xenografts GLPG0187 inhibited the progression of bone metastasis. Maximum efficacy of inhibition of bone metastasis was achieved when GLPG0187 was combined with the standard-of-care metastatic breast cancer treatments.. These findings show that αv integrin is required for efficient TGF-β/Smad signaling and TGF-β-induced breast cancer cell migration, and for maintaining a mesenchymal phenotype of the breast cancer cells. Our results also provide evidence that targeting αv integrin could be an effective therapeutic approach for treatment of breast cancer tumors and/or metastases that overexpress αv integrin.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Integrin alphaV; Mice; Neoplasm Metastasis; Transforming Growth Factor beta; Xenograft Model Antitumor Assays; Zebrafish

T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T-cell activity in patients who naturally develop them. Eliciting T-cell responses to a patient's individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear whether it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFβ activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here, we show that antibody-mediated TGFβ neutralization during radiation therapy effectively generates CD8(+) T-cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and nonirradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNγ and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFβ blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection, resulting in rapid recurrence. Addition of anti-PD-1 antibodies extended survival achieved with radiation and TGFβ blockade. Thus, TGFβ is a fundamental regulator of radiation therapy's ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFβ neutralization offers a novel individualized strategy for vaccinating patients against their tumors.

Topics: Animals; Antibodies, Neutralizing; Antigens, Neoplasm; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Disease Models, Animal; Female; Humans; Immunotherapy; Mice; Neoplasm Recurrence, Local; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Acetylsalicylic acid (ASA), also known as aspirin, a classic, nonsteroidal, anti-inflammatory drug (NSAID), is widely used to relieve minor aches and pains and to reduce fever. Epidemiological studies and other experimental studies suggest that ASA use reduces the risk of different cancers including breast cancer (BC) and may be used as a chemopreventive agent against BC and other cancers. These studies have raised the tempting possibility that ASA could serve as a preventive medicine for BC. However, lack of in-depth knowledge of the mechanism of action of ASA reshapes the debate of risk and benefit of using ASA in prevention of BC. Our studies, using in vitro and in vivo tumor xenograft models, show a strong beneficial effect of ASA in the prevention of breast carcinogenesis. We find that ASA not only prevents breast tumor cell growth in vitro and tumor growth in nude mice xenograft model through the induction of apoptosis, but also significantly reduces the self-renewal capacity and growth of breast tumor-initiating cells (BTICs)/breast cancer stem cells (BCSCs) and delays the formation of a palpable tumor. Moreover, ASA regulates other pathophysiological events in breast carcinogenesis, such as reprogramming the mesenchymal to epithelial transition (MET) and delaying in vitro migration in BC cells. The tumor growth-inhibitory and reprogramming roles of ASA could be mediated through inhibition of TGF-β/SMAD4 signaling pathway that is associated with growth, motility, invasion, and metastasis in advanced BCs. Collectively, ASA has a therapeutic or preventive potential by attacking possible target such as TGF-β in breast carcinogenesis.

Topics: Adenocarcinoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Breast Neoplasms; Cell Movement; Cell Survival; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Mice, Nude; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The molecular mechanisms of hypoxia induced breast cell migration remain incompletely understood. Our results show that hypoxia through hypoxia-inducible factor (HIF) brings about a time-dependent increase in the level of an oncogenic microRNA, miR-191 in various breast cancer cell lines. miR-191 enhances breast cancer aggressiveness by promoting cell proliferation, migration and survival under hypoxia. We further established that miR-191 is a critical regulator of transforming growth factor beta (TGFβ)-signaling and promotes cell migration by inducing TGFβ2 expression under hypoxia through direct binding and indirectly by regulating levels of a RNA binding protein, human antigen R (HuR). The levels of several TGFβ pathway genes (like VEGFA, SMAD3, CTGF and BMP4) were found to be higher in miR-191 overexpressing cells. Lastly, anti-miR-191 treatment given to breast tumor spheroids led to drastic reduction in spheroid tumor volume. This stands as a first report of identification of a microRNA mediator that links hypoxia and the TGFβ signaling pathways, both of which are involved in regulation of breast cancer metastasis. Together, our results show a critical role of miR-191 in hypoxia-induced cancer progression and suggest that miR-191 inhibition may offer a novel therapy for hypoxic breast tumors.

Topics: Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Progression; ELAV-Like Protein 1; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1; MicroRNAs; RNA Interference; RNA, Messenger; Signal Transduction; Spheroids, Cellular; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Cells, Cultured; Tumor Microenvironment

Tenascin-W is a matricellular protein with a dynamically changing expression pattern in development and disease. In adults, tenascin-W is mostly restricted to stem cell niches, and is also expressed in the stroma of solid cancers. Here, we analyzed its expression in the bone microenvironment of breast cancer metastasis. Osteoblasts were isolated from tumor-free or tumor-bearing bones of mice injected with MDA-MB231-1833 breast cancer cells. We found a fourfold upregulation of tenascin-W in the osteoblast population of tumor-bearing mice compared to healthy mice, indicating that tenascin-W is supplied by the bone metastatic niche. Transwell and co-culture studies showed that human bone marrow stromal cells (BMSCs) express tenascin-W protein after exposure to factors secreted by MDA-MB231-1833 breast cancer cells. To study tenascin-W gene regulation, we identified and analyzed the tenascin-W promoter as well as three evolutionary conserved regions in the first intron. 5'RACE analysis of mRNA from human breast cancer, glioblastoma and bone tissue showed a single tenascin-W transcript with a transcription start site at a noncoding first exon followed by exon 2 containing the ATG translation start. Site-directed mutagenesis of a SMAD4-binding element in proximity of the TATA box strongly impaired promoter activity. TGFβ1 induced tenascin-W expression in human BMSCs through activation of the TGFβ1 receptor ALK5, while glucocorticoids were inhibitory. Our experiments show that tenascin-W acts as a niche component for breast cancer metastasis to bone by supporting cell migration and cell proliferation of the cancer cells.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Female; Humans; Mice; Neoplasm Transplantation; Osteoblasts; Signal Transduction; Stromal Cells; Tenascin; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation

Trastuzumab resistance is leading cause of mortality in HER2-positive breast cancers, and the role of TGF-β-induced epithelial-mesenchymal transition (EMT) in trastuzumab resistance is well established, but the involvement of lncRNAs in trastuzumab resistance is still unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-β signaling in these cells. We identified long noncoding RNA activated by TGF-β (lnc-ATB) was the most remarkably upregulated lncRNA in TR SKBR-3 cells and the tissues of TR breast cancer patients. We found that lnc-ATB could promote trastuzumab resistance and invasion-metastasis cascade in breast cancer by competitively biding miR-200c, up-regulating ZEB1 and ZNF-217, and then inducing EMT. In addition, we also found that the high level of lnc-ATB was correlated with trastuzumab resistance of breast cancer patients. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose breast cancer patients to EMT and trastuzumab resistance.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; MicroRNAs; Neoplasm Invasiveness; Phenotype; RNA Interference; RNA, Long Noncoding; Signal Transduction; Time Factors; Trans-Activators; Transcription Factors; Transfection; Transforming Growth Factor beta; Trastuzumab; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration.. Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1-inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor.. A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralising the above growth factors.. These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies.

Topics: Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Cell Movement; Coculture Techniques; Epithelial-Mesenchymal Transition; Female; Fibroblast Growth Factors; Fibroblasts; Hepatocyte Growth Factor; Humans; MCF-7 Cells; Paracrine Communication; RNA, Messenger; RNA, Small Interfering; Stearoyl-CoA Desaturase; Transforming Growth Factor beta

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Topics: Biomechanical Phenomena; Biophysical Phenomena; Birefringence; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Collagen; Disease Progression; Extracellular Matrix; Female; Humans; Macrophages; Microscopy, Atomic Force; Microscopy, Fluorescence, Multiphoton; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta

Ductal carcinoma is one of the most common cancers among women, and the main cause of death is the formation of metastases. The development of metastases is caused by cancer cells that migrate from the primary tumour site (the mammary duct) through the blood vessels and extravasating they initiate metastasis. Here, we propose a multi-compartment model which mimics the dynamics of tumoural cells in the mammary duct, in the circulatory system and in the bone. Through a branching process model, we describe the relation between the survival times and the four markers mainly involved in metastatic breast cancer (EPCAM, CD47, CD44 and MET). In particular, the model takes into account the gene expression profile of circulating tumour cells to predict personalised survival probability. We also include the administration of drugs as bisphosphonates, which reduce the formation of circulating tumour cells and their survival in the blood vessels, in order to analyse the dynamic changes induced by the therapy. We analyse the effects of circulating tumour cells on the progression of the disease providing a quantitative measure of the cell driver mutations needed for invading the bone tissue. Our model allows to design intervention scenarios that alter the patient-specific survival probability by modifying the populations of circulating tumour cells and it could be extended to other cancer metastasis dynamics.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Computer Simulation; Disease Progression; Female; Gene Expression Profiling; Humans; Kaplan-Meier Estimate; Models, Biological; Neoplastic Cells, Circulating; Survival Rate; Transforming Growth Factor beta

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity.

Topics: beta Catenin; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Eukaryotic Initiation Factor-4E; Female; Humans; Integrin alpha3beta1; Integrin beta1; Neoplasm Invasiveness; Phosphorylation; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor-beta (TGF-β) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-β. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-β production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-β production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.

Topics: Breast Neoplasms; CD40 Antigens; CD40 Ligand; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Female; Humans; Interleukin-17; Lymphocyte Activation; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; STAT3 Transcription Factor; T-Lymphocytes; Th17 Cells; Transforming Growth Factor beta; Up-Regulation

All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Receptor, Notch1; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tretinoin

PMEPA1 is induced by TGFβ and inhibits TGFβ signaling to suppress prostate cancer bone metastasis.

Topics: Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Prognosis; Prostatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

The eIF3e protein is a component of the multisubunit eIF3 complex, which is essential for cap-dependent translation initiation. Decreased eIF3e expression is often observed in breast and lung cancer and has been shown to induce epithelial-to-mesenchymal transition (EMT) in breast epithelial cells by an unknown mechanism. Here, we study the effect of decreased eIF3e expression in lung epithelial cells by creating stable clones of lung epithelial cells (A549) that express an eIF3e-targeting shRNA. Our data indicate that decreased eIF3e expression in lung epithelial cells leads to EMT, as it does in breast epithelial cells. Importantly, we show that decreased eIF3e expression in both lung and breast epithelial cells leads to the overproduction of the TGFβ cytokine and that inhibition of TGFβ signaling can reverse eIF3e-regulated EMT in lung epithelial cells. In addition, we discovered that several mRNAs that encode important EMT regulators are translated by a cap-independent mechanism when eIF3e levels are reduced. These findings indicate that EMT mediated by a decrease in eIF3e expression may be a general phenomenon in epithelial cells and that it requires activation and maintenance of the TGFβ signaling pathway.. These results indicate that inhibition of TGFβ signaling could be an efficient way to prevent metastasis in patients with NSCLC that display reduced eIF3e expression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-3; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

Bone metastasis affects more than 70% of advanced breast cancer patients, but the molecular mechanisms of this process remain unclear. Here, we present clinical and experimental evidence to clarify the role of the integrin β-like 1 (ITGBL1) as a key contributor to bone metastasis of breast cancer. In an in vivo model system and in vitro experiments, ITGBL1 expression promoted formation of osteomimetic breast cancers, facilitating recruitment, residence, and growth of cancer cells in bone microenvironment along with osteoclast maturation there to form osteolytic lesions. Mechanistic investigations identified the TGFβ signaling pathway as a downstream effector of ITGBL1 and the transcription factor Runx2 as an upstream activator of ITGBL1 expression. In support of these findings, we also found that ITGBL1 was an essential mediator of Runx2-induced bone metastasis of breast cancer. Overall, our results illuminate how bone metastasis occurs in breast cancer, and they provide functional evidence for new candidate biomarkers and therapeutic targets to identify risk, to prevent, and to treat this dismal feature of advanced breast cancer.

Topics: Animals; Blotting, Western; Bone Neoplasms; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; Cell Survival; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Kaplan-Meier Estimate; Mice, Inbred BALB C; Mice, SCID; Osteoblasts; Osteogenesis; Osteolysis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transforming Growth Factor beta

Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their "M1/M2" activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0 .032), moist desquamation (p = 0 .027), and CTC grade (p = 0 .056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0 .069), moist desquamation (p = 0 .037), and CTC grade (p = 0 .046). To conclude, our findings underline for the first time the biological significance of increased ARG1 mRNA levels as an early independent predictive biomarker of RT-induced acute skin toxicities.

Topics: Arginase; Breast Neoplasms; Carcinoma, Ductal, Breast; Cells, Cultured; Female; Humans; Macrophages; Middle Aged; Nitric Oxide Synthase Type II; Prognosis; Radiation Injuries; Skin Diseases; Transforming Growth Factor beta

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 (Iβ3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype.

Topics: Adenocarcinoma; Animals; Bone Neoplasms; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Elastic Modulus; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin beta3; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Nuclear Proteins; Osteolysis; Pliability; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays; Zinc Finger Protein Gli2

Transforming growth factor beta (TGF-β) readily suppresses the development of early-stage breast cancers, an activity that gives way to tumor promotion in their late-stage counterparts. The molecular mechanisms underlying this mysterious switch in TGF-β function remain murky. In addressing this conundrum, Xu et al. observed aberrant 14-3-3ζ expression to prevent the formation of tumor-suppressive Smad2/3:p53 complexes, while simultaneously driving the generation of oncogenic Smad2/3:Gli2 complexes. Once formed, Smad2/3:Gli2 complexes stimulate the expression of parathyroid hormone-related protein necessary for breast cancer metastasis to bone. This viewpoint highlights 14-3-3ζ as an essential driver of oncogenic signaling by Smad2/3 and TGF-β in metastatic breast cancers.

Topics: 14-3-3 Proteins; Bone Neoplasms; Breast Neoplasms; Female; Humans; Kruppel-Like Transcription Factors; Nuclear Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Transforming growth factor-β (TGF-β) has both tumor suppressive and oncogenic activities. Autocrine TGF-β signaling supports tumor survival and growth in certain types of cancer, and the TGF-β signaling pathway is a potential therapeutic target for these types of cancer. TGF-β induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-β signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-β signaling using siRNAs targeting TGF-β signaling components and SB431542, a TGF-β receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-β signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-β signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-β signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

Topics: Animals; Autocrine Communication; Benzamides; Breast Neoplasms; Caco-2 Cells; Cell Proliferation; Cellular Senescence; Chlorocebus aethiops; COS Cells; Cyclin-Dependent Kinase Inhibitor p21; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; MCF-7 Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Sp1 Transcription Factor; Time Factors; Transfection; Transforming Growth Factor beta

Previous studies suggested that bisphosphonate zoledronic acid exerts an anti-tumor effect by interacting with the microenvironment. In this study, we aimed to elucidate the mechanism behind the anti-breast cancer effect of zoledronic acid.Here we showed that zoledronic acid did not influence in vitro human breast cancer cell survival, but did affect human stromal cell survival. Breast cancer cell death in co-culture with stromal cells was analyzed in vitro by fluorescent microscopy and flowcytometry analysis. In co-culture, the addition of stromal cells to breast cancer cells induced tumor cell death by zoledronic acid, which was abolished by transforming growth factor (TGF)-β. In the in vivo chicken chorioallantoic membrane model, zoledronic acid reduced the breast cancer cells fraction per tumor only in the presence of human stromal cells. Zoledronic acid decreased TGF-β excretion by stromal cells and co-cultures. Moreover, supernatant of zoledronic acid treated stromal cells reduced phospho-Smad2 protein levels in breast cancer cells. Thus, zoledronic acid exerts an anti-breast cancer effect via stromal cells, accompanied by decreased stromal TGF-β excretion and reduced TGF-β signaling in cancer cells.

Topics: Animals; Antineoplastic Agents; Bone Density Conservation Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chickens; Chorioallantoic Membrane; Coculture Techniques; Diphosphonates; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inhibitory Concentration 50; MCF-7 Cells; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; Smad2 Protein; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment; Zoledronic Acid

The contribution of Ca(2+) in TGF-β-induced EMT is poorly understood. We aimed to confirm the effect of TGF-β on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-β-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-β. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-β induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-β induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-β treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP3Rs were significantly changed due to TGF-β-induced EMT. The TGF-β-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-β-treated cells than in control cells. This is the first report to demonstrate that TGF-β-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis.

Topics: Breast Neoplasms; Calcium; Calcium Channels; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; MCF-7 Cells; Organelles; RNA; Transforming Growth Factor beta

Transforming Growth Factor β (TGFβ) has dual functions as both a tumor suppressor and a promoter of cancer progression within the tumor microenvironment, but the molecular mechanisms by which TGFβ signaling switches between these outcomes and the contexts in which this switch occurs remain to be fully elucidated. We previously identified PEAK1 as a new non-receptor tyrosine kinase that associates with the cytoskeleton, and facilitates signaling of HER2/Src complexes. We also showed PEAK1 functions downstream of KRas to promote tumor growth, metastasis and therapy resistance using preclinical in vivo models of human tumor progression. In the current study, we analyzed PEAK1 expression in human breast cancer samples and found PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse. At the cellular level, we also observed that PEAK1 expression was highest in mesenchymal breast cancer cells, correlated with migration potential and increased in response to TGFβ-induced epithelial-mesenchymal transition (EMT). Thus, we sought to evaluate the role of PEAK1 in the switching of TGFβ from a tumor suppressing to tumor promoting factor. Notably, we discovered that high PEAK1 expression causes TGFβ to lose its anti-proliferative effects, and potentiates TGFβ-induced proliferation, EMT, cell migration and tumor metastasis in a fibronectin-dependent fashion. In the presence of fibronectin, PEAK1 caused a switching of TGFβ signaling from its canonical Smad2/3 pathway to non-canonical Src and MAPK signaling. This report is the first to provide evidence that PEAK1 mediates signaling cross talk between TGFβ receptors and integrin/Src/MAPK pathways and that PEAK1 is an important molecular regulator of TGFβ-induced tumor progression and metastasis in breast cancer. Finally, PEAK1 overexpression/upregulation cooperates with TGFβ to reduce breast cancer sensitivity to Src kinase inhibition. These findings provide a rational basis to develop therapeutic agents to target PEAK1 expression/function or upstream/downstream pathways to abrogate breast cancer progression.

Topics: Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line; Cell Movement; Epithelial-Mesenchymal Transition; Humans; MCF-7 Cells; Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

Breast cancer is the most common cause of death among women. KIF3C, a member of kinesin superfamily, functions as a motor protein involved in axonal transport in neuronal cells. To explore the expression, regulation and mechanism of KIF3C in breast cancer, 4 breast cancer cell lines and 93 cases of primary breast cancer and paired adjacent tissues were examined. Immunohistochemistry, Real Time Polymerase Chain Reaction (RT-PCR), Western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation techniques and xenograft mice model were used. We found that KIF3C was over-expressed in breast cancer tissues and such high KIF3C expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3C by shRNA inhibited epithelial-mesenchymal transition and metastasis by inhibiting TGF-β signaling and suppressed breast cancer cell proliferation through inducing G2/M phase arrest. The tumor size was smaller and the number of lung metastatic nodules was less in KIF3C depletion MDA-MB-231 cell xenograft mice than in negative control group. These results suggested that high expression of KIF3C in breast cancer may be associated with the tumor progression and metastasis.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Movement; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; Female; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Kinesins; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; RNA Interference; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Burden

Downregulation of claudin 1, a critical tight junction protein, has been correlated with increased invasiveness in breast cancer. However, recent studies suggest that claudin 1 contributes to the progression of some molecular subtypes of breast cancer. In this study, claudin 1 promotes migration in luminal-like MCF7 human breast cancer cells and increases their sensitivity to tamoxifen, etoposide, and cisplatin. We also observed an inverse relationship between upregulation of claudin 1 and TGFβ. Collectively, our results suggest that claudin 1 has the potential to be used as a predictive marker for treatment efficacy for specific breast cancer patient subgroups.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cisplatin; Claudin-1; Down-Regulation; Etoposide; Female; Gene Expression; Humans; MCF-7 Cells; Tamoxifen; Tight Junctions; Transforming Growth Factor beta

Transforming Growth Factor beta (TGFβ) is the archetypal member of the TGFβ superfamily of ligands and has pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer. In epithelial cancers TGFβ signaling can either suppress tumor growth or promote metastasis via the induction of a well-characterized epithelial-mesenchymal transition (EMT) program. We recently reported that PEAK1 kinase mediates signaling cross talk between TGFβ receptors and integrin/Src/MAPK pathways and functions as a critical molecular regulator of TGFβ-induced breast cancer cell proliferation, migration, EMT and metastasis. Here, we examined the breast cancer cell contexts in which TGFβ induces both EMT and PEAK1, and discovered this event to be unique to oncogene-transformed mammary epithelial cells and triple-negative breast cancer cells. Using the Cancer BioPortal database, we identified PEAK1 co-expressors across multiple malignancies that are also common to the TGFβ response gene signature (TBRS). We then used the ScanSite database to identify predicted protein-protein binding partners of PEAK1 and the PEAK1-TBRS co-expressors. Analysis of the Cytoscape interactome and Babelomics-derived gene ontologies for a novel gene set including PEAK1, CRK, ZEB1, IL11 and COL4A1 enabled us to hypothesize that PEAK1 may be regulating TGFβ-induced EMT via its interaction with or regulation of these other genes. In this regard, we have demonstrated that PEAK1 is necessary for TGFβ to induce ZEB1-mediated EMT in the context of fibronectin/ITGB3 activation. These studies and future mechanistic studies will pave the way toward identifying the context in which TGFβ blockade may significantly improve breast cancer patient outcomes.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Fibronectins; Homeodomain Proteins; Humans; Protein-Tyrosine Kinases; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-β1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications.. Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1β, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-β1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78-0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort.. Our data show that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.

Topics: Animals; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Female; Fibroblast Growth Factors; Fibroblasts; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Interleukin-1beta; Mice; Middle Aged; Prognosis; Real-Time Polymerase Chain Reaction; Retrospective Studies; Survival Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

Microvascular damage is an important component of late radiation-induced morbidity. In our pre-clinical models, we demonstrated that repair of vessel injury is dependent on proper endoglin-mediated transforming growth factor-beta (TGF-β) signalling and that it can be affected by infiltrating macrophages. We now wanted to extend these findings in irradiated patients, using skin as a model system, and assess whether bisphosphonates could modulate the response.. Paired skin biopsies from irradiated and non-irradiated sites were obtained from 48 breast cancer patients. In 8 patients, biopsies were repeated after 4months of bisphosphonate treatment. Immunohistochemistry was used to assess vascular alterations and leucocyte infiltration. Western Blot and qPCR were used to assess expression of growth factors and their receptors.. Decreased blood vessel numbers at early time points were followed by increased endoglin expression and restoration of vessel number. Loss of small lymphatic vessels was associated with increased TGF-β levels, whereas dilation of lymphatic vessels correlated with increased macrophage infiltration. Bisphosphonate treatment reduced leucocyte infiltration, but also prevented restoration of blood vessel numbers after irradiation.. Radiation injury of the microvasculature is mediated through TGF-β, whereas repair is modulated by the co-receptor endoglin and promoted by macrophages.

Topics: Adult; Aged; Antigens, CD; Biopsy; Blotting, Western; Breast Neoplasms; Endoglin; Female; Humans; Immunohistochemistry; Lymphatic System; Macrophages; Microvessels; Middle Aged; Phosphorylation; Radiation Injuries; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Transforming Growth Factor beta

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cytochrome-B(5) Reductase; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Membrane Glycoproteins; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Niemann-Pick C1 Protein; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Nerve Growth Factor; RNA, Small Interfering; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

Topics: ADAM Proteins; ADAM12 Protein; Adult; Aged; Aged, 80 and over; Biocatalysis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cytoplasm; Drug Resistance, Neoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; MAP Kinase Signaling System; Membrane Proteins; Mesoderm; Middle Aged; Phenotype; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.

Topics: Absorptiometry, Photon; Animals; Bone Neoplasms; Breast Neoplasms; Calcium; Calcium Signaling; Camurati-Engelmann Syndrome; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Nude; Mice, SCID; Multiple Myeloma; Muscle Contraction; Muscle Proteins; Muscle Strength; Muscle Weakness; Muscle, Skeletal; NADPH Oxidase 4; NADPH Oxidases; Neoplasms; Osteolysis; Oxidation-Reduction; Prostatic Neoplasms; Ryanodine Receptor Calcium Release Channel; Transforming Growth Factor beta; Up-Regulation; X-Ray Microtomography

Distant relapse after chemotherapy is an important clinical issue for treating breast cancer patients and results from the development of cancer stem-like cells (CSCs) during chemotherapy. Here we report that blocking epithelial-to-mesenchymal transition (EMT) suppresses paclitaxel-induced CSCs properties by using a MDA-MB-231-xenografted mice model (in vivo), and breast cancer cell lines (in vitro). Paclitaxel, one of the cytotoxic taxane-drugs such as docetaxel, increases mesenchymal markers (Vimentin and Fibronectin) and decreases an epithelial marker (Zo-1). Blocking TGF-β signaling with the TGF-β type I receptor kinase (ALK5) inhibitor, EW-7197, suppresses paclitaxel-induced EMT and CSC properties such as mammosphere-forming efficiency (MSFE), aldehyde dehydrogenase (ALDH) activity, CD44+/CD24- ratio, and pluripotency regulators (Oct4, Nanog, Klf4, Myc, and Sox2). The combinatorial treatment of EW-7197 improves the therapeutic effect of paclitaxel by decreasing the lung metastasis and increasing the survival time in vivo. We confirmed that Snail is increased by paclitaxel-induced intracellular reactive oxygen species (ROS) and EW-7197 suppresses the paclitaxel-induced Snail and EMT by attenuating paclitaxel-induced intracellular ROS. Knock-down of SNAI1 suppresses paclitaxel-induced EMT and CSC properties. These data together suggest that blocking the Snail-induced EMT with the ALK5 inhibitor attenuates metastasis after paclitaxel-therapy and that this combinatorial approach could prove useful in treating breast cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Combined Modality Therapy; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Kruppel-Like Factor 4; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Paclitaxel; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

The number of CD3(+)CD56-HLA-G(+) cells in the peripheral blood of breast cancer patients was shown to increase by 2 times. Our results and published data suggest that the increase in the relative content of CD3(+)CD56-HLA-G(+) cells in the circulating blood in breast cancer contributes to tumor development due to suppression of antitumor immunity.

Topics: Adult; Breast Neoplasms; Case-Control Studies; CD3 Complex; CD56 Antigen; Female; Flow Cytometry; Gene Expression; HLA-G Antigens; Humans; Immunity, Innate; Immunophenotyping; Interferon-gamma; Interleukin-10; Lymphocyte Count; Middle Aged; Neoplasm Staging; T-Lymphocyte Subsets; Transforming Growth Factor beta

Disulfiram (DSF), an anti-alcoholism drug, has been reported as an inhibitor of NF-κB. NF-κB is involved in epithelial-mesenchymal transition (EMT) and self-renewal of breast cancer stem cells (CSCs). In this study, we treated MCF-7 and MDA-MB-231 breast cancer cells with TGF-β to induce EMT and cancer stem-like features and studied whether DSF can reverse this process. We found that DSF inhibited TGF-β induced EMT in breast cancer cells in a dose-dependent manner. Also, DSF inhibited EMT-associated stem-like features, migration and invasion of tumor cells as well as tumor growth in xenograft model. The activation of NF-κB was linked with EMT and stem-like cells. We conclude that DSF can suppress NF-κB activity and downregulate ERK/NF-κB/Snail pathway, leading to reverse EMT and stem-like features. Our data suggest that DSF inhibits EMT and stem-like properties in breast cancer cells associated with inhibition of the ERK/NF-κB/Snail pathway.

Topics: Alcohol Deterrents; Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Disulfiram; Electrophoretic Mobility Shift Assay; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; NF-kappa B; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment and influence tumor progression; however, how MSCs induce the invasion of cancer cells is not completely understood. Here, we used a 3D coculture model to determine how MSCs affect the migration of invasive breast cancer cells. Coculture with MSCs increases the elongation, directional migration, and traction generation of breast cancer cells. MSC-induced directional migration directly correlates with traction generation and is mediated by transforming growth factor β (TGF-β) and the migratory proteins rho-associated kinase, focal adhesion kinase, and matrix metalloproteinases. Treatment with MSC conditioned media or recombinant TGF-β1 elicits a similar migration response to coculture. Taken together, this work suggests TGF-β is secreted by MSCs, leading to force-dependent directional migration of invasive breast cancer cells. These pathways may be potential targets for blocking cancer cell invasion and subsequent metastasis.

Topics: Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Enzyme Inhibitors; Female; Focal Adhesion Kinase 1; Humans; Matrix Metalloproteinases, Secreted; MCF-7 Cells; Mesenchymal Stem Cells; Microscopy, Fluorescence; Neoplasm Invasiveness; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; Transforming Growth Factor beta

Better methods to predict prognosis can play a supplementary role in administering individualized treatment for breast cancer patients. Altered expressions of PTHrP and TGF-β have been observed in various types of human cancers. The objective of the current study was to evaluate the association of PTHrP and TGF-β level with the clinicopathological features of the breast cancer patients.. Immunohistochemistry was used to examine PTHrP and TGF-β protein expression in 497 cases of early breast cancer, and Kaplan-Meier method and COX's Proportional Hazard Model were applied to the prognostic value of PTHrP and TGF-β expression.. Both over-expressed TGF-β and PTHrP were correlated with the tumor in larger size, higher proportion of axillary lymph node metastasis and later clinical stage. Additionally, the tumors with a high TGF-β level developed poor differentiation, and only TGF-β expression was associated with disease-free survival (DFS) of the breast cancer patients. Followed up for a median of 48 months, it was found that only the patients with negative TGF-β expression had longer DFS (P < 0.05, log-rank test). Nevertheless, those with higher PTHrP expression tended to show a higher rate of bone metastasis (67.6 % vs. 45.8 %, P = 0.019). In ER negative subgroup, those who developed PTHrP positive expression presented poor prognosis (P < 0.05, log-rank test). The patients with both positive TGF-β and PTHrP expression were significantly associated with the high risk of metastases. As indicated by Cox's regression analysis, TGF-β expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for breast cancer recurrence.. TGF-β and PTHrP were confirmed to be involved in regulating the malignant progression in breast cancer, and PTHrP expression, to be associated with bone metastasis as a potential prognostic marker in ER negative breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Parathyroid Hormone-Related Protein; Prognosis; Transforming Growth Factor beta

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

Topics: Animals; Axl Receptor Tyrosine Kinase; Benzocycloheptenes; Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Lung Neoplasms; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Metastasis; Neoplastic Stem Cells; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; RNA Interference; Signal Transduction; Smad Proteins; Thrombospondins; Transforming Growth Factor beta; Transplantation, Heterologous; Triazoles

Transforming growth factor beta (TGFβ) signalling is involved in both tumour suppression and tumour progression. The mRNA expression levels of the TGFβ isoforms and receptors in breast tumours may have prognostic value and clinical implications.. The mRNA levels of TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 were analysed in primary breast tumours and adjacent normal breast tissues, and the associations with tumour characteristics and patients' overall and relapse-free survival were evaluated, using the public gene expression microarray data from The Cancer Genome Atlas (n = 520) and the Gene Expression Omnibus (four datasets) and our quantitative real-time PCR validation data (n = 71).. Significantly higher TGFB1 and TGFB3 mRNA levels and lower TGFBR2 mRNA levels were observed in primary tumours compared with their paired normal tissues. TGFB1 mRNA expression was seemly lower in triple-negative tumours and in tumours from lymph node-negative patients. TGFB3 mRNA expression was significantly lower in estrogen receptor-negative/progesterone receptor-negative/Basal-like/Grade 3 tumours. High TGFB2, TGFB3 and TGFBR2 mRNA levels in tumours were generally associated with better prognosis for patients, especially those diagnosed with lymph node-negative diseases. High TGFBR1 mRNA levels in tumours were associated with poorer clinical outcomes for patients diagnosed with small (diameter ≤ 2 cm) tumours.. The results indicate a reduced responsiveness of tumour cells to TGFβ, a preferential up-regulation of TGFB1 in malignant tumours and a preferential up-regulation of TGFB3 in premalignant tumours. The results may not only provide prognostic value for patients but also assist in classifying tumours according to their potential responses to TGFβ and selecting patients for TGFβ signalling pathway targeted therapies.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Prognosis; Protein Isoforms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors, the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast, and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-κB (NF-κB) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-κB was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover, both canonical and non-canonical NF-κB signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact, canonical and non-canonical NF-κB signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-κB promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1β and interleukin-6. The results suggest the use of NF-κB inhibitors for clinical therapy of certain breast cancers.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; I-kappa B Kinase; Interleukin-1beta; Interleukin-6; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; Spheroids, Cellular; Transcription Factor RelA; Transforming Growth Factor beta

In the mammary gland, the stromal extracellular matrix (ECM) undergoes dramatic changes during development and in tumorigenesis. For example, normal adult breast tissue is largely devoid of the ECM protein fibronectin (FN) whereas high FN levels have been detected in the stroma of breast tumors. FN is an established marker for epithelial-mesenchymal transition (EMT), which occurs during development and has been linked to cancer. During EMT, epithelial cell adhesion switches from cell-cell contacts to mainly cell-ECM interactions, raising the possibility that FN may have a role in promoting this transition. Using MCF-10A mammary epithelial cells, we show that exposure to exogenous FN induces an EMT response including upregulation of the EMT markers FN, Snail, N-cadherin, vimentin, the matrix metalloprotease MMP2, α-smooth muscle actin and phospho-Smad2, as well as acquisition of cell migratory behavior. FN-induced EMT depends on Src kinase and extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase signaling but not on the immediate early gene EGR-1. FN initiates EMT under serum-free conditions; this response is partially reversed by a transforming growth factor (TGF)β-neutralizing antibody, suggesting that FN enhances the effect of endogenous TGFβ. EMT marker expression is upregulated in cells on a fragment of FN containing the integrin-binding domain but not other domains. Differences in gene expression between FN and Matrigel are maintained with addition of a subthreshold level of TGFβ1. Together, these results show that cells interacting with FN are primed to respond to TGFβ. The ability of FN to induce EMT shows an active role for the stromal ECM in this process and supports the notion that the increased levels of FN observed in breast tumors facilitate tumorigenesis.

Topics: Breast; Breast Neoplasms; Cell Differentiation; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Fibronectins; Humans; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS).. Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFβ signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography.. Expression of αvβ6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvβ6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvβ6-positive myoepithelial cells is dependent on TGFβ-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway.. These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvβ6 on myoepithelial cells generates a tumor promoter function through TGFβ upregulation of MMP-9. These data suggest that expression of αvβ6 may be used to stratify patients with DCIS.

Topics: Animals; Antigens, Neoplasm; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Case-Control Studies; Cell Line; Cell Line, Tumor; Disease Progression; Epithelial Cells; Female; Follow-Up Studies; Humans; Immunohistochemistry; Integrins; Matrix Metalloproteinase 9; Mice; Mink; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Prognosis; Transforming Growth Factor beta; Tumor Burden; Tumor Microenvironment

In view of the fact that insufficiency in immune response often correlates with poor prognosis, research in recent years has focused on the task of describing the precise status and function of the immune system and its possible effect on cancer patients. Although more than two thirds of treated patients respond to endocrine therapy, most patients with metastatic breast cancer develop a resistance to it. Estrogen modulates angiogenesis, partially through its effects on vascular endothelial growth factor (VEGF). It also appears that transforming growth factor-beta (TGF beta) could be another factor contributing to this resistance. TGF beta is a highly immunosuppressive factor that inhibits natural and specific immunity against tumors and stimulates the production of VEGF. The purpose of the study was to monitor immune responses in patients with hormone receptor-positive breast cancer who were resistant to hormone therapy. The examination of cellular components (CD4, CD8, HLA-DR, NK cells) and humoral immunity (IgG, IgG subclasses, IgA, IgM,). TGF beta and VEGF production were monitored with special attention, along with an analysis of the changes that occurred during the hormonal treatment. 68 patients included in the research project were implemented with routine cancer treatment with endocrine therapy. Basic parameters (the histological type and grade, the degree of expression of estrogen receptors (ER) and progesterone receptors (PR), human epidermal growth factor receptor 2 (HER2), and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. TGF beta and VEGF were measured by ELISA and antitumor cellular immunity (CD4, CD8) was measured by flow cytometry. Patients who failed in the first line of hormone therapy treatment were considered as resistant to hormone therapy.Depression in cellular immunity was found especially in patients with resistance to endocrine therapy. In addition, immunoglobulin plasma levels were decreased (mainly IgG4 subtype). Most patients showed clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections). Significant increases in TGF beta and VEGF plasma were also detected.The correlation of these factors with resistance to hormonal therapy and the state of anticancer immunity could be helpful in the task of predicting resistance to hormonal therapy and could contribute to t

Topics: Adult; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Immunity, Cellular; Immunity, Humoral; Middle Aged; Tamoxifen; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

In late stages of cancer, TGF-β promotes the metastasis process by enhancing the invasiveness of cancer cells and inducing the epithelial-to-mesenchymal transition (EMT), a process that is concomitantly associated with breast cancer metastasis. Metastasis comprises of multiple steps with the regulation of complex network of signaling. Metastasis is associated with both the EMT and cell proliferation, but yet it has not been clearly distinguished how the balance between the cell proliferation and EMT is maintained together. Recently, it has been accounted that a transcription factor, NFAT has an important role for switching tumor suppressive to progressive effect of TGF-β and NFAT has a role in TGF-β mediated EMT by regulating N-cadherin. CDC 25A phosphatase, an important cell cycle regulator is overexpressed in breast cancer. Our results demonstrate that TGF-β regulating the CDC 25A in a Smad2 dependent way, translocates NFAT to nucleus and NFAT in co-operation with Smad2 promotes the tumor progression by upregulating the CDK2, CDK4, and cyclin E. This result signifies that TGF-β by regulating NFAT in different ways maintains the balance between EMT and cell proliferation mechanism concurrently during the late stage of breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cadherins; cdc25 Phosphatases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; NFATC Transcription Factors; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.. Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.. The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.. Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.

Topics: Actins; Breast Neoplasms; Cell Movement; Chemokine CXCL12; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Muscle, Smooth; Paracrine Communication; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

The TGF-β signaling pathway regulates cellular proliferation and differentiation. We evaluated genetic variation in this pathway, its association with breast cancer survival, and survival differences by genetic ancestry and self-reported ethnicity. The Breast Cancer Health Disparities Study includes participants from the 4-Corners Breast Cancer Study (n = 1,391 cases) and the San Francisco Bay Area Breast Cancer Study (n = 946 cases) who have been followed for survival. We evaluated 28 genes in the TGF-β signaling pathway using a tagSNP approach. Adaptive rank truncated product (ARTP) was used to test the gene and pathway significance by Native American (NA) ancestry and by self-reported ethnicity (non-Hispanic white (NHW) and Hispanic/NA). Genetic variation in the TGF-β signaling pathway was associated with overall breast cancer survival (P ARTP = 0.05), especially for women with low NA ancestry (P ARTP = 0.007) and NHW women (P ARTP = 0.006). BMP2, BMP4, RUNX1, and TGFBR3 were significantly associated with breast cancer survival overall (P ARTP = 0.04, 0.02, 0.002, and 0.04, respectively). Among women with low NA, ancestry associations were as follows: BMP4 (P ARTP = 0.007), BMP6 (P ARTP = 0.001), GDF10 (P ARTP = 0.05), RUNX1 (P ARTP = 0.002), SMAD1 (P ARTP = 0.05), and TGFBR2 (P ARTP = 0.02). A polygenic risk model showed that women with low NA ancestry and high numbers of at-risk alleles had twice the risk of dying from breast cancer as did women with high NA ancestry. Our data suggest that genetic variation in the TGF-β signaling pathway influences breast cancer survival. Associations were similar when the analyses were stratified by genetic ancestry or by self-reported ethnicity.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; DNA, Neoplasm; Female; Genetic Predisposition to Disease; Healthcare Disparities; Hispanic or Latino; Humans; Middle Aged; Polymorphism, Single Nucleotide; San Francisco; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; White People

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-β) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-β and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/β-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.

Topics: Bone Neoplasms; Breast Neoplasms; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplasm Invasiveness; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

It has been proposed that the epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features. WISP2 (Wnt-1-induced signaling protein-2) plays an important role in maintenance of the differentiated phenotype of estrogen receptor-positive breast cancer cells and loss of WISP2 is associated with EMT. We now report that loss of WISP2 in MCF7 breast cancer cells can also promote the emergence of a cancer stem-like cell phenotype characterized by high expression of CD44, increased aldehyde dehydrogenase activity and mammosphere formation. Higher levels of the stem cell markers Nanog and Oct3/4 were observed in those mammospheres. In addition we show that low-cell inoculums are capable of tumor formation in the mammary fat pad of immunodeficient mice. Gene expression analysis show an enrichment of markers linked to stem cell function such as SOX9 and IGFBP7 which is linked to TGF-β inducible, SMAD3-dependent transcription. Taken together, our data demonstrate that WISP2 loss promotes both EMT and the stem-like cell phenotype.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; CCN Intercellular Signaling Proteins; Cells, Cultured; Epithelial-Mesenchymal Transition; Estrogens; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Hyaluronan Receptors; Mice; Mice, Nude; Neoplastic Stem Cells; Phenotype; Repressor Proteins; Signal Transduction; Transforming Growth Factor beta

Hyaluronan synthase 2 (HAS2) is an enzyme in hyaluronan synthesis. Several studies have demonstrated that HAS2 plays a critical role in tumour progression in breast cancer cells. The in-situ expression patterns of HAS2 remain unclear, and the aim of this study was to determine these in order to elucidate the role of HAS2 in breast cancer.. We examined HAS2 expression using immunohistochemistry in 244 breast carcinomas of various subtypes. We found expression of HAS2 in 30.6% of invasive ductal carcinomas (IDCs); in IDCs, HAS2 expression was correlated significantly with the triple-negative phenotype and the basal-like phenotype, and univariate and multivariate analyses indicated that it was associated with poorer overall survival. In contrast to other carcinoma subtypes, HAS2 expression was observed in up to 72.7% of metaplastic carcinomas of breast (MCB), a carcinoma subtype related to the epithelial-mesenchymal transition (EMT). Consistently, we noted up-regulated levels of HAS2 RNA and protein in TGF-β-induced EMT in MCF-10A mammary epithelial cells.. Our findings demonstrate that HAS2 plays a role in aggressive phenotypes of primary breast carcinoma. The strong expression of HAS2 in MCB and the up-regulation of HAS2 in breast cells induced to exhibit EMT implicates an association between HAS2 expression and EMT in breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Immunohistochemistry; Kaplan-Meier Estimate; Metaplasia; Prognosis; RNA, Neoplasm; Transforming Growth Factor beta; Up-Regulation

Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation, and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signaling to promote breast cancer cell proliferation. We used small interfering RNA knockdown strategies to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1-dependent mechanisms. We identified key genes involved in TGF-β signaling to be highly responsive to LRH-1 knockdown. This relationship was validated in 2 breast cancer cell lines overexpressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 overexpression in mammary epithelial cells. Notably, TGF-β signaling was activated in LRH-1-overexpressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 overexpression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-β expression. The regulation of TGF-β isoforms and SMAD2/3-mediated downstream signaling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively, these data demonstrate that LRH-1 regulates TGF-β expression and downstream signaling in mouse mammary glands.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Mammary Glands, Animal; MCF-7 Cells; Mice; Mice, Transgenic; Morphogenesis; Neoplasm Proteins; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta; Up-Regulation

Recent works have highlighted a double role for the Transforming Growth Factor β (TGF-β): it inhibits cancer in healthy cells and potentiates tumor progression during late stage of tumorigenicity, respectively; therefore it has been termed the "Jekyll and Hyde" of cancer or, alternatively, an "excellent servant but a bad master". It remains unclear how this molecule could have the two opposite behaviours. In this work, we propose a TGF-β multi scale mathematical model at molecular, cellular and tissue scales. The multi scalar behaviours of the TGF-β are described by three coupled models built up together which can approximatively be related to distinct microscopic, mesoscopic, and macroscopic scales, respectively. We first model the dynamics of TGF-β at the single-cell level by taking into account the intracellular and extracellular balance and the autocrine and paracrine behaviour of TGF-β. Then we use the average estimates of the TGF-β from the first model to understand its dynamics in a model of duct breast tissue. Although the cellular model and the tissue model describe phenomena at different time scales, their cumulative dynamics explain the changes in the role of TGF-β in the progression from healthy to pre-tumoral to cancer. We estimate various parameters by using available gene expression datasets. Despite the fact that our model does not describe an explicit tissue geometry, it provides quantitative inference on the stage and progression of breast cancer tissue invasion that could be compared with epidemiological data in literature. Finally in the last model, we investigated the invasion of breast cancer cells in the bone niches and the subsequent disregulation of bone remodeling processes. The bone model provides an effective description of the bone dynamics in healthy and early stages cancer conditions and offers an evolutionary ecological perspective of the dynamics of the competition between cancer and healthy cells.

Topics: Autocrine Communication; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; Models, Biological; Molecular Dynamics Simulation; Neoplasm Invasiveness; Neoplasm Metastasis; Paracrine Communication; Transforming Growth Factor beta

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β-induced expression of parathyroid hormone-like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho-TGF-β crosstalk in osteolytic bone metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Female; Gene Knockdown Techniques; GTPase-Activating Proteins; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Osteoclasts; Parathyroid Hormone-Related Protein; rho GTP-Binding Proteins; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Suppressor Proteins

Metastasis represents a major problem in the treatment of patients with advanced primary breast cancer. Both Transforming Growth Factor-Beta (TGF-β) signaling and Plasminogen Activator (PA) components, urokinase-type Plasminogen Activator (uPA) and Plasminogen Activator Inhibitor-1 (PAI-1) represent a complex network crucial for such enhanced invasiveness of tumors and imply high prognostic/predictive and promising therapeutic potential. Therefore, protein expression of specific effector molecules comprising the main parts of the TGF-β signaling pathway were determined in HOPE-fixed human tumor tissues through IHC (Scoring) using tissue microarray (TMA) technique and correlated with respective uPA and PAI-1 levels determined earlier in the same TMAs through optimized IHC and semi-quantitative image analysis. TGF-β signaling was active in vast majority (96 %) of the tumor samples and 88 % of all cases were significantly correlated with established metastasis markers uPA and PAI-1. In addition, TGF-β was also closely associated with tumor size, nodal status and two steroid hormone receptors. Consistent interrelationships between TGF-β, PA components and additional tumor characteristics underline the superiority of such more comprising data with regards to confirming TGF-β signaling as a promising target system to inhibit metastasis in advanced breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Plasminogen Activator Inhibitor 1; Prognosis; Signal Transduction; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

Resistance to trastuzumab and concomitantly distal metastasis are leading causes of mortality in HER2-positive breast cancers, the molecular basis of which remains largely unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased tumorigenicity and invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-β signaling in these cells. MiR-200c, which was the most significantly downregulated miRNA in trastuzumab-resistant cells, restored trastuzumab sensitivity and suppressed invasion of breast cancer cells by concurrently targeting ZNF217, a transcriptional activator of TGF-β, and ZEB1, a known mediator of TGF-β signaling. Given the reported backward inhibition of miR-200c by ZEB1, ZNF217 also exerts a feedback suppression of miR-200c via TGF-β/ZEB1 signaling. Restoration of miR-200c, silencing of ZEB1 or ZNF217 or blockade of TGF-β signaling increased trastuzumab sensitivity and suppressed invasiveness of breast cancer cells. Therefore, our study unraveled nested regulatory circuits of miR-200c/ZEB1 and miR-200c/ZNF217/TGF-β/ZEB1 in synergistically promoting trastuzumab resistance and metastasis of breast cancer cells. These findings provide novel insights into the common role of EMT and related molecular machinery in mediating the malignant phenotypes of breast cancers.

Topics: Animals; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Trastuzumab; Xenograft Model Antitumor Assays; Zinc Finger E-box-Binding Homeobox 1

It has been proposed that epithelial cells can acquire invasive properties through exposure to paracrine signals originated from mesenchymal cells within the tumor microenvironment. Transforming growth factor-β (TGF-β) has been revealed as an active factor that mediates the epithelial-stroma cross-talk that facilitates cell invasion and metastasis. TGF-β signaling is modulated by the coreceptor Endoglin (Eng), which shows a tumor suppressor activity in epithelial cells and regulates the ALK1-Smad1,5,8 as well as the ALK5-Smad2,3 signaling pathways. In the current work, we present evidence showing that cell surface Eng abundance in epithelial MCF-7 breast cancer cells is inversely related with cell motility. Shedding of Eng in MCF-7 cell surface by soluble matrix metalloproteinase-14 (MMP-14) derived from the HS-5 bone-marrow-derived cell line induces a motile epithelial phenotype. On the other hand, restoration of full-length Eng expression blocks the stromal stimulus on migration. Processing of surface Eng by stromal factors was demonstrated by biotin-neutravidin labeling of cell surface proteins and this processing generated a shift in TGF-β signaling through the activation of Smad2,3 pathway. Stromal MMP-14 abundance was stimulated by TGF-β secreted by MCF-7 cells acting in a paracrine manner. In turn, the stromal proteolytic activity of soluble MMP-14, by inducing Eng shedding, promoted malignant progression. From these data, and due to the capacity of TGF-β to regulate malignancy in epithelial cancer, we propose that stromal-dependent epithelial Eng shedding constitutes a putative mechanism that exerts an environmental control of cell malignancy.

Topics: Antigens, CD; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Endoglin; Epithelial Cells; Female; Flow Cytometry; Humans; Immunoprecipitation; Matrix Metalloproteinase 14; Mesenchymal Stem Cells; Microscopy, Fluorescence; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Dab2 is a multifunctional adapter protein which is frequently under-expressed in a variety of cancers. It is implicated in many critical functions, including several signaling pathways, cell arrangement, differentiation of stem cells, and receptor endocytosis. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and pathogenesis of many diseases. It has been documented that Dab2 played an important role in TGF-β receptors endocytosis. Here, we present evidence that re-expression of Dab2 in SK-BR-3 cell partially restored its ability to deplete TGF-β in surrounding medium by normalizing the trafficking of TGF-β receptors. We also demonstrate that the difference in TGF-β depletions produced by Dab2 expression was sufficient to impact on the conversion of naive CD4+ T cells to regulatory T cells (Tregs), and thus inhibited the proliferation of T cells. This work revealed a critical result that breast cancer cell was deficient in Dab2 expression and related receptor endocytosis-mediated TGF-β depletion, which may contribute to the accumulation of TGF-β in tumor microenvironment and the induction of immune tolerance.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymphocyte Activation; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Suppressor Proteins

Uncontrolled transforming growth factor-β (TGFβ) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFβ-mediated cues are directed to induce tumorigenic events is poorly understood, particularly given that TGFβ has clear tumor suppressing activity in other contexts. Here, we demonstrate that the transcriptional regulators TAZ and YAP (TAZ/YAP), key effectors of the Hippo pathway, are necessary to promote and maintain TGFβ-induced tumorigenic phenotypes in breast cancer cells. Interactions between TAZ/YAP, TGFβ-activated SMAD2/3, and TEAD transcription factors reveal convergent roles for these factors in the nucleus. Genome-wide expression analyses indicate that TAZ/YAP, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by TAZ/YAP, TEAD, and TGFβ, such as the novel targets NEGR1 and UCA1, are necessary for maintaining tumorigenic activity in metastatic breast cancer cells. Nuclear TAZ/YAP also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in nontumorigenic cells to overcome TGFβ-repressive effects. Our work thus identifies cross-talk between nuclear TAZ/YAP and TGFβ signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms.

Topics: Acyltransferases; Breast Neoplasms; Cell Adhesion Molecules, Neuronal; Cell Cycle Proteins; Cell Line, Tumor; Female; Genome-Wide Association Study; GPI-Linked Proteins; Humans; Neoplasm Metastasis; Neoplasm Proteins; Nuclear Proteins; RNA, Long Noncoding; RNA, Neoplasm; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Hypoxia; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; MicroRNAs; Neoplasm Metastasis; Oxygen; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration.. We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53.. We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-β-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. Remarkably, immunofluorescence analysis revealed MCF-10A cells expressing p53-R280K mutant showed an upregulation of Nox4 in both confluent and migrating cells.. Collectively, our findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signalling in TGF-β-mediated cell motility.

Topics: Breast; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Enzyme Induction; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Lung; Lung Neoplasms; Male; Mutation, Missense; NADPH Oxidase 4; NADPH Oxidases; Neoplasm Metastasis; Neoplasm Proteins; Reactive Oxygen Species; RNA Interference; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Tumor Suppressor Protein p53

HER2, an oncogenic receptor is overexpressed in about 25-30% of breast cancer patients. HER2 has been shown to play role in tumor promotion by having cross-talk with multiple oncogenic pathways in cancer cells. Our results show that Cucurbitacin B (CuB), a triterpenoid steroidal compound inhibited the growth of various breast cancer cells with an IC50 ranging from 18-50nM after 48 and 72 h of treatment. Our study also revealed the significant inhibitory effects of CuB on HER2 and integrin signaling in breast cancer. Notably, CuB inhibited ITGA6 and ITGB4 (integrin α6 and integrin β4), which are overexpressed in breast cancer. Furthermore, CuB also induced the expression of major ITGB1and ITGB3, which are known to cause integrin-mediated cell death. In addition, we observed that TGFβ treatment resulted in the increased association of HER2 with ITGA6 and this association was inhibited by CuB treatment. Efficacy of CuB was tested in vivo using two different orthotopic models of breast cancer. MDA-MB-231 and 4T-1 cells were injected orthotopically in the mammary fat pad of female athymic nude mice or BALB/c mice respectively. Our results showed that CuB administration inhibited MDA-MB-231 orthotopic tumors by 55%, and 4T-1 tumors by 40%. The 4T-1 cells represent stage IV breast cancer and form very aggressive tumors. CuB mediated breast tumor growth suppression was associated with the inhibition of HER2/integrin signaling. Our results suggest novel targets of CuB in breast cancer in vitro and in vivo.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; ErbB Receptors; Female; Humans; Inhibitory Concentration 50; Integrin alpha6; Integrin alpha6beta4; Integrin beta1; Integrin beta3; Integrin beta4; MAP Kinase Signaling System; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta; Triterpenes; Twist-Related Protein 1

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+ CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+ CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection.

Topics: Animals; beta-Glucans; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Dendritic Cells; Disease Models, Animal; Female; Humans; Lectins, C-Type; Mice; Mucous Membrane; Signal Transduction; T-Lymphocyte Subsets; Th2 Cells; Transforming Growth Factor beta

The relationship between tumor-associated macrophages (TAM) and epithelial-to-mesenchymal transition (EMT) during the initiation and progression of metastasis is still unclear. Here, a role for the vitamin D receptor (VDR) in metastasis was identified, as well as a role in the relationship between TAMs and EMT. First, the expression level of VDR was examined in clinical tissue from human patients with breast cancer or a mouse model of breast cancer with differential metastasis. These results revealed that VDR expression negatively correlates with metastasis in breast cancer. Second, coculture of VDR-overexpressing breast cancer cells with a macrophage cell line demonstrated that overexpression of VDR alleviated the prometastatic effect of cocultured macrophages on breast cancer cells. Furthermore, VDR overexpression abrogated the induction of EMT in breast cancer cells by cocultured macrophage cells, as measured by a loss of E-cadherin (CDH1) and induction of α-smooth muscle actin (α-SMA). TNFα in macrophage conditioned media inhibited VDR expression, whereas downregulation of VDR further mediated the promotion of TGFβ-induced EMT by TNFα. In addition, β-catenin expression was inhibited in VDR-overexpressing breast cancer cells and tumor xenografts. Finally, administration of calcitriol [1,25-(OH)2D3], an active vitamin D metabolite, exerted similar antimetastatic effects in breast cancer cells in vitro and a mouse model of breast cancer in vivo with preservation of VDR and suppression of β-catenin.. VDR suppression by TNFα mediates the prometastatic effect of TAMs through enhancement of the β-catenin pathway.

Topics: Actins; Animals; beta Catenin; Breast Neoplasms; Cadherins; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Macrophages; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Receptors, Calcitriol; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The majority of breast cancers originate from the highly polarized luminal epithelial cells lining the breast ducts. However, cell polarity is often lost during breast cancer progression. The type III transforming growth factor-β cell surface receptor (TβRIII) functions as a suppressor of breast cancer progression and also regulates the process of epithelial-to-mesenchymal transition (EMT), a consequence of which is the loss of cell polarity. Many cell surface proteins exhibit polarized expression, being targeted specifically to the apical or basolateral domains. Here we demonstrate that TβRIII is basolaterally localized in polarized breast epithelial cells and that disruption of the basolateral targeting of TβRIII through a single amino acid mutation of proline 826 in the cytosolic domain results in global loss of cell polarity through enhanced EMT. In addition, the mistargeting of TβRIII results in enhanced proliferation, migration, and invasion in vitro and enhanced tumor formation and invasion in an in vivo mouse model of breast carcinoma. These results suggest that proper localization of TβRIII is critical for maintenance of epithelial cell polarity and phenotype and expand the mechanisms by which TβRIII prevents breast cancer initiation and progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Polarity; Cell Proliferation; Disease Progression; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Mice, Nude; Mutation, Missense; Neoplasm Transplantation; Protein Transport; Proteoglycans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Transforming growth factor-βs (TGF-βs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-β antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-β are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses.. Using a breast cancer progression model that exemplifies the dual role of TGF-β, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-β-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts.. TGF-β-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-β action. An in vivo-weighted TGF-β/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-β/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression.. Tumor-suppressive effects of TGF-β persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-β antagonists.

Topics: Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Ephrins; Female; Humans; Promoter Regions, Genetic; Receptor, EphA2; RNA Interference; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.

Topics: Breast Neoplasms; CD24 Antigen; Cell Differentiation; Chromatin; Chromatin Assembly and Disassembly; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Isoenzymes; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; NF-kappa B p50 Subunit; Protein Kinase C; Protein Kinase C-theta; Receptors, Urokinase Plasminogen Activator; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Transcription Factor RelA; Transforming Growth Factor beta

Recurrent mutations in histone-modifying enzymes imply key roles in tumorigenesis, yet their functional relevance is largely unknown. Here, we show that JARID1B, encoding a histone H3 lysine 4 (H3K4) demethylase, is frequently amplified and overexpressed in luminal breast tumors and a somatic mutation in a basal-like breast cancer results in the gain of unique chromatin binding and luminal expression and splicing patterns. Downregulation of JARID1B in luminal cells induces basal genes expression and growth arrest, which is rescued by TGFβ pathway inhibitors. Integrated JARID1B chromatin binding, H3K4 methylation, and expression profiles suggest a key function for JARID1B in luminal cell-specific expression programs. High luminal JARID1B activity is associated with poor outcome in patients with hormone receptor-positive breast tumors.

Topics: Breast Neoplasms; CCCTC-Binding Factor; Cell Growth Processes; Cell Line, Tumor; Cell Lineage; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; MCF-7 Cells; Mutation; Nuclear Proteins; Oncogenes; Promoter Regions, Genetic; Pyrazoles; Pyrroles; Repressor Proteins; RNA, Small Interfering; Transfection; Transforming Growth Factor beta

The impact of combinations of anti-cancer drugs and growth factors on tumour cells may differ from the assumed sum of the effects of each factor separately. Therefore it is important to study the effects of different combinations of various drugs and treatments. Our aim was to study the effects on breast cancer cell proliferation of EGF, TGFβ and 17β-oestradiol, three important regulators of breast tumourigenesis, and their respective inhibitors in different combinations.. We screened the effects on proliferation of MCF7 and MDA-MB-231 cells of ninety different combinations of EGF, TGFβ and 17β-oestradiol, Iressa, SB431542 and Tamoxifen. Meta-data analysis of available clinical data was performed to validate observed proliferation data.. In MDA-MB-231 cells, TGFβ1 was found inhibitory when cells were simultaneously treated with EGF and 17β-oestradiol, with the effect potentiated by addition of all inhibitors combined. In the same cells, Iressa when combined with EGF was paradoxically stimulatory. Tamoxifen inhibited MCF7 cells co-treated with EGF or oestrogen, and enhanced the inhibitory effect of TGFβ in MDA-MB-231 cells. Meta-analysis of clinical gene expression studies confirmed several of these points, showing enhanced TGFβ and EGF expression in Tamoxifen-treated patients to correlate with decreased tumour size and grade respectively, and combined TGFβ-EGF expression to decrease the risk of metastasis.. Our study shows significant differences in proliferation response to drugs and growth factors between MCF7 cells which do not have propensity to form metastases in animal models and MDA-MB-231 cells which may form metastases upon inoculation into animals. Several of these differences are unexpected and confirmed by clinical observations.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Combinations; Epidermal Growth Factor; Estradiol; Female; Humans; Signal Transduction; Transforming Growth Factor beta

Inflammation and inflammatory mediators are inextricably linked with epithelial-mesenchymal transition (EMT) through complex pathways in the tumor microenvironment. However, the mechanism by which inflammatory mediators, such as the lipid inflammatory mediators, eicosanoids, contribute to EMT is largely unknown. In the present study we observed that BLT2, leukotriene B4 receptor-2, is markedly up-regulated by oncogenic Ras and promotes EMT in response to transforming growth factor-β (TGF-β) in mammary epithelial cells. Blockade of BLT2 by the BLT2 inhibitor LY255283 or by siRNA reduced EMT induced by Ras in the presence of TGF-β. In addition, stimulation of BLT2 by the addition of a BLT2 ligand, such as leukotriene B4, restored EMT in the presence of TGF-β in human immortalized mammary epithelial MCF-10A cells. We further searched BLT2 downstream components and identified reactive oxygen species and nuclear factor κB as critical components that contribute to EMT. Taken together, these results demonstrate for the first time that a BLT2-linked inflammatory pathway contributes to EMT. This provides valuable insight into the mechanism of EMT in mammary epithelial cells. In addition, considering the implications of EMT with the stemness of cancer cells, our finding may contribute to a better understanding of tumor progression.

Topics: Breast Neoplasms; Cell Line; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Glands, Human; NADPH Oxidase 1; NADPH Oxidases; NF-kappa B; Proto-Oncogene Proteins p21(ras); Reactive Oxygen Species; Receptors, Leukotriene B4; RNA, Small Interfering; Signal Transduction; Tetrazoles; Transforming Growth Factor beta

HER2 is an important determinant of poor prognosis in breast cancer patients. Studies indicate that HER2 positive tumors are mostly resistant to therapy and have high metastatic potential however, the underlying mechanisms remain unknown. In this study, MDA-MB-231 and MCF-7 breast cancer cells with their HER2 overexpressing syngeneic variants were used to delineate the role of HER2 in EMT and metastasis. Our results demonstrated that HER2 overexpression increased the invasive potential of cells. Our results also showed that HER2 overexpression lead to the production of TGFβ resulting in the activation of TGFβ/SMAD signaling. Furthermore, activation of SNAIL, SLUG and ZEB-1, the transcriptional repressors of E-cadherin and increased mesenchymal characteristics were observed in high HER2 cells. Interestingly, EMT by HER2 was mediated through TGFβ. Intravenous injection of high HER2 MDA-MB-231 (HH) cells in athymic nude mice showed early and substantial metastasis as compared to the parent cells establishing the direct role of HER2 in metastasis. Our results showed that inhibition of HER2 mediated EMT by cucurbitacin B a triterpenoid, resulted in the suppression of brain metastasis of breast cancer cells. Taken together, our results identify a novel mechanism of HER2 in promoting breast cancer metastasis through de novo synthesis of TGFβ leading to EMT, an initial and essential step of metastasis.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; Enzyme-Linked Immunosorbent Assay; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Self-renewal and differentiation are two epigenetic programs that regulate stem cells fate. Dysregulation of these two programs leads to the development of cancer stem cells (CSCs). Recent evidence suggests that CSCs are relatively resistant to conventional therapies and responsible for metastasis formation. Deciphering these processes will help understand oncogenesis and allow the development of new targeted therapies. Here, we have used a whole genome promoter microarray to establish the DNA methylation portraits of breast cancer stem cells (bCSCs) and non-bCSCs. A total of 68 differentially methylated regions (DMRs) were more hypomethylated in bCSCs than in non-bCSCs. Using a differentiation assay we demonstrated that DMRs are rapidly hypermethylated within the first 6 hours following induction of CSC differentiation whereas the cells reached the steady-state within 6 days, suggesting that these DMRs are linked to early CSC epigenetic regulation. These DMRs were significantly enriched in genes coding for TGF-β signaling-related proteins. Interestingly, DMRs hypomethylation was correlated to an overexpression of TGF-β signaling genes in a series of 109 breast tumors. Moreover, patients with tumors harboring the bCSC DMRs signature had a worse prognosis than those with non-bCSC DMRs signature. Our results show that bCSCs have a distinct DNA methylation landscape with TGF-β signaling as a key epigenetic regulator of bCSCs differentiation.

Topics: Breast Neoplasms; Cell Differentiation; Cell Transformation, Neoplastic; DNA Methylation; Embryonic Stem Cells; Epigenesis, Genetic; Female; Humans; Neoplastic Stem Cells; Transforming Growth Factor beta

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-β and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.

Topics: Animals; Breast Neoplasms; Chemokine CXCL12; DNA-Binding Proteins; Fibroblasts; Heat Shock Transcription Factors; Heterografts; Humans; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor beta (TGFβ) proteins are multitasking cytokines, in which high levels at tumor sites generally correlate with poor prognosis in human patients with cancer. Previously, it was reported that TGFβ downregulates the expression of ataxia telangiectasia-mutated (ATM) and mutS homolog 2 (MSH2) in breast cancer cells through an miRNA-mediated mechanism. In this study, expression of a panel of DNA-repair genes was examined, identifying breast cancer 1, early onset (BRCA1) as a target downregulated by TGFβ through the miR181 family. Correlations between the expression levels of TGFβ1 and the miR181/BRCA1 axis were observed in primary breast tumor specimens. By downregulating BRCA1, ATM, and MSH2, TGFβ orchestrates DNA damage response in certain breast cancer cells to induce a "BRCAness" phenotype, including impaired DNA-repair efficiency and synthetic lethality to the inhibition of poly (ADP-ribose) polymerase (PARP). Xenograft tumors with active TGFβ signaling exhibited resistance to the DNA-damaging agent doxorubicin but increased sensitivity to the PARP inhibitor ABT-888. Combination of doxorubicin with ABT-888 significantly improved the treatment efficacy in TGFβ-active tumors. Thus, TGFβ can induce "BRCAness" in certain breast cancers carrying wild-type BRCA genes and enhance the responsiveness to PARP inhibition, and the molecular mechanism behind this is characterized.. These findings enable better selection of patients with sporadic breast cancer for PARP interventions, which have exhibited beneficial effects in patients carrying BRCA mutations.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Disease Progression; DNA Repair; Down-Regulation; Doxorubicin; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Mice; MicroRNAs; MutS Homolog 2 Protein; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

NR2F2, a member of nuclear receptor subfamily 2, was shown to be associated with cancer, but its role in breast malignancy remains unclear. In this study, we aimed to explore the function of NR2F2 in breast cancer. We browsed GEO and TCGA databases and used Kaplan-Meier survival analysis to explore the association between NR2F2 transcript level and patient survival in breast cancer. NR2F2 expression in breast cancer tissues was evaluated by immunohistochemistry staining. NR2F2-related functions and its role in Epithelial-Mesenchymal Transition (EMT) were predicted by Gene Set Enrichment Analysis (GSEA) and validated by in vitro assays with NR2F2 knockdown MDA-MB231 and MCF7 cells. We found high NR2F2 transcript level was correlated with favorable overall survival and distant metastasis-free survival. Positive rate of NR2F2 protein tended to be decreased with the progression of malignancy. Results of in vitro migration and invasion assays suggested NR2F2's potential in inhibiting invasiveness. NR2F2 was predicted to be negatively linked with EMT and TGF-β-pathway related genes, which was supported by observation of EMT-like morphology and changes in EMT-markers E-cadherin and Slug. Additionally, we found TGF-β inhibited the expression of NR2F2. GSEA also predicted that NR2F2 could be inversely associated with chemoresistance, which was verified by results of in vitro growth inhibition assays using chemotherapeutic agents. Our results demonstrated high NR2F2 transcript level was associated with favorable clinical outcome, which might be due to NR2F2's inhibitory effect on TGF-β-dependent EMT and its role in inhibiting chemoresistance.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; COUP Transcription Factor II; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Middle Aged; Neoplasm Metastasis; RNA, Messenger; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3-Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15-16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.

Topics: Amino Acid Sequence; Animals; Antibodies; Breast; Breast Neoplasms; Cell Line, Tumor; Female; Histocytological Preparation Techniques; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Molecular Sequence Data; Protein Interaction Mapping; Sequence Alignment; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

TGFβ signaling is known to drive metastasis in human cancer. Under physiologic conditions, the level of TGFβ activity is tightly controlled by a regulatory network involving multiple negative regulators. At metastasis, however, these inhibitory mechanisms are usually overridden so that oncogenic TGFβ signaling can be overactivated and sustained. To better understand how the TGFβ inhibitors are suppressed in metastatic breast cancer cells, we compared miRNA expression profiles between breast cancers with or without metastasis and found that the miR424-503 cluster was markedly overexpressed in metastatic breast cancer. Mechanistic studies revealed that miR424 and miR503 simultaneously suppressed Smad7 and Smurf2, two key inhibitory factors of TGFβ signaling, leading to enhanced TGFβ signaling and metastatic capability of breast cancer cells. Moreover, antagonizing miR424-503 in breast cancer cells suppressed metastasis in vivo and increased overall host survival. Interestingly, our study also found that heterogeneous expression of the miR424-503 cluster contributed to the heterogeneity of TGFβ activity levels in, and metastatic potential of, breast cancer cell subsets. Overall, our findings demonstrate a novel mechanism, mediated by elevated expression of the miR424-503 cluster, underlying TGFβ activation and metastasis of human breast cancer.

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genetic Heterogeneity; Humans; MicroRNAs; Neoplasm Metastasis; Smad7 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neoplasm Metastasis; Phosphorylation; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cells, Cultured; Female; Humans; Mice; Mice, Transgenic; Receptors, Notch; Repressor Proteins; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Oct-4 and Nanog in regulating the epithelial-mesenchymal transition (EMT) and metastasis of breast cancer has not been clarified. We found that both Oct-4 and Nanog expression were significantly associated with tumor pathology and poor prognosis in 126 breast cancer patients. Characterization of CD44+CD24-Cancer stem cell(CSC) derived from breast cancer cells indicated that CSC rapidly formed mammospheres and had potent tumorigenicity in vivo. Furthermore, TGF-β up-regulated the expression of Oct-4, Nanog, N-cadherin, vimentin, Slug, and Snail, but down-regulated E-cadherin and cytokeratin 18 expression, demonstrating that CSC underwent EMT. Knockdown of both Oct-4 and Nanog expression inhibited spontaneous changes in the expression of EMT-related genes, while induction of both Oct-4 and Nanog over-expression enhanced spontaneous changes in the expression of EMT-related genes in CSC. However, perturbing alternation of Oct-4 and Nanog expression also modulated TGF-β-induced EMT-related gene expression in CSC. Induction of Oct-4 and Nanog over-expression enhanced the invasiveness of CSC, but knockdown of both Oct-4 and Nanog inhibited the migration of CSC in vitro. Our data suggest that both Oct-4 and Nanog may serve as biomarkers for evaluating breast cancer prognosis. Our findings indicate that Oct-4 and Nanog positively regulate the EMT process, contributing to breast cancer metastasis.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cadherins; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Mice, SCID; Nanog Homeobox Protein; Neoplastic Stem Cells; Octamer Transcription Factor-3; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin; Xenograft Model Antitumor Assays

Women diagnosed with pregnancy associated breast cancer often have advanced cancer with metastases and reduced expression of ERα compared to non-pregnant women. Nevertheless, metastases to the placenta are uncommon. Previously, we demonstrated that breast cancer cells (MCF-7/T47D) migrated from ex vivo human placental explant implantation sites. We aimed to analyze the effect of factors produced during placental implantation or as a result of the interaction between the implanted placentae to cancer cells on cancer cells migration and aggressiveness. We collected supernatants from implanted placentae and placental-breast cancer cells cocultures and analyzed their effects on cancer cells phenotype and pathways. Supernatants collected from breast cancer cells served as controls. We found that supernatants collected from implanted placentae induced modest cancer cells migration that was not accompanied by epithelial to mesenchymal transition (EMT), supported breast cancer cells survival and elevated MCF-7 cell number. The coculture supernatant induced excessive motility and EMT of the MCF-7 cells. This EMT was mediated by Smad3 and JNK/ERK activation. Both placenta and coculture supernatants reduced ERα expression in the cancer cells. Finally, we showed that MCF-7 cocultured with the human placental explants underwent continuous activation of JNK and Smad3 pathways and the EMT process, which led to their migration away from the placental implantation sites. These findings may explain the reduced ERα and elevated metastases found in breast cancer during pregnancy and highlights pathways involved in it.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Communication; Cell Cycle; Cell Movement; Cell Proliferation; Coculture Techniques; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; JNK Mitogen-Activated Protein Kinases; Placenta; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

CXCL1 is a chemotactic cytokine shown to regulate breast cancer progression and chemo-resistance. However, the prognostic significance of CXCL1 expression in breast cancer has not been fully characterized. Fibroblasts are important cellular components of the breast tumor microenvironment, and recent studies indicate that this cell type is a potential source of CXCL1 expression in breast tumors. The goal of this study was to further characterize the expression patterns of CXCL1 in breast cancer stroma, determine the prognostic significance of stromal CXCL1 expression, and identify factors affecting stromal CXCL1 expression.. Stromal CXCL1 protein expression was analyzed in 54 normal and 83 breast carcinomas by immunohistochemistry staining. RNA expression of CXCL1 in breast cancer stroma was analyzed through data mining in http://www.Oncomine.org. The relationships between CXCL1 expression and prognostic factors were analyzed by univariate analysis. Co-immunofluorescence staining for CXCL1, α-Smooth Muscle Actin (α-SMA) and Fibroblast Specific Protein 1 (FSP1) expression was performed to analyze expression of CXCL1 in fibroblasts. By candidate profiling, the TGF-β signaling pathway was identified as a regulator of CXCL1 expression in fibroblasts. Expression of TGF-β and SMAD gene products were analyzed by immunohistochemistry and data mining analysis. The relationships between stromal CXCL1 and TGF-β signaling components were analyzed by univariate analysis. Carcinoma associated fibroblasts isolated from MMTV-PyVmT mammary tumors were treated with recombinant TGF-β and analyzed for CXCL1 promoter activity by luciferase assay, and protein secretion by ELISA.. Elevated CXCL1 expression in breast cancer stroma correlated with tumor grade, disease recurrence and decreased patient survival. By co-immunofluorescence staining, CXCL1 expression overlapped with expression of α-SMA and FSP1 proteins. Expression of stromal CXCL1 protein expression inversely correlated with expression of TGF-β signaling components. Treatment of fibroblasts with TGF-β suppressed CXCL1 secretion and promoter activity.. Increased CXCL1 expression in breast cancer stroma correlates with poor patient prognosis. Furthermore, CXCL1 expression is localized to α-SMA and FSP1 positive fibroblasts, and is negatively regulated by TGF-β signaling. These studies indicate that decreased TGF-β signaling in carcinoma associated fibroblasts enhances CXCL1 expression in fibroblasts, which could contribute to breast cancer progression.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Chemokine CXCL1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Patient Outcome Assessment; Prognosis; Protein Binding; Risk Factors; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

β-Galactoside α2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal α2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and α2,6-sialylated N-glycans are up-regulated during TGF-β-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-β-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-β-induced EMT. Overexpressing β-galactoside α2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for α2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-β-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.

Topics: Antigens, CD; Binding Sites; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Gene Silencing; Humans; Phenotype; Promoter Regions, Genetic; Sialyltransferases; Sp1 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Topics: Animals; beta Catenin; Blotting, Western; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation; Humans; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice; Mice, Nude; Nuclear Proteins; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Wnt3A Protein; Xenograft Model Antitumor Assays; Zinc Finger Protein Gli2

Deregulated cell cycle progression can often be traced to intrinsic defects in specific regulatory proteins in cancer cells. Knowledge of these primary defects has led to targeted approaches that exploit the defects and spare normal cells. However, the success of such targeted approaches is still hit-or-miss. Genetic and epigenetic variability inherent in most tumors often results in phenotypic heterogeneity that, in turn, results in de novo or acquired resistance to therapeutic agents. The ability of cells to compensate and adapt to the inhibition of a specific cell cycle mediator is not remarkable. What is novel and of great potential importance is that the ability of cells to exhibit such adaptability varies markedly. "Phenotypic stability factors" that restrict the ability of cells to undergo epithelial-mesenchymal transitions (EMT) may dictate the success or failure of targeted therapies by interfering with compensatory changes such as deregulation of CDK2 activity. Identification of existing and new agents that induce and maintain phenotypic stability factors will inform and enable synergistic approaches to the eradication of even the most aggressive tumors.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D; Cyclin E; Cyclin-Dependent Kinase 2; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Neoplastic Stem Cells; NF-kappa B; Oncogene Proteins; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFβ conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TβRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFβ also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TβRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFβ that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Neoplasm Invasiveness; Promoter Regions, Genetic; Prostatic Neoplasms; Serine; Snail Family Transcription Factors; TNF Receptor-Associated Factor 6; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

This study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor β (TGFβ) pathway, which induces potent migration and invasion responses in breast cancer cells.. We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFβ/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFβ-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging-based matrix degradation assay, respectively.. Relatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFβ through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFβ/Smad signaling pathway.. BCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFβ/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3's molecular mechanism of action and highlight BCAR3 as a novel TGFβ/Smad antagonist in breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Disease-Free Survival; Female; Guanine Nucleotide Exchange Factors; Humans; MCF-7 Cells; Prognosis; RNA, Messenger; Signal Transduction; Smad Proteins; Tamoxifen; Transforming Growth Factor beta

We validated a model of the TGF-β signaling pathway using reactions from Reactome. Using a patentpending technique, gene expression profiles from individual patients are used to determine model parameters. Gene expression profiles from 45 women, normal, or benign tumor and malignant breast cancer were used as training and validating sets for assessing clinical sensitivity and specificity. Biomarkers were identified from the biosimulation results using sensitivity analyses and derivative properties from the model. A membrane signaling marker had sensitivity of 80% and specificity of 60%; while a nuclear transcription factor marker had sensitivity of 80% and specificity of 90% to predict malignancy. Use of Fagan's nomogram increased probability from 7.5% for positive mammogram to 39% with positive results of the biosimulation for the nuclear marker. Our technology will allow researchers to identify and develop biomarkers and assist clinicians in diagnostic and treatment decision making.

Topics: Biomarkers, Tumor; Breast Neoplasms; Computer Simulation; Female; Humans; Precision Medicine; Reproducibility of Results; Signal Transduction; Time Factors; Transcriptome; Transforming Growth Factor beta

The establishment of lethal metastases depends on the capacity of a small number of cancer cells to regenerate a tumor after entering a target organ. Stankic and colleagues have identified a role for the inhibitor of differentiation protein, ID1, as a critical regulator of breast cancer stem-like properties and metastatic colonization. Under the control of tumor growth factor-beta signaling, ID1 induces mesenchymal-epithelial transition at the metastatic site by antagonizing the activity of the basic helix-loop-helix transcription factor Twist1. This study sheds light on mechanisms that initiate metastatic outgrowth, and strengthens the concept that epithelial-mesenchymal plasticity is crucial at different stages of metastasis.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; Nuclear Proteins; Transforming Growth Factor beta; Twist-Related Protein 1

Deletion of tumor suppressor genes in stromal fibroblasts induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. However, the underlying mechanisms remain to be elucidated. Here we report that deletion of the gene encoding TGFβ receptor 2 (Tgfbr2) in the stromal fibroblasts (Tgfbr2(fspKO)) induces inflammation and significant DNA damage in the neighboring epithelia of the forestomach. This results in loss or down-regulation of cyclin-dependent kinase inhibitors p15, p16, and p21, which contribute to the development of invasive squamous cell carcinoma (SCC). Anti-inflammation treatment restored p21 expression, delayed tumorigenesis, and increased survival of Tgfbr2(fspKO) mice. Our data demonstrate for the first time that inflammation is a critical player in the epigenetic silencing of p21 in tumor progression. Examination of human esophageal SCC showed a down-regulation of TGFβ receptor 2 (TβRII) in the stromal fibroblasts, as well as increased inflammation, DNA damage, and loss or decreased p15/p16 expression. Our study suggests anti-inflammation may be a new therapeutic option in treating human SCCs with down-regulation of TβRII in the stroma.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor Proteins; Down-Regulation; Epigenesis, Genetic; Epithelial Cells; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Fibroblasts; Humans; Inflammation; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

SnoN is a negative regulator of TGF-β signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the snoN gene, a strong correlation between increased SnoN copy number and inactivation of p53 was detected, suggesting that the tumor suppressor SnoN-p53 pathway must be inactivated, either through downregulation of SnoN or inactivation of p53, in order to allow cancer cell to proliferate and survive. These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues.

Topics: Adult; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Progression; Esophageal Neoplasms; Esophagus; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Middle Aged; Ovarian Neoplasms; Ovary; Pancreas; Pancreatic Neoplasms; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

TGF-β promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). Understanding the molecular and epigenetic mechanisms by which TGF-β induces EMT may facilitate the development of new therapeutic strategies for metastasis. Here, we report that TGF-β induced SNAI2 to promote EMT by repressing miR-203. Although miR-203 targeted SNAI2, SNAI2 induced by TGF-β could directly bind to the miR-203 promoter to inhibit its transcription. SNAI2 and miR-203 formed a double negative feedback loop to inhibit each other's expression, thereby controlling EMT. Moreover, we found that miR-203 was significantly down-regulated in highly metastatic breast cancer cells. The restoration of miR-203 in highly metastatic breast cancer cells inhibited tumor cell invasion in vitro and lung metastatic colonization in vivo by repressing SNAI2. Taken together, our results suggest that the SNAI2 and miR-203 regulatory loop plays important roles in EMT and tumor metastasis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Dogs; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; RNA, Neoplasm; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) is a potent modifier of the malignant phenotype in ErbB2-expressing breast cancers. We demonstrate that epithelial-derived breast cancer cells, which undergo a TGFβ-induced epithelial-to-mesenchymal transition (EMT), engage signaling molecules that normally facilitate cellular migration and invasion of mesenchymal cells. We identify lipoma preferred partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We show that LPP re-localizes to focal adhesion complexes upon TGFβ stimulation and is a critical determinant in TGFβ-mediated focal adhesion turnover. Finally, we have determined that the interaction between LPP and α-actinin, an actin cross-linking protein, is necessary for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Thus, our data reveal that LPP, which is normally operative in cells of mesenchymal origin, can be co-opted by breast cancer cells during an EMT to promote their migration and invasion.

Topics: Actinin; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; LIM Domain Proteins; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Multiprotein Complexes; Neoplasm Invasiveness; Receptor, ErbB-2; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) has received noteworthy attention in the recent past due to its unique characteristic of functionally switching roles from tumor suppressor to metastasis promoter. To uncover the black box surrounding the mechanisms of TGF-β, Taylor and colleagues performed global miRNA expression analyses using a murine mammary carcinoma progression model. They discovered multiple miRNA regulated by TGF-β and matrix stiffness. Focusing on miR-181a, they uncovered an intricate pathway regulating breast cancer metastasis that sheds new insight into metastasis regulation that may prove useful in clinical settings.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; MicroRNAs; Transforming Growth Factor beta

The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor's metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, -435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These findings qualify the RTCA assay as an in vitro migration/invasion testing system for patient-specific primary breast cancer cells.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cytological Techniques; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Time Factors; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

TGF-β plays an important role in breast cancer progression as a prometastatic factor, notably through enhancement of cell migration. It is becoming clear that microRNAs, a new class of small regulatory molecules, also play crucial roles in mediating tumor formation and progression. We found TGF-β to down-regulate the expression of the microRNA miR-584 in breast cancer cells. Furthermore, we identified PHACTR1, an actin-binding protein, to be positively regulated by TGF-β in a miR-584-dependent manner. Moreover, we found TGF-β-mediated down-regulation of miR-584 and increased expression of PHACTR1 to be required for TGF-β-induced cell migration of breast cancer cells. Indeed, both overexpression of miR-584 and knockdown of PHACTR1 resulted in a drastic reorganization of the actin cytoskeleton and reduced TGF-β-induced cell migration. Our data highlight a novel signaling route whereby TGF-β silences the expression of miR-584, resulting in enhanced PHACTR1 expression, and further leading to actin rearrangement and breast cancer cell migration.

Topics: Actins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cytoskeleton; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Microfilament Proteins; MicroRNAs; Neoplasm Proteins; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a potent inhibitor of the growth of normal mammary epithelial cells, and has a pleiotropic, context-dependent, concentration-dependent action. We found attenuation of TGF-β signalling in mammary adenoma carcinoma cells. Phosphorylation at the linker site of Smad2 occurred in a cooperative way during the attenuation of TGF-β signalling, and was associated with upregulation of CDK2 and cyclin D1. CDK2 inhibitor restored the anti-proliferative effect of TGF-β by upregulating p21, with inhibition of linker phosphorylation of Smad2. CDK2-mediated linker phosphorylation of Smad2 may be a plausible mechanism for the attenuation of TGF-β signalling in breast cancer.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phosphorylation; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

MicroRNA-181 (miR-181) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. While miR-181 is often overexpressed in human tumors, a direct role for this miRNA in breast cancer progression has not yet been characterized. In this study, we found this miRNA to be regulated by both activin and TGFβ. While we found no effect of miR-181 modulation on activin/TGFβ-mediated tumor suppression, our data clearly indicate that miR-181 plays a critical and prominent role downstream of two growth factors, in mediating their pro-migratory and pro-invasive effects in breast cancer cells miR-181 acts as a metastamir in breast cancer. Thus, our findings define a novel role for miR-181 downstream of activin/TGFβ in regulating their tumor promoting functions. Having defined miR-181 as a critical regulator of tumor progression in vitro, our results thus, highlight miR-181 as an important potential therapeutic target in breast cancer.

Topics: Activins; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Invasiveness; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. Transforming growth factor-βeta (TGF-β) and Chemokine (C-X-C Motif) Ligand-1 (CXCL1) are cytokines involved in the colonization of distant sites by CTCs in several pre-clinical animal models. However, their role is poorly-investigated in patients with metastatic cancer. Here, we investigated whether circulating levels of TGF-β and CXCL1 are predictors of CTC seeding in preferential distant sites in patients with metastatic breast cancer.. CTCs were isolated from the peripheral blood of 61 patients with metastatic breast cancer by immunomagnetic separation. Plasma samples were collected from the same patients and assayed for TGF-β and CXCL1 by enzyme-linked immunoassay.. Patients were grouped in CK1+/- (N<10), CK2+ (N ≥ 10<50) and CK3+ (N ≥ 50), according to the number (N) of cytokeratin 7/8-positive CTCs: the highest number of CK7/8-positive CTCs was detected in patients with negative Human epidermal growth factor receptor-2 (HER-2/NEU) status (p<0.0001) antigen, identified by the monoclonal antibody Ki-67 (Ki-67) ≥ 15% (p=0.003), Carcinoma antigen 15-3 (CA-15.3) ≥ 40 U/ml (p=0.004) and those with lung metastases (p=0.01). We found that elevated plasma concentrations of TGF-β and CXCL1 are predictive for the detection of CTCs. In particular, patients with CK3+ CTCs and plasma concentrations of TGF-β and CXCL1 higher than the median value had a poor prognosis in comparison to patients with CK1+/- CTCs and TGF-β and CXCL1 concentrations below the median value.. Our study shows that elevated circulating levels of TGF-β and CXCL1 are associated with a poor prognosis, and higher detection of CTCs and propensity of these cells to seed lung metastases in patients with breast cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Chemokine CXCL1; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Lung Neoplasms; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Prognosis; Survival Rate; Transforming Growth Factor beta; Young Adult

Heparansulfate-proteoglycans (HSPGs) interact via their polyanionic heparansulfate (HS) side chains with a variety of proteins on the cell surface or within the extracellular matrix membrane. The large number of heparin/HS binding proteins form a highly interconnected functional network, which has been termed as the heparin/HS interactome and is functionally linked to physiological and pathological processes. The aim of this study was to investigate the global effect of these protein-HSPG interactions on the tumourigenicity of two breast cancer cell lines (MCF-7 and MDA-MB-231). Cancer cells were cultured in serum-free medium and treated with a concentration of heparin which was capable of modulating HS/ligand interaction. Microarray analysis of MCF-7 cells cultured under these conditions showed that expression of 105 of 1,357 genes potentially related to the pathogenesis of breast neoplasm was significantly altered by heparin treatment. The changes in gene expression correlated with a less tumourigenic phenotype, including reduction of cell adhesive, invasive and migratory properties. These effects were associated with an inhibition of the PI3K/Akt and Raf/MEK/ERK signalling pathways. The modulatory effect of heparin on HS-associated activity was confirmed with one example of heparin/HS interactomes, transforming growth factor β (TGFβ). The innate TGFβ activity of MCF-7 cells was reduced by heparin treatment, with specific interruption of the TGFβ-Smad signalling pathway. The pro-tumourigenic contribution of the heparin/HS interactomes was verified in cells in which HSPG synthesis was blocked using β-xyloside. In conclusion, the interaction between cell surface HPSGs and innate heparin/HS interactomes makes a significant contribution to the tumourigenicity.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Culture Media, Serum-Free; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Heparin; Heparitin Sulfate; Humans; Ligands; MAP Kinase Signaling System; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Protein Binding; Proteoglycans; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (ß1TGFß1) can promote proliferation in late stage cancers but acts as a tumor suppressor in normal epithelial cells and in early stage cancers. Although, the TGFß pathway has been shown to play a key role in tumorigenesis and metastasis, only a limited number of models have been developed to understand this process. Here, we present a novel model system to discern this paradoxical role of TGFß1 using the MDA-MB-231 (MB-231) cell line. The MB-231 triple-negative breast cancer cell line has been extensively characterized and has been shown to continue to proliferate and undergo epithelial-to-mesenchymal transition (EMT) upon TGFß1 stimulation. We have previously shown by microarray analysis that expression of GATA3 in MB-231 cells results in reprogramming of these cells from a basal to a luminal subtype associated with a reduction of metastasis and tumorigenesis when implanted as xenografts. We now demonstrate that GATA3 overexpression in these cells results in a reduction of TGFß1 response, reversal of EMT, and most importantly, restoration of sensitivity to the inhibitory effects on proliferation of TGFß1. Microarray analysis revealed that TGFß1 treatment resulted in reduction of several cell cycle effectors in 231-GATA3 cells but not in control cells. Furthermore, our microarray analysis revealed a significant increase of BMP5 in 231-GATA3 cells. We demonstrate that combined treatment of MB-231 control cells with TGFß1 and BMP5 results in a significant reduction of cellular proliferation. Thus, this model offers a means to further investigate potentially novel mechanisms involved in the switch in response to TGFß1 from tumor promoter to tumor suppressor through the reprogramming of a triple-negative breast cancer cell line by the GATA3 transcription factor.

Topics: Biomarkers, Tumor; Bone Morphogenetic Protein 5; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

The epithelial-to-mesenchymal transition (EMT) is important for the development of cancer metastases and organ fibrosis, conditions prevalent in aging. Because sirtuins affect the pathology of aging, we tested the effect of SirT1 on EMT. Reduced SIRT1 levels in HMLER breast cancer cells led to increased metastases in nude mice, and the loss of SIRT1 in kidney tubular epithelial cells exacerbated injury-induced kidney fibrosis. SIRT1 reduces EMT in cancer and fibrosis by deacetylating Smad4 and repressing the effect of TGF-β signaling on MMP7, a Smad4 target gene. Consequently, less E-cadherin is cleaved from the cell surface and β-catenin remains bound to E-cadherin at the cell-cell junctions. Our findings suggest that the SIRT1/Smad4/β-catenin axis may be a target for diseases driven by EMT.

Topics: Animals; beta Catenin; Breast Neoplasms; Cadherins; Cell Line; Cell Movement; Epithelial-Mesenchymal Transition; Female; Fibrosis; Humans; Kidney; Matrix Metalloproteinase 7; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Neoplasm Metastasis; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirtuin 1; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

In this study, two invasive mammary carcinoma cells (MDA-MB-231 and 4T1) were utilized to evaluate the inhibitory activities of platycodin D, osthole, and the two in combination. The anti-proliferative effect was tested using the MTT and BrdU assay, and the combination of 15μM osthole and 75μM platycodin D was used for subsequent analyses. The anti-invasive effect was evaluated by the transwell assay. The results showed that the combination treatment reduced both cell proliferation and invasion. Western blot and real-time PCR revealed that the platycodin D-osthole combination significantly decreased TβRII, Smad2, Smad3 and Smad4 gene or protein expressions, as well as effectively blocked TGF-β-induced phosphorylation of Smad2 and Smad3. Thus, this study demonstrates that the anti-cancer effects of the platycodin D-osthole combination in breast cancer cells involve proliferation inhibition and invasion blockade, both of which may be mediated by perturbations in the TGF-β/Smads pathway.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Coumarins; Gene Expression Regulation, Neoplastic; Humans; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Saponins; Smad Proteins; Transforming Growth Factor beta; Triterpenes

In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumour cells (DTCs) are kept dormant, and what wakes them up, are fundamental problems in tumour biology. To address these questions, we used metastasis assays in mice and showed that dormant DTCs reside on microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumour-promoting factors derived from endothelial tip cells. Our work reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth.

Topics: Animals; Bone Marrow Neoplasms; Brain Neoplasms; Breast Neoplasms; Cell Adhesion Molecules; Endothelium, Vascular; Female; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Mice; Neoplasm, Residual; Neovascularization, Pathologic; Pericytes; Stem Cell Niche; Thrombospondin 1; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment; Zebrafish

Activated cancer-associated fibroblasts (CAFs) or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16(INK4A) and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-β, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated β-galactosidase (SA-β-gal) activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16(INK4A)-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts.

Topics: Actins; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Movement; Cellular Senescence; Curcumin; Cyclin-Dependent Kinase Inhibitor p16; DNA Damage; Dose-Response Relationship, Drug; Female; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-6; Ki-67 Antigen; Lamin Type B; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; STAT3 Transcription Factor; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Multifunctional Y-box Binding Protein-1 (YB1) is correlated with a poor outcome in breast cancer. We found YB1 expression to be regulated by antiestrogens commonly used in the hormonal therapy of breast cancer and known as activators of Transforming Growth Factor-β (TGFβ). Thus, a putative influence of YB1 on TGFβ signaling should be investigated.. The effect of YB1 on TGFβ signaling was monitored by expression analysis and reporter gene assays in breast cancer cells overexpressing YB1 and treated with antiestrogens.. Antiestrogen-mediated inhibition of estrogen receptor-α led to a suppression of YB1 protein synthesis. On the other hand, YB1 was found to be an enhancer of TGFβ signaling.. High levels of YB1 expression lead to a stimulation of TGFβ pathways, thereby counteracting antihormonal breast cancer therapy and representing a putative resistance mechanism.

Topics: Breast Neoplasms; Cell Line, Tumor; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor Modulators; Estrogens; Female; Fulvestrant; Humans; Signal Transduction; Survival Rate; Tamoxifen; Transforming Growth Factor beta; Y-Box-Binding Protein 1

Mesenchymal stem cells (MSCs) are known to migrate to tumor tissues. This behavior of MSCs has been exploited as a tumor-targeting strategy for cell-based cancer therapy. However, the effects of MSCs on tumor growth are controversial. This study was designed to determine the effect of MSCs on the growth of breast and prostate tumors.. Bone marrow-derived MSCs (BM-MSCs) were isolated and characterized. Effects of BM-MSCs on tumor cell proliferation were analyzed in a co-culture system with mouse breast cancer cell 4T1 or human prostate cancer cell DU145. Tumor cells were injected into nude mice subcutaneously either alone or coupled with BM-MSCs. The expression of cell proliferation and angiogenesis-related proteins in tumor tissues were immunofluorescence analyzed. The angiogenic effect of BM-MSCs was detected using a tube formation assay. The effects of the crosstalk between tumor cells and BM-MSCs on expression of angiogenesis related markers were examined by immunofluorescence and real-time PCR.. Both co-culturing with mice BM-MSCs (mBM-MSCs) and treatment with mBM-MSC-conditioned medium enhanced the growth of 4T1 cells. Co-injection of 4T1 cells and mBM-MSCs into nude mice led to increased tumor size compared with injection of 4T1 cells alone. Similar experiments using DU145 cells and human BM-MSCs (hBM-MSCs) instead of 4T1 cells and mBM-MSCs obtained consistent results. Compared with tumors induced by injection of tumor cells alone, the blood vessel area was greater in tumors from co-injection of tumor cells with BM-MSCs, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. Furthermore, both conditioned medium from hBM-MSCs alone and co-cultures of hBM-MSCs with DU145 cells were able to promote tube formation ability of human umbilical vein endothelial cells. When hBM-MSCs are exposed to the DU145 cell environment, the expression of markers associated with neovascularization (macrophage inflammatory protein-2, vascular endothelial growth factor, transforming growth factor-beta and IL-6) was increased.. These results indicate that BM-MSCs promote tumor growth and suggest that the crosstalk between tumor cells and BM-MSCs increased the expression of pro-angiogenic factors, which may have induced tumor cell proliferation and angiogenesis thereby increasing solid tumor growth.

Topics: Animals; Bone Marrow Cells; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Female; Humans; Interleukin-6; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Nude; Neovascularization, Pathologic; Prostatic Neoplasms; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To evaluate the relationship between IL-11 and bone metastasis in patients with breast cancer and explore the potential molecular mechanism, total serum samples were collected from 180 breast cancer patients and 20 women without breast cancer. The serum expression level of interleukin (IL)-11, connective tissue growth factor (CTGF), transforming growth factor-β, and Tracp5b was determined by enzyme-linked immunosorbent assay, and mRNA expression of IL-11 in fresh breast cancer tissue was determined by RT-PCR. Immunohistochemical staining was used to detect the expression of IL-11 and CTGF in breast cancer tissue, and Western blot was used to detect the expression of p-38, p-C-JUN, p-STAT3, and p-gp130 in fresh breast cancer tissue. DNA-binding activity of AP-1 was examined by electrophoretic mobility shift assay. Differences were statistically analyzed between the group with breast cancer metastatic to bone (MBC-B) and the group with only primary breast cancer (PBC). Serum level and mRNA expression of IL-11 in the MBC-B group were significantly higher than those in the PBC group. IL-11 immunohistochemical staining showed that the percentage of positively stained cells in the MBC-B group (57.5 %) was significantly higher than that in the PBC group (14.29 %). Western blot analysis showed higher expression of p-p38, p-C-JUN, p-STAT3, and p-gp130 in the MBC-B group than in the PBC group. DNA-binding activity of AP-1 was significantly higher in the MBC-B group than in the PBC group. These data suggest that IL-11 is associated with bone metastasis and may be of value for predicting bone metastasis from breast cancer.

Topics: Acid Phosphatase; Adult; Aged; Bone Neoplasms; Breast Neoplasms; Connective Tissue Growth Factor; Cytokine Receptor gp130; Female; Humans; Interleukin-11; Isoenzymes; Middle Aged; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-jun; RNA, Messenger; STAT3 Transcription Factor; Tartrate-Resistant Acid Phosphatase; Transcription Factor AP-1; Transforming Growth Factor beta

Histone deacetylases (HDACs) have been shown to play important roles in the regulation of chromatin remodeling by histone deacetylation, and their expression is induced in several types of cancer. In addition, they are known to be associated with resistance to anticancer drugs. However, the relevance of HDAC4 in chemoresistance remains unclear. Therefore, we investigated the interaction between HDAC4 expression and chemoresistance in breast cancer cells. We found that increased HDAC4 expression in MDA-MB-231 cells was associated with resistance to the anticancer drug 5-fluorouracil (5-FU). To verify these results, a cell line stably overexpressing HDAC4 was generated using MCF-7 cells (HDAC4OE). This cell line displayed increased 5-FU resistance, and HDAC4 knockdown in HDAC4OE cells restored 5-FU sensitivity. Consequently, we concluded that HDAC4 is a critical gene associated with 5‑FU chemoresistance. Further investigation using a microarray approach revealed that 355 genes were differentially expressed following HDAC4 overexpression. Based on functional annotation of the array results, HDAC4 overexpression was found to downregulate genes related to the transforming growth factor (TGF) β signaling pathway, including SMAD4, SMAD6, bone morphogenetic protein 6, inhibitor of DNA binding 1 and TGFβ2. We also found that HDAC4 expression regulates SMAD4 expression by inducing deacetylation of histone H3 in the SMAD4 promoter region. In addition, SMAD4 knockdown in MCF‑7 cells increased 5-FU resistance. In summary, our data suggest that HDAC4‑mediated deacetylation of the SMAD4 promoter may lead to 5-FU resistance in breast cancer cells.

Topics: Acetylation; Antimetabolites, Antineoplastic; Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Chromatin Immunoprecipitation; Drug Resistance, Neoplasm; Female; Fluorouracil; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Histones; Humans; MCF-7 Cells; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFβ. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.

Topics: Animals; Breast Neoplasms; Chromatin; Epithelial Cells; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Hyaluronan Receptors; Mice; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Neoplastic Stem Cells; Promoter Regions, Genetic; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Epithelial to mesenchymal transition (EMT) is a secondary phenomenon concomitantly associated with the tumor progression. The regulatory signals and mechanistic details of EMT are not fully elucidated. Here, we shared a TGF-β mediated mechanism of EMT in breast cancer (MDA-MB 231) cells. Initial exposure of TGF-β for 48 h, enhanced the rate of cell proliferation and associated with EMT of MDA-MB 231 cells. The EMT was characterized by observing the increased N-cadherin, fibronectin, Snail expression and associated with the morphological change with a reduced E-cadherin expression. NFAT, a transcription factor, alters tumor suppressive function of TGF-β towards tumor progression. Up regulation of NFAT, coupled with a foremost translocation of one oncogenic protein SnoN from cytoplasm to nucleus was noticed during this TGF-β mediated EMT. Silencing of NFAT also showed the inhibition of TGF-β mediated EMT characterized by down regulation of N-cadherin and associated with reduced expression of SnoN. In addition, it was also observed that NFAT sequestering the Smad3 prevents the proteasome mediated degradation of SnoN and this SnoN has a role on the regulation of MMP-2, MMP-9 activity. Increased Smad3-SnoN interaction and proteasome mediated degradation of SnoN were detected after silencing of NFAT with a reduced MMP-2, MMP-9 activity. All of these observations provide a fresh mechanism in which by a twofold involvement of NFAT and SnoN plays a crucial role in TGF-β mediated EMT by recruiting the effector molecules N-cadherin and MMP-2, MMP-9.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cadherins; Cell Adhesion; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Fibronectins; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; NFATC Transcription Factors; Proto-Oncogene Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

Normal cellular phenotypes that serve an oncogenic function during tumorigenesis are potential candidates for cancer targeting drugs. Within a subset of invasive primary breast carcinoma, we observed relatively abundant expression of Tetherin, a cell surface protein encoded by the Bone Marrow Stromal Cell Antigen (BST2) known to play an inhibitory role in viral release from infected immune cells of the host. Using breast cancer cell lines derived from low and intermediate histopathologic grade invasive primary tumors that maintain growth-suppressive TGFβ signaling, we demonstrate that BST2 is negatively regulated by the TGFβ axis in epithelial cells. Binding of the transcription factor AP2 to the BST2 promoter was attenuated by inhibition of the TGFβ pathway thereby increasing BST2 expression in tumor cells. In contrast, inherent TGFβ resistance characteristic of high grade breast tumors is a key factor underlying compromised BST2 regulation, and consequently its constitutive overexpression relative to non-malignant breast epithelium, and to most low and intermediate grade cancer cells. In both 2-dimensional and 3-dimensional growth conditions, BST2-silenced tumor cells displayed an enhancement in tamoxifen or staurosporine-induced apoptotic cell death together with a reduction in the S-phase fraction compared to BST2 overexpressing counterparts. In a subset of breast cancer patients treated with pro apoptotic hormonal therapy, BST2 expression correlated with a trend for poor clinical outcome, further supporting its role in conferring an anti apoptotic phenotype. Similar to the effects of gene manipulation, declining levels of endogenous BST2 induced by the phytoalexin - resveratrol, restored apoptotic function, and curbed cell proliferation. We provide evidence for a direct approach that diminishes aberrant BST2 expression in cancer cells as an early targeting strategy to assist in surmounting resistance to pro apoptotic therapies.

Topics: Antigens, CD; Antineoplastic Agents, Hormonal; Apoptosis; Base Sequence; Binding Sites; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Fatty Acid-Binding Proteins; Female; Gene Expression; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Kaplan-Meier Estimate; Molecular Sequence Data; Promoter Regions, Genetic; Proportional Hazards Models; Protein Binding; Resveratrol; Stilbenes; Tamoxifen; Transforming Growth Factor beta

Activation-induced cytidine deaminase (AID), which functions in antibody diversification, is also expressed in a variety of germ and somatic cells. Evidence that AID promotes DNA demethylation in epigenetic reprogramming phenomena, and that it is induced by inflammatory signals, led us to investigate its role in the epithelial-mesenchymal transition (EMT), a critical process in normal morphogenesis and tumor metastasis. We find that expression of AID is induced by inflammatory signals that induce the EMT in nontransformed mammary epithelial cells and in ZR75.1 breast cancer cells. shRNA-mediated knockdown of AID blocks induction of the EMT and prevents cells from acquiring invasive properties. Knockdown of AID suppresses expression of several key EMT transcriptional regulators and is associated with increased methylation of CpG islands proximal to the promoters of these genes; furthermore, the DNA demethylating agent 5 aza-2'deoxycytidine (5-Aza-dC) antagonizes the effects of AID knockdown on the expression of EMT factors. We conclude that AID is necessary for the EMT in this breast cancer cell model and in nontransformed mammary epithelial cells. Our results suggest that AID may act near the apex of a hierarchy of regulatory steps that drive the EMT, and are consistent with this effect being mediated by cytosine demethylation. This evidence links our findings to other reports of a role for AID in epigenetic reprogramming and control of gene expression.

Topics: Animals; Azacitidine; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; CpG Islands; Cytidine Deaminase; Decitabine; DNA Methylation; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Genetic Complementation Test; HEK293 Cells; Humans; Mammary Glands, Human; Matrix Metalloproteinases; Mice; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Tamoxifen resistance remains to be a huge obstacle in the treatment of hormone-dependent breast cancer, and this therefore highlights the dire need to explore the underlying mechanisms. The epithelial-mesenchymal transition (EMT) is a molecular process through which an epithelial cell transfers into a mesenchymal phenotype. Roles of EMT in embryo development, cancer invasion and metastasis have been extensively reported. Herein, we established tamoxifen-resistant MCF-7/TR breast cancer cells and showed that MCF-7/TR cells underwent EMT driven by enhanced endogenous TGF-β/Smad signaling. Ectopic supplement of TGF-β promoted in MCF-7 cells a mesenchymal and resistant phenotype. In parallel, we demonstrated that resveratrol was capable of synergizing with tamoxifen and triggering apoptosis in MCF-7/TR cells. Further Western blot analysis indicated that the chemosensitizing effects of resveratrol were conferred with its modulation on endogenous TGF-β production and Smad phosphorylation. In particular, 50 μM resveratrol had minor effects on MCF-7/TR cell proliferation, but could significantly attenuate endogenous TGF-β production and the Smad pathway, ultimately leading to reversion of EMT. Collectively, our study highlighted distinct roles of EMT in tamoxifen resistance and resveratrol as a potential agent to overcome acquired tamoxifen resistance. The molecular mechanism of resveratrol chemosensitizing effects is, at least in part, TGF-β/Smad-dependent.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Estrogen Antagonists; Female; Humans; MCF-7 Cells; Resveratrol; Signal Transduction; Smad Proteins; Stilbenes; Tamoxifen; Transforming Growth Factor beta

The homeodomain protein TGIF (TG-interacting factor) restricts TGF-β/Smad cytostatic signaling by interfering with the nucleocytoplasmic transit of the tumor suppressor cPML. Here, we identify PHRF1 as a ubiquitin ligase that enforces TGIF decay by driving its ubiquitination at lysine 130. In so doing, PHRF1 ensures redistribution of cPML into the cytoplasm, where it associates with SARA and coordinates activation of Smad2 by the TGF-β receptor. The PHRF1 gene resides within the tumor suppressor locus 11p15.5, which displays frequent loss in a wide variety of malignancies, including breast cancer. Remarkably, we found that the PHRF1 gene is deleted or silenced in a high proportion of human breast cancer samples and cancer cell lines. Reconstitution of PHRF1 into deficient cells impeded their propensity to form tumors in vivo, most likely because of the reemergence of TGF-β responsiveness. These findings unveil a paradigm behind inactivation of the cPML tumor suppressor network in human malignancies.

Topics: Animals; Breast Neoplasms; Dogs; Female; Genes, Tumor Suppressor; Hep G2 Cells; Homeodomain Proteins; Humans; Interferon Regulatory Factor-7; Madin Darby Canine Kidney Cells; Nuclear Proteins; Phosphorylation; Promyelocytic Leukemia Protein; Repressor Proteins; RNA, Messenger; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitination

Cordyceps militaris (C. militaris) and its main functional component, cordycepin, has been shown to possess a number of pharmacological activities including immunological stimulation and antitumor effects. However, the pharmacological mechanisms of C. militaris on tumor immunity underlying its antitumor effect have yet to be elucidated. In the present study, we evaluated the antitumor and immunomodulatory effects of C. militaris on FM3A tumor-bearing C3H/He mice, comparing wild-type C. militaris and cordycepin-enriched C. militaris (JLM 0636). The concentration of cordycepin produced by crossbred JLM 0636 was 7.42 mg/g dry weight, which was 7-fold higher than that of wild-type C. militaris. Dietary administration of C. militaris revealed retardation of tumor growth as well as elongation of survival rates of tumor-bearing mice. This effect was more pronounced in JLM 0636. There was a cordycepin-dependent decrease in IL-2 and TGF-β secretion and an increase in IL-4 secretion without changes in the proliferative responses of concanavalin A-stimulated lymphocytes, which suggested that C. militaris feeding might induce changes in the subpopulations of tumor-derived T lymphocytes. CD4+CD25+ cell population was significantly reduced in the total splenocytes from JLM 0636-administered mice, while CD4+ T cell population remained unchanged. FoxP3+-expressing Treg cells among CD4+CD25+ population showed a similar pattern. On the contrary, CD8+ T cells as well as the IFN-γ expressing CD8+ T cells from tumor-bearing mice were significantly upregulated by the administration of JLM 0636. These results demonstrated the suppressive role of JLM 0636 on the function of Treg cells contributing to tumor specific IFN-γ-expressing CD8+ T cell responses in tumor-bearing mice, which explained the underlying mechanism of the antitumor immunity of cordycepin. Therefore, cordycepin-enriched C. militaris is a promising candidate for an adjuvant in cancer immunotherapy.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Cordyceps; Deoxyadenosines; Female; Forkhead Transcription Factors; Immunomodulation; Immunotherapy; Interferon-gamma; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Mice; Mice, Inbred C3H; Survival Rate; Transforming Growth Factor beta

The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFβ/Smad signalling activity linked to epithelial-mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFβ, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFβ-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFβ/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFβ-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Case-Control Studies; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Humans; Immunoprecipitation; Luciferases; Male; Melanoma; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Prognosis; Prostatic Neoplasms; Protein Isoforms; RNA, Small Interfering; Smad3 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

During breast cancer progression, transforming growth factor-beta (TGF-β) switches from acting as a growth inhibitor to become a major promoter of epithelial-mesenchymal transition (EMT), invasion and metastasis. However, the mechanisms involved in this switch are not clear. We found that loss of CCAAT-enhancer binding protein beta (C/EBPβ), a differentiation factor for the mammary epithelium, was associated with signs of EMT in triple-negative human breast cancer, and in invasive areas of mammary tumors in MMTV-PyMT mice. Using an established model of TGF-β-induced EMT in mouse mammary gland epithelial cells, we discovered that C/EBPβ was repressed during EMT by miR-155, an oncomiR in breast cancer. Depletion of C/EBPβ potentiated the TGF-β response towards EMT, and contributed to evasion of the growth inhibitory response to TGF-β. Furthermore, loss of C/EBPβ enhanced invasion and metastatic dissemination of the mouse mammary tumor cells to the lungs after subcutaneous injection into mice. The mechanism by which loss of C/EBPβ promoted the TGF-β response towards EMT, invasion and metastasis, was traced to a previously uncharacterized role of C/EBPβ as a transcriptional activator of genes encoding the epithelial junction proteins E-cadherin and coxsackie virus and adenovirus receptor. The results identify miR-155-mediated loss of C/EBPβ as a mechanism, which promotes breast cancer progression by shifting the TGF-β response from growth inhibition to EMT, invasion and metastasis.

Topics: Animals; Base Sequence; Binding Sites; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Mice; MicroRNAs; Models, Biological; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Binding; RNA Interference; Transcriptional Activation; Transforming Growth Factor beta

Cancer stem cells (CSCs) are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, the identification of molecules involved in CSC self-renewal is a necessary step toward more effective therapies. To this aim, through the transcription profiling of the murine ErbB2(+) tumor cell line TUBO vs. derived CSC-enriched mammospheres, Toll-like receptor 2 (TLR2) was identified as 2-fold overexpressed in CSCs, as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA-MB-231 (50%), HCC1806 (60%), and MCF7 (50%) cells. In CSC, TLR2 was activated by endogenous high-mobility-group box 1 (HMGB1), inducing IκBα phosphorylation, IL-6 and TGFβ secretion, and, consequently, STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2-fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively, these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self-renewal, tumorigenesis, and metastatic ability. These findings, while providing evidence against the controversial use of TLR2 agonists in antitumor therapy, lay out new paths toward the design of anticancer treatments.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Female; HMGB1 Protein; Humans; I-kappa B Kinase; Interleukin-6; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Neovascularization, Pathologic; Smad3 Protein; STAT3 Transcription Factor; Toll-Like Receptor 2; Transcription, Genetic; Transforming Growth Factor beta

TGF-β signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-β-induced SMAD and non-SMAD signaling. Upon TGF-β stimulation, TRAF4 is recruited to the active TGF-β receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-β type I receptor (TβRI). Both processes contribute to TβRI stabilization on the plasma membrane and thereby enhance TGF-β signaling. In addition, the TGF-β receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-β-activated kinase (TAK)1. TRAF4 is required for efficient TGF-β-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-β pathway and is a key determinant in breast cancer pathogenesis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Mice; Phosphorylation; Polyubiquitin; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; TNF Receptor-Associated Factor 4; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin-specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

Topics: Animals; Breast Neoplasms; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Humans; Immunoblotting; Integrin beta1; Integrin beta3; Lung Neoplasms; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; RNA Interference; Transforming Growth Factor beta; Tumor Burden

Recently, the scientific community has begun to establish the health benefits of deep-sea water (DSW) due to its enrichment in nutrients and minerals. In this study, we investigated the effects of deep-sea water (DSW) on the metastatic potential of two human breast cancer cell lines exhibiting highly different phenotypes. MDA-MB-231 cells exhibit invasive/metastatic tumor features with rapid migration ability and high endogenous expression of TGF-β and Wnt5a. DSW treatment significantly inhibits their migratory ability in a wound-healing assay. This inhibitory effect of DSW appears to be mediated through TGF-β and Wnt5a signaling, resulting in attenuated expression of CD44. We further investigated the preventive effect of DSW on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasive/metastatic tumor features in non-invasive MCF-7 cells. Similar to the inhibitory effects shown in MDA-MB-231 cells, we observed that DSW treatment resulted in the inhibition of TPA-induced migration and MMP-9 activity with a concomitant decrease in mRNA levels of MMP-9, TGF-β, Wnt5a and Wnt3a. Taken together, our data show that DSW has inhibitory effects on breast cancer invasion/metastasis, suggesting that DSW has some promise in improving cancer survival by preventing tumor metastasis.

Topics: Blotting, Western; Breast Neoplasms; Cell Movement; Female; Flow Cytometry; Humans; Matrix Metalloproteinase 9; Mineral Waters; Neoplasm Metastasis; Proto-Oncogene Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Seawater; Transforming Growth Factor beta; Tumor Cells, Cultured; Wnt Proteins; Wnt-5a Protein; Wound Healing

Epithelial-to-mesenchymal transition (EMT) facilitates the escape of epithelial cancer cells from the primary tumor site, which is a key event early in metastasis. Here, we explore how extrinsic, tumor microenvironmental cytokines cooperate with intrinsic, genetic changes to promote EMT in human mammary epithelial cells (HMECs). Viral transduction of transforming genetic events into HMECs routinely generated two distinct cell populations. One population retained epithelial characteristics, while an emergent population spontaneously acquired a mesenchymal morphology and properties associated with cancer stem cells (CSCs). Interestingly, the spontaneous mesenchymal/CSCs were unable to differentiate and lacked epithelial-mesenchymal plasticity. In contrast, exposure of the transformed HMECs retaining epithelial characteristics to exogenous transforming growth factor-β (TGF-β) generated a mesenchymal/CSC population with remarkable plasticity. The TGF-β-induced mesenchymal/CSC population was dependent on the continued presence of TGF-β. Removal of TGF-β or pharmacologic or genetic inhibition of TGF-β/SMAD signaling led to the reversion of mesenchymal/CSC to epithelial/non-CSC. Our results demonstrate that targeting exogenous cytokine signaling disrupts epithelial-mesenchymal plasticity and may be an effective strategy to inhibit the emergence of circulating tumor cells. The model of epithelial-mesenchymal plasticity we describe here can be used to identify novel tumor microenvironmental factors and downstream signaling that cooperate with intrinsic genetic changes to drive metastasis. Understanding the interaction between extrinsic and intrinsic factors that regulate epithelial-mesenchymal plasticity will allow the development of new therapies that target tumor microenvironmental signals to reduce metastasis.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Glands, Human; Mesenchymal Stem Cells; Mice; Mice, Nude; Neoplasm Transplantation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

The TGF-β signaling pathway has a significant role in breast cancer initiation and promotion by regulating various cellular processes. We evaluated whether genetic variation in eight genes (TGF-β1, TGF-β2, TGF-βR1, TGF-βR2, TGF-βR3, RUNX1, RUNX2, and RUNX3) is associated with breast cancer risk in women from the Breast Cancer Health Disparities Study. A total of 3,524 cases (1,431 non-Hispanic whites (NHW); 2,093 Hispanics/Native Americans(NA)) and 4,209 population-based controls (1,599 NHWs; 2,610 Hispanics/NAs) were included in analyses. Genotypes for 47 single nucleotide polymorphisms (SNPs) were determined. Additionally, 104 ancestral informative markers estimated proportion of NA ancestry. Associations with breast cancer risk overall, by menopausal status, NA ancestry, and estrogen receptor (ER)/progesterone receptor tumor phenotype were evaluated. After adjustment for multiple comparisons, two SNPs were significantly associated with breast cancer risk: RUNX3 (rs906296 ORCG/GG = 1.15 95 % CI 1.04-1.26) and TGF-β1 (rs4803455 ORCA/AA = 0.89 95 % CI 0.81-0.98). RUNX3 (rs906296) and TGF-βR2 (rs3773644) were associated with risk in pre-menopausal women (p adj = 0.002 and 0.02, respectively) and in those with intermediate to high NA ancestry (p adj = 0.04 and 0.01, respectively). Self-reported race was strongly correlated with NA ancestry (r = 0.86). There was a significant interaction between NA ancestry and RUNX1 (rs7279383, p adj = 0.04). Four RUNX SNPs were associated with increased risk of ER- tumors. Results provide evidence that genetic variation in TGF-β and RUNX genes are associated with breast cancer risk. This is the first report of significant associations between genetic variants in TGF-β and RUNX genes and breast cancer risk among women of NA ancestry.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Breast Neoplasms; Case-Control Studies; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Genotype; Hispanic or Latino; Humans; Menopause; Middle Aged; Polymorphism, Single Nucleotide; Receptors, Estrogen; Receptors, Progesterone; Risk; Signal Transduction; Southwestern United States; Transforming Growth Factor beta; White People; Young Adult

Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitory Concentration 50; Mice; Neoplasm Metastasis; Plant Extracts; Plant Roots; Solubility; Transforming Growth Factor beta; Tumor Burden; Withania; Xenograft Model Antitumor Assays

Tamoxifen is the most widely used anti-estrogen for the treatment of breast cancer. Studies show that the combination therapy with other substances that helps the activity of tamoxifen. The objective of this study was to evaluate the effect of tamoxifen when used in combination with tranilast on human breast cancer cells.. Two MCF-7 and MDA-MB-231 human breast cancer cell lines were treated with tamoxifen and/or tranilast. The cell viability and cytotoxicity was assessed using MTT and LDH assays; the apoptotic effects were examined by TUNEL assay, acridine orange/ethidium bromide staining and DNA laddering, also the expression levels of bax and bcl-2 genes were detected by real-time RT-PCR. The mRNA expression of TGF-β ligands and receptors examined using real-time RT-PCR and TGF-β1 protein secretion levels were also evaluated by ELISA assay. Inhibitory effect of these drugs on invasion and metastasis were tested by wound healing and matrigel invasion assay.. These findings indicate that tranilast, by synergistic effect, enhances the activity of tamoxifen and the TGF-β pathway is a target for this combination therapy, therefore; we propose that this combined treatment may be suitable selection in prevention of breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Proliferation; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; ortho-Aminobenzoates; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Transforming Growth Factor beta

Carcino-embryonic antigen-related cell adhesion molecule 6 (CEACAM6), one of the members of human carcino-embryonic antigens, is a multifunctional regulatory protein involved in various cellular processes in cancers. Its role in malignant transformation and the clinical significance has been extensively studied in colonic and pancreatic cancers. However, relatively few studies have been done on breast cancers. In the current study, CEACAM6 expression in two independent cohorts of invasive breast cancers were evaluated immunohistochemically and correlated with clinico-pathological features, biomarker profiles and patient survival. In the primary cohort, CEACAM6 expression was detected in 37.1 % (312/840) of primary invasive cancers. It was positively correlated with HER2 (p < 0.001). Concordantly, HER2-OE subtype showed the highest CEACAM6 expression (62.7 %) among all molecular subtypes; whereas, other subtypes also showed substantial CEACAM6 expression (21.8-37.5 %). Interestingly, a significantly worse overall survival was found in high pN stage HER2 positive cancers with CEACAM6 positivity (log-rank = 4.452, p = 0.035) and this could be validated in an independent cohort. Additionally, HER2 signaling was found to induce SMAD3 phosphorylation and CEACAM6 expression in a cell line model. Likewise, in the primary tumors, a positive association was found between HER2 and SMAD3 phosphorylation in CEACAM6 positive cancers (p = 0.012). Overall, CEACAM6 was widely expressed in different molecular subtypes, but highest and significantly in HER2-OE breast cancer. Within this group, CEACAM6 was associated with adverse high nodal stage patient outcome. Given the wide expression of CEACAM6 in all breast cancers, its roles as prognostic marker and therapeutic target warrant further evaluation.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers, Tumor; Breast Neoplasms; Cell Adhesion Molecules; Cohort Studies; Disease-Free Survival; Female; GPI-Linked Proteins; Humans; Middle Aged; Prognosis; Receptor, ErbB-2; Smad3 Protein; Transforming Growth Factor beta; Young Adult

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

Topics: Animals; Benzamides; Breast Neoplasms; Chromones; Dioxoles; Dipeptides; Disease Models, Animal; Drug Screening Assays, Antitumor; Embryo, Nonmammalian; Enzyme Inhibitors; Female; Humans; Matrix Metalloproteinase Inhibitors; Morpholines; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad4 Protein; Small Molecule Libraries; Transforming Growth Factor beta; Xenograft Model Antitumor Assays; Zebrafish; Zebrafish Proteins

Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (P = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (P = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity in vitro, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.

Topics: Animals; Antibodies; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Survival; Disease Models, Animal; Doxorubicin; Female; Mice; Osteoblasts; Oxidative Stress; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) is a potent and context-dependent regulator of tumor progression. TGFβ promotes the lung metastasis of basal-like (but not the luminal-like) breast cancer. Here, we demonstrated that fascin, a pro-metastasis actin bundling protein, was a direct target of the canonical TGFβ-Smad4 signaling pathway in basal-like breast cancer cells. TGFβ and Smad4 induced fascin overexpression by directly binding to a Smad binding element on the fascin promoter. We identified GATA3, a transcription factor crucial for mammary gland morphogenesis and luminal differentiation, as a negative regulator of TGFβ- and Smad4-induced fascin overexpression. When ectopically expressed in basal-like breast cancer cells, GATA-3 abrogated TGFβ- and Smad4-mediated overexpression of fascin and other TGFβ response genes, invadopodium formation, cell migration, and invasion, suggesting suppression of the canonical TGFβ-Smad signaling axis. Mechanistically, GATA3 abrogated the canonical TGFβ-Smad signaling by abolishing interactions between Smad4 and its DNA binding elements, potentially through physical interactions between the N-terminal of GATA3 and Smad3/4 proteins. Our findings provide mechanistic insight into how TGFβ-mediated cell motility and invasiveness are differentially regulated in breast cancer.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Female; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Microfilament Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Response Elements; Signal Transduction; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

Triple-negative breast cancer (TNBC) is defined by a lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor 2 (HER 2). Therefore, targeted therapy agents may not be used, and therapy is largely limited to chemotherapy. Doxorubicin treatment consequently acquires undesired malignance characteristics [i.e., epithelial-mesenchymal transition (EMT) and multi-drug resistance]. Our results illustrated that doxorubicin triggered EMT and resulted in the acquisition of a mesenchymal phenotype in TNBC cells. Moreover, we found that transforming growth factor-β (TGF-β) and PI3K/AKT signaling pathways were acquired for doxorubicin-induced EMT. Interestingly, we found that curcumin suppressed doxorubicin-induced EMT. Curcumin reversed doxorubicin-induced morphological changes, inhibited doxorubicin-induced downregulation of E-cadherin expressions, and inhibited doxorubicin-induced upregulation of vimentin expression. We also found that curcumin inhibited doxorubicin-induced EMT by inhibiting the TGF-β and PI3K/AKT signaling pathways. Moreover, curcumin enhanced the antiproliferative effects of doxorubicin in TNBC cells. In summary, our results suggest that doxorubicin in combination with curcumin may be a potential therapy for TNBC.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Curcumin; Down-Regulation; Doxorubicin; Epithelial-Mesenchymal Transition; Female; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

To investigate the effect and the relevant molecular mechanisms of leptin on the migration and invasion of human breast cancer MCF-7 cells.. The expression of OB-R in MCF-7 cells was measured by RT-PCR and Western blotting. The effects of leptin (100 ng/mL) on the the phosphorylation of a few key cell signaling proteins, p-ERK1/2, p-STAT3, p-AKT in MCF-7 cells were examined by Western blotting. Cell scratch assay and Transwell(TM); assay were utilized to measure the effects of leptin on the migration and invasion capability of MCF-7 cells, respectively. The effects of leptin on the mRNA and protein expression of matrix metalloproteinas 9 (MMP-9) and transforming growth factor β (TGF-β) were measured by RT-PCR and Western blotting.. Both OB-Rb and OB-Rt were expressed in MCF-7 cells. This indicated that leptin may have significant activities in MCF7 cells. Indeed, leptin increased the phosphorylation of p-ERK1/2, p-STAT3, and p-AKT in MCF-7 cells (P < 0.05). Further, leptin promoted migration and invasion of MCF-7 cells, which were attenuated by the JAK/STAT inhibitor AG490 (50 μmol/L), and the PI3K/AKT inhibitor LY294002 (10 μmol/L) (P < 0.05). Similarly, leptin also increased the mRNA and protein expression of MMP-9 and TGF-β, and these effects were blocked by AG490 and LY294002 as well (P < 0.05).. Leptin promoted the migration and invasion capabilities of MCF-7 cells. These activities may be achieved by the upregulation of MMP-9 and TGF-β through JAK/STAT and PI3K/AKT signaling pathways.

Topics: Breast Neoplasms; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Leptin; Matrix Metalloproteinase 9; MCF-7 Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Phosphoproteins; Proto-Oncogene Proteins c-akt; Receptors, Leptin; STAT3 Transcription Factor; Transforming Growth Factor beta

ID genes are required for breast cancer colonization of the lungs, but the mechanism remains poorly understood. Here, we show that Id1 expression induces a stem-like phenotype in breast cancer cells while retaining epithelial properties, contrary to the notion that cancer stem-like properties are inextricably linked to the mesenchymal state. During metastatic colonization, Id1 induces a mesenchymal-to-epithelial transition (MET), specifically in cells whose mesenchymal state is dependent on the Id1 target protein Twist1, but not at the primary site, where this state is controlled by the zinc finger protein Snail1. Knockdown of Id expression in metastasizing cells prevents MET and dramatically reduces lung colonization. Furthermore, Id1 is induced by transforming growth factor (TGF)-β only in cells that have first undergone epithelial-to-mesenchymal transition (EMT), demonstrating that EMT is a prerequisite for subsequent Id1-induced MET during lung colonization. Collectively, these studies underscore the importance of Id-mediated phenotypic switching during distinct stages of breast cancer metastasis.

Topics: Breast Neoplasms; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; MCF-7 Cells; Neoplastic Stem Cells; Nuclear Proteins; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

Topics: Animals; Breast Neoplasms; Cell Line; Disease Progression; Female; Heterografts; Imidazoles; Luciferases; Luciferases, Firefly; Luminescent Measurements; Mice; Molecular Imaging; Neoplasm Transplantation; Pyrazines; Signal Transduction; Substrate Specificity; Transfection; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) programs are essential in promoting breast cancer invasion, systemic dissemination and in arousing proliferative programs in breast cancer micrometastases, a reaction that is partially dependent on focal adhesion kinase (FAK). Many functions of FAK are shared by its homolog, protein tyrosine kinase 2 (Pyk2), raising the question as to whether Pyk2 also participates in driving the metastatic outgrowth of disseminated breast cancer cells. In addressing this question, we observed Pyk2 expression to be (i) significantly upregulated in recurrent human breast cancers; (ii) differentially expressed across clonal isolates of human MDA-MB-231 breast cancer cells in a manner predictive for metastatic outgrowth, but not for invasiveness; and (iii) dramatically elevated in ex vivo cultures of breast cancer cells isolated from metastatic lesions as compared with cells that produced the primary tumor. We further show that metastatic human and murine breast cancer cells robustly upregulate their expression of Pyk2 during EMT programs stimulated by transforming growth factor-β (TGF-β). Genetic and pharmacological inhibition of Pyk2 demonstrated that the activity of this protein tyrosine kinase was dispensable for the ability of breast cancer cells to undergo invasion in response to TGF-β, and to form orthotopic mammary tumors in mice. In stark contrast, Pyk2-deficiency prevented TGF-β from stimulating the growth of breast cancer cells in 3D-organotypic cultures that recapitulated pulmonary microenvironments, as well as inhibited the metastatic outgrowth of disseminated breast cancer cells in the lungs of mice. Mechanistically, Pyk2 expression was inversely related to that of E-cadherin, such that elevated Pyk2 levels stabilized β1 integrin expression necessary to initiate the metastatic outgrowth of breast cancer cells. Thus, we have delineated novel functions for Pyk2 in mediating distinct elements of the EMT program and metastatic cascade regulated by TGF-β, particularly the initiation of secondary tumor outgrowth by disseminated cells.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Kinase 2; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Signal Transduction; Smad4 Protein; src-Family Kinases; Transforming Growth Factor beta

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program.

Topics: Breast Neoplasms; Cell Line, Tumor; Enzyme Activation; Humans; Matrix Metalloproteinases; Mesoderm; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Spheroids, Cellular; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Mediator Complex; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-β signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-β signaling biomarkers.. The expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-β receptors I and II (TβRI and TβRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-β receptors.. Tumors with high expression of TβRII, TβRI and TβRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-β receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390-6.658] and 2.20 (95% CI 1.464-3.307), respectively, for disease relapse.. Combining TGF-β biomarkers provides prognostic information for patients with stage I-III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.

Topics: Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Female; Humans; Receptor, ErbB-2; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad4 Protein; Tissue Array Analysis; Transforming Growth Factor beta

TGF-β is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-β production or signaling plays a pivotal role in a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-β inhibitors should be promising therapeutic agents for the suppression of cancer progression and metastasis as well as fibrotic disorders. In a screening program of natural compounds from fungi inhibiting the TGF-β dependent expression of a reporter gene in HepG2 cells, we found that the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-β including reporter gene activation and expression of TGF-β induced genes in HepG2 and MDA-MB-231 cells. The most active compound oxacyclododecindione inhibited TGF-β dependent reporter activity with IC50-values of 190-217 nM. In an in vitro angiogenesis assay, the fungal lactones strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Genes, Reporter; Hep G2 Cells; Humans; Lactones; Macrocyclic Compounds; Neovascularization, Physiologic; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Zearalenone

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-β-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 μM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-β signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-β induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Curcumin; Disease Models, Animal; Female; Humans; Mice; Molecular Structure; Osteolysis; Parathyroid Hormone-Related Protein; Signal Transduction; Transforming Growth Factor beta

In vitro and in vivo study.. To evaluate the role of recombinant human bone morphogenetic protein-2 (rhBMP2) on breast cancer cell (MDA-MB-231 cells) growth.. Bone morphogenetic proteins (BMPs) are expressed in a variety of human carcinoma cell lines and are known to promote tumor invasion and metastasis. However, their roles in tumor progression have not been fully clarified. In addition, there is no in vivo study regarding the inhibitory effect of BMP2 on breast cancer cell proliferation.. Cell proliferation was determined by BrdU incorporation assay and flow cytometry. BMP2 signal transduction pathways were estimated on Western blot. Fifteen animals were divided into 2 groups; 1 (control = 5) was breast cancer cells alone, while the other (experiment = 5) was rhBMP2 + breast cancer cells. Cancer cells were injected into 2 sites (subcutaneous and femur) of nude mice with or without BMP2. Tumor size was determined by direct measurements for subcutaneous tumor formation and by femur radiographs. Histological and immunohistochemical analyses were performed.. RhBMP2 inhibited the proliferation of MDA-MB-231 cells in vitro. Inhibition was associated with changes in both the Smad and Wnt signaling pathways and was ultimately mediated through effects on various cell cycle proteins. Furthermore, rhBMP2 inhibited the growth of MDA-MB-231 cells injected both subcutaneously and intrafemorally.. In this model using human breast adenocarcinoma cell line, rhBMP2 has no stimulatory effect of tumor growth. Therefore, we can provide the basic science data to support the utilization in the management of patients with spine tumor in the future.

Topics: Animals; Blotting, Western; Bone Morphogenetic Protein 2; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Humans; Mice; Mice, Nude; Recombinant Proteins; Smad Proteins; Transforming Growth Factor beta; Tumor Burden; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Ductal carcinoma in situ (DCIS) is an early stage noninvasive breast cancer that originates in the epithelial lining of the milk ducts, but it can evolve into comedo DCIS and ultimately, into the most common type of breast cancer, invasive ductal carcinoma. Understanding the progression and how to effectively intervene in it presents a major scientific challenge. The extracellular matrix (ECM) surrounding a duct contains several types of cells and several types of growth factors that are known to individually affect tumor growth, but at present the complex biochemical and mechanical interactions of these stromal cells and growth factors with tumor cells is poorly understood. Here we develop a mathematical model that incorporates the cross-talk between stromal and tumor cells, which can predict how perturbations of the local biochemical and mechanical state influence tumor evolution. We focus on the EGF and TGF-β signaling pathways and show how up- or down-regulation of components in these pathways affects cell growth and proliferation. We then study a hybrid model for the interaction of cells with the tumor microenvironment (TME), in which epithelial cells (ECs) are modeled individually while the ECM is treated as a continuum, and show how these interactions affect the early development of tumors. Finally, we incorporate breakdown of the epithelium into the model and predict the early stages of tumor invasion into the stroma. Our results shed light on the interactions between growth factors, mechanical properties of the ECM, and feedback signaling loops between stromal and tumor cells, and suggest how epigenetic changes in transformed cells affect tumor progression.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Epidermal Growth Factor; Female; Humans; Mathematical Concepts; Models, Biological; Neoplasm Invasiveness; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

Vimentin is an intermediate filament protein, with a key role in the epithelial to mesenchymal transition as well as cell invasion, and it is often upregulated during cancer progression. However, relatively little is known about its regulation in cancer cells. Here, we performed an RNA interference screen followed by protein lysate microarray analysis in bone metastatic MDA-MB-231(SA) breast cancer cells to identify novel regulators of vimentin expression. Out of the 596 genes investigated, three novel vimentin regulators EPHB4, WIPF2 and MTHFD2 were identified. The reduced vimentin expression in response to EPHB4, WIPF2 and MTHFD2 silencing was observed at mRNA and protein levels. Bioinformatic analysis of gene expression data across cancers indicated overexpression of EPHB4 and MTHFD2 in breast cancer and high expression associated with poor clinical characteristics. Analysis of 96 cDNA samples derived from both normal and malignant human tissues suggested putative association with metastatic disease. MTHFD2 knockdown resulted in impaired cell migration and invasion into extracellular matrix as well as decreased the fraction of cells with a high CD44 expression, a marker of cancer stem cells. Furthermore, MTHFD2 expression was induced in response to TGF-β stimulation in breast cancer cells. Our results show that MTHFD2 is overexpressed in breast cancer, associates with poor clinical characteristics and promotes cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion.

Topics: Aminohydrolases; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cadherins; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Methylenetetrahydrofolate Dehydrogenase (NADP); Microfilament Proteins; Microscopy, Confocal; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Receptor, EphB4; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transforming Growth Factor beta; Vimentin

The APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation.. The localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity.. APC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton.. These findings indicate that membrane protrusions with APC/β-catenin-containing puncta control the migratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later stages of tumor progression might impact tumor cell dissemination or colonization.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Cell Shape; Cell Surface Extensions; Cytoskeleton; Dogs; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Genes, Reporter; HCT116 Cells; Humans; Madin Darby Canine Kidney Cells; Mice; Microscopy, Phase-Contrast; Multiprotein Complexes; Neoplasm Invasiveness; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Time Factors; Transfection; Transforming Growth Factor beta

Expression of the Notch ligand Jagged 1 (JAG1) and Notch activation promote poor-prognosis in breast cancer. We used high throughput screens to identify elements responsible for Notch activation in this context. Chemical kinase inhibitor and kinase-specific small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK-ERK signaling to be the predominant JAG1/Notch regulator and this was supported by gene set enrichment analyses in 51 breast cancer cell lines. In accordance with the chemical screen, kinome small interfering RNA high throughput screens identified Tribbles homolog 3 (TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrate that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Complementary in vitro and in vivo studies underscore the importance of TRB3 for tumor growth. These data demonstrate a dominant role for TRB3 and MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer, establishing TRB3 as a potential therapeutic target.

Topics: Animals; Blotting, Western; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Female; Hep G2 Cells; Humans; Intercellular Signaling Peptides and Proteins; Interleukin Receptor Common gamma Subunit; Jagged-1 Protein; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Receptor, Notch1; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Serrate-Jagged Proteins; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

A recent Cell publication demonstrates that the secreted antagonist of transforming growth factor-beta ligands, Coco, can re-activate previously dormant metastatic breast cancer cells specifically in the lung by inhibiting bone morphogenetic protein (BMP) signaling. The authors provided evidence for a connection between Coco/BMP signaling and molecular and cellular traits of cancer stem cells. Their findings represent a significant advance in our understanding of metastatic dormancy, an extremely important clinical issue that remains understudied. Equally as important, this study also opens interesting avenues for future research.

Topics: Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Intercellular Signaling Peptides and Proteins; Signal Transduction; Transforming Growth Factor beta

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

Topics: Adaptor Proteins, Signal Transducing; Blotting, Western; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Colforsin; Cyclic AMP; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Humans; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins

Epithelial-mesenchymal transition (EMT) of adherent epithelial cells to a migratory mesenchymal state has been implicated in tumor metastasis in preclinical models. To investigate its role in human cancer, we characterized EMT in circulating tumor cells (CTCs) from breast cancer patients. Rare primary tumor cells simultaneously expressed mesenchymal and epithelial markers, but mesenchymal cells were highly enriched in CTCs. Serial CTC monitoring in 11 patients suggested an association of mesenchymal CTCs with disease progression. In an index patient, reversible shifts between these cell fates accompanied each cycle of response to therapy and disease progression. Mesenchymal CTCs occurred as both single cells and multicellular clusters, expressing known EMT regulators, including transforming growth factor (TGF)-β pathway components and the FOXC1 transcription factor. These data support a role for EMT in the blood-borne dissemination of human breast cancer.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Count; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Mice; Neoplasm Transplantation; Neoplastic Cells, Circulating; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon-α (IFN-α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor-associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN-α, the production by Toll-like receptor (TLR)-activated healthy pDC of IFN-β and TNF-α but not IP-10/CXCL10 nor MIP1-α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF-β and TNF-α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF-β1 and TNF-α synergistically blocked IFN-α production of TLR-activated pDC, and neutralization of TGF-β and TNF-α in tumor-derived supernatants restored pDCs' IFN-α production. The involvment of tumor-derived TGF-β was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF-7 expression and nuclear translocation in pDC after their exposure to tumor-derived supernatants or recombinant TGF-β1 and TNF-α. Our findings indicate that targeting TApDC to restore their IFN-α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9-based immunotherapy with TGF-β and TNF-α antagonists.

Topics: Breast Neoplasms; Chemokine CCL3; Chemokine CXCL10; Dendritic Cells; Female; Humans; Interferon Regulatory Factor-7; Interferon-alpha; Interferon-beta; Phosphorylation; Protein Transport; Recombinant Proteins; Smad2 Protein; Toll-Like Receptor 7; Toll-Like Receptor 9; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Gene Expression; Gene Knockdown Techniques; Humans; Interleukin-8; Mice; Mice, Nude; Neoplastic Stem Cells; Paclitaxel; Protein Serine-Threonine Kinases; Pyrazoles; Quinolines; Receptor, ErbB-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, Estrogen; Receptors, Progesterone; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Spheroids, Cellular; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor beta-1 (TGF-β1) participation in breast cancer development and metastasis is well-established, however, the clinical meaning of its circulating levels in women with breast cancer is poorly understood.. To characterize the levels of TGF-β1 in plasma from women with breast cancer and to associate them with the main clinical factors associated with disease prognosis.. TGF-β1 levels were measured by Enzyme-linked immunoassay (ELISA). Clinicopathological data were also assessed.. Women bearing triple-negative tumors presented significantly reduced levels of this cytokine when compared to the other subtypes (p=0.0338). Patients with metastases exhibited lower levels of TGF-β1 than the non-metastatic cohort (p=0.0442). Patients with early-onset disease had the highest plasma TGF-β1 levels (p=0.0036). Doxorubicin chemotherapy induced a reduction in TGF-β1 level, promptly after drug infusion (p=0.0494). Patients with TGF-β1 levels lower than 20 pg/ml exhibited a tendency to have a reduced overall survival in a 40-month follow-up.. Lower levels of circulating TGF-β1 are associated with a poor disease prognosis.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Enzyme-Linked Immunosorbent Assay; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Prognosis; Transforming Growth Factor beta

Epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (IDC) were found to express the ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP; ABCG2). Also, low BCRP expression was present on CD34(+) blood DC precursors and expression was increased upon their differentiation to LC. The CD34(+) acute myeloid leukemia-derived DC cell line MUTZ3 can be cultured into LC or IDC, depending on the cytokine cocktail used. Introduction of functional BCRP in MUTZ3 progenitor cells through retroviral transduction resulted in the emergence of typical LC-characteristics in IDC cultures; the majority of cells remained negative for the IDC-specific C-type lectin DC-SIGN, but rather displayed enhanced expression of the LC-specific C-type lectin Langerin and characteristic high expression levels of CD1a. BCRP-induced skewing toward LC-like differentiation coincided with early RelB expression in 'IDC', derived from MUTZ3-BCRP, and depended on endogenous transforming growth factor beta (TGF-β) production. Intriguingly, cellular BCRP localization differed between skin LC and IDC, and a more cytoplasmic BCRP localization, as observed in primary skin LC, seemed to relate to LC-like differentiation in IDC cultures upon BCRP introduction in MUTZ3 progenitors. Together these data support a role for BCRP in preferential LC differentiation from CD34(+) myeloid DC progenitors.

Topics: Antigens, CD1; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Dendritic Cells; Female; Hematopoietic Stem Cells; Humans; Langerhans Cells; Lectins, C-Type; Leukemia, Myeloid, Acute; Neoplasm Proteins; Skin; Transcription Factor RelB; Transforming Growth Factor beta

Six1 is a critical regulator of embryonic development that requires interaction with the Eya family of proteins (Eya1-4) to activate the transcription of genes involved in neurogenesis, myogenesis and nephrogenesis. Although expression of Six1 and Eya family members is predominantly observed in development, their overexpression is observed in numerous cancers. Importantly, both Six1 and Eya have independently been shown to mediate breast cancer metastasis, but whether they functionally interact during tumor progression has not been explored. Herein, we demonstrate that knockdown of Eya2 in MCF7 mammary carcinoma cells reverses the ability of Six1 to induce transforming growth factor-β signaling, as well as to induce characteristics associated with epithelial-mesenchymal transition and cancer stem cells, suggesting that Six1 is dependent on Eya2 to mediate numerous pro-metastatic characteristics. The importance of the Six1-Eya interaction in human breast cancer is underscored by the finding that high levels of Six1 correlate with shortened time to relapse and metastasis as well as decreased survival only when co-expressed with high levels of Eya2. Overall, these data implicate Eya2 as a necessary co-factor for many of the metastasis promoting functions of Six1, suggesting that targeting the Six1-Eya interaction may inhibit breast cancer progression. As Six1 and Eya2 are not highly expressed in most adult tissues, the Six1-Eya interaction may be a valuable future therapeutic target whose inhibition would be expected to impair breast cancer progression while conferring limited side effects.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Neoplasm Metastasis; Neoplastic Stem Cells; Nuclear Proteins; Prognosis; Protein Tyrosine Phosphatases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta

The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.. In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay.. BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion.. Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).

Topics: Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha5; Integrin beta3; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

A hallmark of human cancer is heterogeneity, reflecting the complex series of changes resulting in the activation of oncogenes coupled with inactivation of tumor suppressor genes. Breast cancer is no exception and indeed, many studies have revealed considerable complexity and heterogeneity in the population of primary breast tumors and substantial changes in a recurrent breast tumor that has acquired metastatic properties and drug resistance. We have made use of a Myc-inducible transgenic mouse model of breast cancer in which elimination of Myc activity following tumor development initially leads to a regression of a subset of tumors generally followed by de novo Myc-independent growth. We have observed that tumors that grow independent of Myc expression have gene profiles that are distinct from the primary tumors with characteristics indicative of an epithelial-mesenchymal transition (EMT) phenotype. Phenotypic analyses of Myc-independent tumors confirm the acquisition of an EMT phenotype suggested to be associated with invasive and migratory properties in human cancer cells. Further genomic analyses reveal mouse mammary tumors growing independent of myc have a higher probability of exhibiting a gene signature similar to that observed for human 'tumor-initiating' cells. Collectively, the data reveal genetic alterations that underlie tumor progression and an escape from Myc-dependent growth in a transgenic mouse model that can provide insights to what occurs in human cancers as they acquire drug resistance and metastatic properties.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha; Transforming Growth Factor beta

Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49f(high)CD61(high)-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-β (TGFβ) signaling and exhibited gene expression signatures of stemness, TGFβ signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Female; Humans; Integrin alpha6; Integrin beta3; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Neoplastic Stem Cells; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair and cancer progression in adult tissues. We have recently shown that transforming growth factor (TGF)-β-induced EMT involves isoform switching of fibroblast growth factor receptors by alternative splicing. We performed a microarray-based analysis at single exon level to elucidate changes in splicing variants generated during TGF-β-induced EMT, and found that TGF-β induces broad alteration of splicing patterns by downregulating epithelial splicing regulatory proteins (ESRPs). This was achieved by TGF-β-mediated upregulation of δEF1 family proteins, δEF1 and SIP1. δEF1 and SIP1 each remarkably repressed ESRP2 transcription through binding to the ESRP2 promoter in NMuMG cells. Silencing of both δEF1 and SIP1, but not either alone, abolished the TGF-β-induced ESRP repression. The expression profiles of ESRPs were inversely related to those of δEF1 and SIP in human breast cancer cell lines and primary tumor specimens. Further, overexpression of ESRPs in TGF-β-treated cells resulted in restoration of the epithelial splicing profiles as well as attenuation of certain phenotypes of EMT. Therefore, δEF1 family proteins repress the expression of ESRPs to regulate alternative splicing during TGF-β-induced EMT and the progression of breast cancers.

Topics: Alternative Splicing; Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Nerve Tissue Proteins; Phenotype; Protein Isoforms; Receptors, Fibroblast Growth Factor; RNA-Binding Proteins; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoid-based protocols. Here, we demonstrate that 23-32% of the human ERBB2(+) breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARα. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARα agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib+ATRA. Induction of the retinoid-dependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFβ pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARα.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Synergism; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Amplification; Humans; Lapatinib; Phosphorylation; Quinazolines; Receptor, ErbB-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Smad3 Protein; Transcription, Genetic; Transcriptome; Transforming Growth Factor beta; Tretinoin

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor β (TGF-β)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.

Topics: Breast Neoplasms; Cardiac Myosins; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Down-Regulation; Female; Fetal Proteins; Formins; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Myosin Light Chains; Neoplasm Invasiveness; Nuclear Proteins; Oncogene Proteins, Fusion; Phosphoprotein Phosphatases; Serum Response Factor; Stress Fibers; Trans-Activators; Transforming Growth Factor beta

MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, little is known about how they regulate nuclear factor κB (NF-κB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-κB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-κB activity. Among those, the miR-520/373 family inhibited NF-κB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-β (TGF-β) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-κB and TGF-β pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Interleukin-8; MicroRNAs; Neoplasm Metastasis; NF-kappa B; Parathyroid Hormone-Related Protein; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Estrogen; Receptors, Transforming Growth Factor beta; Signal Transduction; Transcription Factor RelA; Transforming Growth Factor beta

In this work we created electrospun fibrous scaffolds with random and aligned fiber orientations in order to mimic the three-dimensional structure of the natural extracellular matrix (ECM). The rigidity and topography of the ECM environment have been reported to alter cancer cell behavior. However, the complexity of the in vivo system makes it difficult to isolate and study such extracellular topographical cues that trigger cancer cells' response. Breast cancer cells were cultured on these fibrous scaffolds for 3-5 days. The cells showed elongated spindle-like morphology in the aligned fibers, whereas they maintained a mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding showed up-regulation of transforming growth factor β-1 (TGFβ-1) along with other mesenchymal biomarkers, suggesting that these cells undergo epithelial-mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Matrix; Humans; Mice; Nanotechnology; Phenotype; Polyesters; Signal Transduction; Tissue Scaffolds; Transforming Growth Factor beta; Tumor Microenvironment

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-myb; Transforming Growth Factor beta

The aim of our study was to investigate the expression of Smad-7 and Ski proteins in invasive breast carcinomas, to determine their clinicopathological value and their influence on carcinomas biologic behavior. Immunohistochemistry was applied on 150 invasive breast carcinomas to detect the expression of Smad-7 and Ski. Their correlation to clinicopathologic parameters and markers of metastasis was statistically processed using chi-squared test. Overall and disease-free survival was assessed using Kaplan-Meier test and log-rank statistics. Smad-7 was immunodetected in the cytoplasm of cancer cells in 60%, whereas Ski was immunodetected in the cytoplasm and nuclei in 44.5% and 17.6% of the cases, respectively. Smad-7 expression was positively correlated with tumor size, stage, matrix metalloproteinase (MMP)-9, and MMP-14. Cytoplasmic Ski expression was negatively associated with tumor size, stage, and lymph node status, and its nuclear expression was negatively related to histologic grade. Cytoplasmic Ski expression was associated with longer overall and disease-free survival. It appears that two negative regulators of the transforming growth factor-β pathway, Smad-7 and Ski, behave differentially in invasive breast carcinomas. Smad-7 appears to be related with an aggressive phenotype, whereas Ski expression is related to a less aggressive behavior and positively influences patients' survival.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chi-Square Distribution; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Invasiveness; Nuclear Receptor Coactivators; Smad7 Protein; Transforming Growth Factor beta

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Topics: Activins; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mad2 Proteins; Nocodazole; Nuclear Proteins; Plasmids; Poly-ADP-Ribose Binding Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Repressor Proteins; Signal Transduction; Transfection; Transforming Growth Factor beta

The role of TGF-β signaling in tumorigenesis is paradoxical: it can be tumor suppressive or tumor promotional, depending on context. The metastatic regulator, Six1, was recently shown to mediate this switch, providing a novel means to explain this elusive 'TGF-β paradox'. Herein, we identify a mechanism by which Six1 activates the tumor promotional arm of TGF-β signaling, via its ability to upregulate the miR-106b-25 microRNA cluster, and further identify a novel function for this cluster of microRNAs. Although expression of the miR-106b-25 cluster is known to overcome TGF-β-mediated growth suppression via targeting p21 and BIM, we demonstrate for the first time that this same cluster can additionally target the inhibitory Smad7 protein, resulting in increased levels of the TGF-β type I receptor and downstream activation of TGF-β signaling. We further show that the miR-106b-25 cluster is sufficient to induce an epithelial-to-mesenchymal transition and a tumor initiating cell phenotype, and that it is required downstream of Six1 to induce these phenotypes. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-β signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promoting.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; MCF-7 Cells; MicroRNAs; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-myb; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs.Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells.Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated.In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.

Topics: Animals; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Health; HEK293 Cells; HeLa Cells; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; MicroRNAs; Neoplasm Invasiveness; Transforming Growth Factor beta

Casitas B-lineage lymphoma (CBL) protein family functions as multifunctional adaptor proteins and E3 ubiquitin ligases that are implicated as regulators of signaling in various cell types. Recent discovery revealed mutations of proto-oncogenic CBL in the linker region and RING finger domain in human acute myeloid neoplasm, and these transforming mutations induced carcinogenesis. However, the adaptor function of CBL mediated signaling pathway during tumorigenesis has not been well characterized. Here, we show that CBL is highly expressed in breast cancer cells and significantly inhibits transforming growth factor-β (TGF-β) tumor suppressive activity. Knockdown of CBL expression resulted in the increased expression of TGF-β target genes, PAI-I and CDK inhibitors such as p15(INK4b) and p21(Cip1). Furthermore, we demonstrate that CBL is frequently overexpressed in human breast cancer tissues, and the loss of CBL decreases the tumorigenic activity of breast cancer cells in vivo. CBL directly binds to Smad3 through its proline-rich motif, thereby preventing Smad3 from interacting with Smad4 and blocking nuclear translocation of Smad3. CBL-b, one of CBL protein family, also interacted with Smad3 and knockdown of both CBL and CBL-b further enhanced TGF-β transcriptional activity. Our findings provide evidence for a previously undescribed mechanism by which oncogenic CBL can block TGF-β tumor suppressor activity.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin-Dependent Kinases; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Tumor Suppressor; Humans; Mice; Mice, Nude; Plasminogen Activator Inhibitor 1; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-cbl; Signal Transduction; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Transplantation, Heterologous

The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.. On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity.. The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality.. Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Cyclooxygenase 2; Female; Gene Expression Regulation, Neoplastic; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Interleukin-10; Ki-67 Antigen; Lymphatic Metastasis; Middle Aged; Prognosis; Risk; Signal Transduction; Stromal Cells; Survival Rate; Transforming Growth Factor beta; Tumor Microenvironment

The molecular mechanisms that drive triple-negative, basal-like breast cancer progression are elusive. Few molecular targets have been identified for the prevention or treatment of this disease. Here we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered transforming growth factor-β (TGF-β) sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative type II receptor (DNRII) of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-Ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, coexpression of DNRII and H-Ras-V12 rendered HMECs highly tumorigenic and metastatic in vivo in comparison with H-Ras-V12-transformed HMECs that spontaneously escaped H-Ras-V12-induced SLGA. Microarray analysis revealed that p21 was the major player mediating Ras-induced SLGA, and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling was blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-Ras-V12-induced SLGA. Our results identify that autocrine TGF-β signaling is an integral part of the cellular anti-transformation network by suppressing the expression of a host of genes, including p21-regulated genes, that mediate oncogene-induced transformation in basal-like breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Cellular Senescence; Epithelial Cells; ErbB Receptors; Female; Humans; Mammary Glands, Human; Mice; Mice, Nude; Oncogene Protein p21(ras); ras Proteins; Receptors, Estrogen; Receptors, Progesterone; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFβ signaling is controlled by the serine/threonine kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFβ type I receptor and with the inhibitory Smad7 and down-regulates the type I receptor. We now demonstrate that TGFβ induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFβ that leads to epithelial growth arrest. In conclusion, TGFβ induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.

Topics: Animals; Breast Neoplasms; Cell Line, Transformed; Chlorocebus aethiops; COS Cells; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Keratinocytes; Mink; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Respiratory Mucosa; RNA, Small Interfering; Smad7 Protein; Transcriptional Activation; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-β dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-β receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

Topics: Anoikis; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Luminal breast cancer is the most frequently encountered type of human breast cancer and accounts for half of all breast cancer deaths due to metastatic disease. We have developed new in vivo models of disseminated human luminal breast cancer that closely mimic the human disease. From initial lesions in the tibia, locoregional metastases develop predictably along the iliac and retroperitoneal lymph node chains. Tumors cells retain their epithelioid phenotype throughout the process of dissemination. In addition, systemically injected metastatic MCF-7 cells consistently give rise to metastases in the skeleton, floor of mouth, adrenal glands, as well as in the lungs, liver, brain and mammary fat pad. We show that growth of luminal breast cancer metastases is highly dependent on estrogen in a dose-dependent manner and that estrogen withdrawal induces rapid growth arrest of metastatic disease. On the other hand, even though micrometastases at secondary sites remain viable in the absence of estrogen, they are dormant and do not progress to macrometastases. Thus, homing to and seeding of secondary sites do not require estrogen. Moreover, in sharp contrast to basal-like breast cancer metastasis in which transforming growth factor-β signaling plays a key role, luminal breast cancer metastasis is independent of this cytokine. These findings have important implications for the development of targeted anti-metastatic therapy for luminal breast cancer.

Topics: Animals; Biomarkers, Tumor; Blotting, Western; Bone Neoplasms; Breast Neoplasms; Cell Communication; Estrogens; Female; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Neoplasm Micrometastasis; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

A gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.. Gene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.. Extratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.. These results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Claudins; Female; Fibroblasts; Fibrosis; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Nuclear Proteins; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Microenvironment; Twist-Related Protein 1

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Female; Fibroblasts; GTPase-Activating Proteins; HEK293 Cells; Humans; Mutation; Phosphatidylinositol Phosphates; Phosphoproteins; Protein Structure, Tertiary; rac1 GTP-Binding Protein; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

Topics: Animals; Bacterial Capsules; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Heparin; Humans; Interleukin-11; Mice; Mice, Nude; Osteoclasts; RNA, Small Interfering; Sulfotransferases; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is a pleiotropic secretory protein which inhibits and potentiates tumour progression during early and late stage of tumourigenicity, respectively. However, it still remains veiled how TGF-β signalling reveals its two faces. Hoshino et al. (Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim, J. Biochem. 2011;149:55-65) demonstrated a new aspect of TGF-β as a survival factor in highly metastatic breast cancer cells from which TGF-β1 and TGF-β3 are abundantly expressed. They found that TGF-β suppressed the expression of BH3-only protein Bim which promotes programmed death signalling via release of cytochrome c from mitochondria. Further interestingly, forkhead box C1 (Foxc1) whose expression is suppressed upon TGF-β stimulation is involved in the expression of Bim. Based on their results, autocrine TGF-β signalling in certain breast cancers promotes cell survival via inhibition of apoptotic signalling. Thus, the inhibitors for activin receptor-like kinase (ALK)5 kinase might exert a curative influence on certain types of metastatic breast cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autocrine Communication; Breast Neoplasms; Cell Survival; Disease Progression; Female; Humans; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Metastatic breast tumor cells display an epithelial-mesenchymal transition (EMT) that increases cell motility, invasion, and dissemination. Although the transcription factor Twist1 has been shown to contribute to EMT and cancer metastasis, the signaling pathways regulating Twist1 activity are poorly understood. Here, we show that Twist1 is ubiquitously phosphorylated in 90% of 1,532 invasive human breast tumors. Akt/protein kinase B (PKB)-mediated Twist1 phosphorylation promotes EMT and breast cancer metastasis by modulating its transcriptional target TGF-β2, leading to enhanced TGF-β receptor signaling, which in turn maintains hyperactive phosphoinositide 3-kinase (PI3K)/Akt signaling. Preventing phosphorylation of Twist1, as well as depletion of TGF-β2, significantly impaired the metastatic potential of cancer cells in vivo, indicating a key role of phosphorylated Twist1 (phospho-Twist1) in mediating cross-talk between the PI3K/Akt and TGF-β/Smad signaling axes that supports metastatic tumor development. Our results describe a novel signaling event linking PI3K/Akt hyperactivation in tumor cells to direct regulation of Twist1 activation and tumor metastasis.. We identified the first phospho-Twist1 transcriptional target TGF-β2, which mediates cross-talk between PI3K/Akt and TGF-β signaling and promotes tumor metastasis. Our results thus illustrate a direct role of PI3K/Akt signaling in metastatic cancer development and suggest that Twist1 phosphorylation could be a potential therapeutic target in clinical cancer treatment.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Twist-Related Protein 1

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3' UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

Topics: Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-11; MicroRNAs; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-β signaling, with increased transcription of TGF-β target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-β / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

Topics: Animals; Autophagy; Breast Neoplasms; Caveolin 1; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Connective Tissue Growth Factor; Female; Fibroblasts; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Oxidative Stress; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Microenvironment

N-myc downstream-regulated gene 2 (NDRG2) has been studied for its inhibitory effects against growth and metastasis of many tumor cell types. In this study, we showed NDRG2 expression was correlated with favorable recurrence-free survival of patients with breast cancer and inhibited metastasis of breast cancer cells (4T1). NDRG2 expression was examined in 189 breast carcinoma tissues and paired normal breast tissues using immunohistochemistry. Histological and clinicopathological data were correlated using Pearson's chi-square test of independence. NDRG2 expression in human breast cancer tissues was inversely associated with lymph node metastasis and pTNM stage. Furthermore, patients with breast cancer with a high level of NDRG2 expression showed favorable recurrence-free survival (P = 0.038). To study the effect of NDRG2 on metastasis in vivo, we established an NDRG2-overexpressing mouse breast cancer cell line (4T1-NDRG2) and measured the metastasis and survival of 4T1-NDRG2 tumor-bearing mice. To test whether transforming growth factor β (TGF-β)- mediated metastasis of 4T1 was inhibited by NDRG2 expression, TGF-Smad-binding element (SBE)-luciferase activity and/or measurement of active TGF-β were performed in cell or tumor tissue level. 4T1-NDRG2 cells grew gradually and showed less metastatic activity in vivo and low invasiveness in vitro. 4T1-NDRG2 cells showed lower SBE-luciferase activity and lower level of active autocrine TGF-β than 4T1-Mock did. Correctly, our data show that NDRG2 significantly suppress tumor metastasis by attenuating active autocrine TGF-β production, and the attenuation might be typically associated with the favorable recurrence-free survival of patients clinically.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Line, Tumor; Disease-Free Survival; Female; Humans; Lymphatic Metastasis; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Proteins; Recurrence; Transforming Growth Factor beta; Tumor Suppressor Proteins

The stability and membrane localization of the transforming growth factor-β (TGF-β) type I receptor (TβRI) determines the levels of TGF-β signalling. TβRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signalling. USP4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby controlling TβRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TβRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-β and AKT signalling pathways.

Topics: Animals; Breast Neoplasms; Cell Membrane; Cell Movement; Enzyme Stability; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; HEK293 Cells; HeLa Cells; Humans; Mice; Mice, Knockout; Mutation; Neoplasm Invasiveness; Oncogene Proteins; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases; Ubiquitination; Zebrafish

Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs.. We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers.. High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers.. Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterografts; Homeodomain Proteins; Humans; Mice; Neoplastic Stem Cells; Prognosis; Signal Transduction; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with breast cancer progression and metastasis. Here, we report that ectopic overexpression of SOX4 in immortalized human mammary epithelial cells is sufficient for acquisition of mesenchymal traits, enhanced cell migration, and invasion, along with epithelial stem cell properties defined by the presence of a CD44(high)/CD24(low) cell subpopulation. SOX4 positively regulated expression of known EMT inducers, also activating the TGF-β pathway to contribute to EMT. SOX4 itself was induced by TGF-β in mammary epithelial cells and was required for TGF-β-induced EMT. Murine xenograft experiments showed that SOX4 cooperated with oncogenic Ras to promote tumorigenesis in vivo. Finally, in clinical specimens of human breast cancer, we found that SOX4 was abnormally overexpressed and correlated with the triple-negative breast cancer subtype (ER(-)/PR(-)/HER2(-)). Our findings define an important function for SOX4 in the progression of breast cancer by orchestrating EMT, and they implicate this gene product as a marker of poor prognosis in this disease.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Disease Progression; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplastic Stem Cells; Prognosis; SOXC Transcription Factors; Transforming Growth Factor beta

The T box transcription factor TBX2, a master regulator of organogenesis, is aberrantly amplified in aggressive human epithelial cancers. While it has been shown that overexpression of TBX2 can bypass senescence, a failsafe mechanism against cancer, its potential role in tumor invasion has remained obscure. Here we demonstrate that TBX2 is a strong cell-autonomous inducer of the epithelial-mesenchymal transition (EMT), a latent morphogenetic program that is key to tumor progression from noninvasive to invasive malignant states. Ectopic expression of TBX2 in normal HC11 and MCF10A mammary epithelial cells was sufficient to induce morphological, molecular, and behavioral changes characteristic of EMT. These changes included loss of epithelial adhesion and polarity gene (E-cadherin, ß-catenin, ZO1) expression, and abnormal gain of mesenchymal markers (N-cadherin, Vimentin), as well as increased cell motility and invasion. Conversely, abrogation of endogenous TBX2 overexpression in the malignant human breast carcinoma cell lines MDA-MB-435 and MDA-MB-157 led to a restitution of epithelial characteristics with reciprocal loss of mesenchymal markers. Importantly, TBX2 inhibition abolished tumor cell invasion and the capacity to form lung metastases in a Xenograft mouse model. Meta-analysis of gene expression in over one thousand primary human breast tumors further showed that high TBX2 expression was significantly associated with reduced metastasis-free survival in patients, and with tumor subtypes enriched in EMT gene signatures, consistent with a role of TBX2 in oncogenic EMT. ChIP analysis and cell-based reporter assays further revealed that TBX2 directly represses transcription of E-cadherin, a tumor suppressor gene, whose loss is crucial for malignant tumor progression. Collectively, our results uncover an unanticipated link between TBX2 deregulation in cancer and the acquisition of EMT and invasive features of epithelial tumor cells.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Silencing; Humans; Mammary Glands, Human; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; T-Box Domain Proteins; Transcription, Genetic; Transforming Growth Factor beta

We have established several breast cancer cell lines that exhibit a permanent ER-depleted phenotype, induced by shRNA transfection of MCF-7 cells, which afford a useful model for studying acquired endocrine resistance. Previously we showed that MDA-231 as well as ER-silenced cells could invade through simulated extracellular matrix components. However, the contribution of individual serum components responsible for cell invasion was not determined. In the present study, an under-agarose gel assay was used to quantitatively assess the invasive movement of two ER-silenced cell lines (pII and YS2.5) in comparison to the parental MCF-7, the ER negative MDA-231, and normal HBL100 cells, as well as a line that was ER-shRNA transfected but failed to exhibit ER down-regulation (YS1.2). We also examined the effect of the growth factors EGF, IGF-1, TGFβ, PDGFC and RANTES on pII cell invasion and proliferation. All breast cancer cell lines which had reduced ER expression exhibited a serum-dependent invasive ability related to the degree of induced ER loss. TGFβ treatment inhibited pII cell proliferation and enhanced their invasive ability but at a relatively high dose. IGF-1 and EGF enhanced pII cell proliferation, with the latter playing the major role in promoting cell invasion. PDGFC did not affect either process although it is highly expressed in pII cells. Differential effects were observed on activation of Akt and ERK1/2 suggesting their involvement as intracellular mediators of EGF induced invasion, in part through the regulation of matrix metalloproteinase activity. Targeting EGF receptor tyrosine kinase activity by erlotinib resulted in significant inhibition of both pII cell proliferation and directional invasion towards EGF suggesting that this drug has potential therapeutic usefulness for preventing spread of particularly endocrine resistant breast cancer.

Topics: Benzamides; Breast Neoplasms; Cell Movement; Cell Proliferation; Chemokine CCL5; Chromones; Collagenases; Diphenylamine; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Female; Gene Knockdown Techniques; Humans; Insulin-Like Growth Factor I; Lymphokines; MCF-7 Cells; Morpholines; Phosphorylation; Platelet-Derived Growth Factor; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quinazolines; Receptors, Estrogen; RNA, Small Interfering; Transforming Growth Factor beta

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.

Topics: Animals; Autocrine Communication; Autophagy; Breast Neoplasms; Caveolin 1; Cell Line, Tumor; Cell Transformation, Neoplastic; Cellular Reprogramming; Coculture Techniques; Female; Fibroblasts; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Immunohistochemistry; Lactic Acid; Ligands; Mice; Mice, Nude; Mitochondria; Neovascularization, Pathologic; Oxidative Phosphorylation; Oxidative Stress; Paracrine Communication; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays

TGF-β plays a dual role in epithelial carcinogenesis with the potential to either suppress or promote tumor progression. We found that levels of Smad3 mRNA, a critical mediator of TGF-β signaling, are reduced by approximately 60% in human breast cancer. We therefore used conditionally immortalized mammary epithelial cells (IMEC) of differing Smad3 genotypes to quantitatively address the Smad3 requirement for different biologic responses to TGF-β. We found that a two-fold reduction in Smad3 gene dosage led to complex effects on TGF-β responses; the growth-inhibitory response was retained, the pro-apoptotic response was lost, the migratory response was reduced, and the invasion response was enhanced. Loss of the pro-apoptotic response in the Smad3(+/-) IMECs correlated with loss of Smad3 binding to the Bcl-2 locus, whereas retention of the growth-inhibitory response in Smad3 IMECs correlated with retention of Smad3 binding to the c-Myc locus. Addressing the integrated outcome of these changes in vivo, we showed that reduced Smad3 levels enhanced metastasis in two independent models of metastatic breast cancer. Our results suggest that different biologic responses to TGF-β in the mammary epithelium are differentially affected by Smad3 dosage and that a mere two-fold reduction in Smad3 is sufficient to promote metastasis.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Disease Progression; Enhancer Elements, Genetic; Epithelium; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mice; Neoplasm Metastasis; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Smad3 Protein; Transforming Growth Factor beta

Infiltration of immune cells, specifically macrophages, into the tumor microenvironment has been linked to increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathways within mammary epithelial cells, such as the fibroblast growth factor receptor 1 (FGFR1) pathway, induces recruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cell proliferation and angiogenesis. However, the specific mechanisms by which these macrophages are regulated by the preneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1-driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 in mammary glands leads to decreased activity of the TGFβ/Smad3 pathway in macrophages associated with early stage lesions. Further studies show that macrophages have increased expression of inflammatory chemokines that bind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGF pathway had been activated. The increase in these ligands is inhibited following activation of the TGFβ pathway, suggesting that decreased TGFβ signaling contributes to the upregulation of these chemokines. Using coculture studies, we further show that macrophages are capable of promoting epithelial and tumor cell migration and invasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may be important for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these results suggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated with high levels of infiltrating macrophages.

Topics: Animals; Breast Neoplasms; Cell Movement; Cell Separation; Coculture Techniques; Culture Media, Conditioned; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Inflammation Mediators; Ligands; Macrophages; Mammary Glands, Animal; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice; Mice, Transgenic; Neoplasm Invasiveness; Protein Binding; Receptors, Fibroblast Growth Factor; Receptors, Interleukin-8B; Smad3 Protein; Transforming Growth Factor beta

The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion.. We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting.. We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Insulin-Like Growth Factor I; Protein Isoforms; Proto-Oncogene Proteins c-akt; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.

Topics: Breast Neoplasms; Cluster Analysis; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Interleukin-6; MicroRNAs; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

The role of transforming growth factor-beta (TGFβ) in the progression of different molecular subtypes of breast cancer has not been clarified. Here we show that TGFβ increases breast tumour-initiating cell (BTIC) numbers but only in claudin(low) breast cancer cell lines by orchestrating a specific gene signature enriched in stem cell processes that predicts worse clinical outcome in breast cancer patients. NEDD9, a member of the Cas family of integrin scaffold proteins, is necessary to mediate these TGFβ-specific effects through a positive feedback loop that integrates TGFβ/Smad and Rho-actin-SRF-dependent signals. In normal human mammary epithelium, TGFβ induces progenitor activity only in the basal/stem cell compartment, where claudin(low) cancers are presumed to arise. These data show opposing responses to TGFβ in both breast malignant cell subtypes and normal mammary epithelial cell subpopulations and suggest therapeutic strategies for a subset of human breast cancers.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Chromatin Immunoprecipitation; Claudins; Female; Humans; Mammary Glands, Human; Mice; Neoplastic Stem Cells; Phosphoproteins; Stem Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.. A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.. We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.. Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lymphatic Metastasis; Mice; Neoplasm Invasiveness; p300-CBP Transcription Factors; Prognosis; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

Although the role of TGF-β in tumor progression has been studied extensively, its impact on drug delivery in tumors remains far from understood. In this study, we examined the effect of TGF-β blockade on the delivery and efficacy of conventional therapeutics and nanotherapeutics in orthotopic mammary carcinoma mouse models. We used both genetic (overexpression of sTβRII, a soluble TGF-β type II receptor) and pharmacologic (1D11, a TGF-β neutralizing antibody) approaches to block TGF-β signaling. In two orthotopic mammary carcinoma models (human MDA-MB-231 and murine 4T1 cell lines), TGF-β blockade significantly decreased tumor growth and metastasis. TGF-β blockade also increased the recruitment and incorporation of perivascular cells into tumor blood vessels and increased the fraction of perfused vessels. Moreover, TGF-β blockade normalized the tumor interstitial matrix by decreasing collagen I content. As a result of this vessel and interstitial matrix normalization, TGF-β blockade improved the intratumoral penetration of both a low-molecular-weight conventional chemotherapeutic drug and a nanotherapeutic agent, leading to better control of tumor growth.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Neutralizing; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Doxorubicin; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Tissue Distribution; Transforming Growth Factor beta; Treatment Outcome; Tumor Burden; Xenograft Model Antitumor Assays

A discontinuous pattern of LOH at chromosome 3p has been reported in 87% of primary breast cancers. Despite the identification of several tumor suppressor genes in this region, there has yet to be a detailed analysis of noncoding RNAs including miRNAs in this region. In this study, we identified 16 aberrant miRNAs in this region and determined several that are frequently lost or amplified in breast cancer. miR-128-2 was the most commonly deleted miRNA. Embedded in the intron of the ARPP21 gene at chromosome 3p22.3, miR-128-2 was frequently downregulated along with ARPP21 in breast cancer, where it was negatively associated with clinicopathologic characteristics and survival outcome. Forced expression of miR-128 impeded several oncogenic traits of mammary carcinoma cells, whereas depleting miR-128-2 expression was sufficient for oncogenic transformation and stem cell-like behaviors in immortalized nontumorigenic mammary epithelial cells, both in vitro and in vivo. miR-128-2 silencing enabled transforming capacity partly by derepressing a cohort of direct targets (BMI1, CSF1, KLF4, LIN28A, NANOG, and SNAIL), which together acted to stimulate the PI3K/AKT and STAT3 signaling pathways. We also found that miR-128-2 was directly downregulated by SNAIL and repressed by TGF-β signaling, adding 2 additional negative feedback loops to this network. In summary, we have identified a novel TGF-β/SNAIL/miR-128 axis that provides a new avenue to understand the basis for oncogenic transformation of mammary epithelial cells.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes, Human, Pair 3; Down-Regulation; Female; Humans; Kruppel-Like Factor 4; Loss of Heterozygosity; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplastic Stem Cells; Oncogene Protein v-akt; Snail Family Transcription Factors; STAT3 Transcription Factor; Stem Cell Factor; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous

Bone marrow mesenchymal stem cells (BM-MSCs) have multiple therapeutic potentials for regenerative, anti-inflammatory, and immunomodulatory purposes and also show promise as vehicles for gene therapy of various metastatic cancers based on their tumor-tropic capacity. However, BM-MSCs are also a source of carcinoma-associated fibroblasts (CAFs) and may promote growth and metastasis of cancer. Transforming growth factor β (TGF-β) signaling is required to induce CAF differentiation of mouse BM-MSCs in vivo and can induce expression of some CAF markers in human BM-MSCs in vitro. To determine whether inhibiting TGF-β signaling in human BM-MSCs can block their differentiation to CAFs induced by tumor microenvironments and the consequent protumor effects, we transduced human BM-MSCs with a lentiviral vector encoding bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a decoy TGF-β receptor. BAMBI transduction significantly inhibited TGF-β/Smad signaling and expression of CAF markers in human BM-MSCs treated with TGF-β1 or tumor-conditioned medium or cocultured with cancer cells, but did not alter the stem cell properties and the tumor-tropic property of MSCs. In addition, BAMBI transduction disrupted the cytokine network mediating the interaction between MSCs and breast cancer cells. Consequently, BAMBI transduction abolished protumor effects of BM-MSCs in vitro and in an orthotopic breast cancer xenograft model, and instead significantly inhibited growth and metastasis of coinoculated cancer. These results indicated that TGF-β signaling is essential for differentiation of human BM-MSCs to CAFs in tumor microenvironments and the consequent protumor effects, and inhibiting TGF-β/Smad pathway may improve the safety of MSC-based therapies in cancer patients.

Topics: Animals; Bone Marrow Cells; Breast Neoplasms; Cell Culture Techniques; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Female; Fibroblasts; Humans; Membrane Proteins; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Signal Transduction; Smad Proteins; Transduction, Genetic; Transforming Growth Factor beta; Transplantation, Heterologous

TGF-β1 is a multifunctional cytokine that mediates diverse biological processes. However, the mechanisms by which the intracellular signals of TGF-β1 are terminated are not well understood. Here, we demonstrate that DRAK2 serves as a TGF-β1-inducible antagonist of TGF-β signaling. TGF-β1 stimulation rapidly induces DRAK2 expression and enhances endogenous interaction of the type I TGF-β receptor with DRAK2, thereby blocking R-Smads recruitment. Depletion of DRAK2 expression markedly augmented the intensity and the extent of TGF-β1 responses. Furthermore, a high level of DRAK2 expression was observed in basal-like and HER2-enriched breast tumors and cell lines, and depletion of DRAK2 expression suppressed the tumorigenic ability of breast cancer cells. Thus, these studies define a function for DRAK2 as an intrinsic intracellular antagonist participating in the negative feedback loop to control TGF-β1 responses, and aberrant expression of DRAK2 increases tumorigenic potential, in part, through the inhibition of TGF-β1 tumor suppressor activity.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis Regulatory Proteins; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Female; HEK293 Cells; HeLa Cells; Humans; Mice; Mice, SCID; Molecular Sequence Data; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous; Tumor Suppressor Proteins

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.

Topics: Animals; Breast Neoplasms; Epithelial Cells; Epithelial-Mesenchymal Transition; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Mesoderm; Mice; Protein Binding; Transforming Growth Factor beta

Topics: Animals; Breast Neoplasms; Female; Fibroblasts; Humans; Lactic Acid; Paracrine Communication; Transforming Growth Factor beta

TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß.. We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis.. TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis.. TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.

Topics: Amino Acid Sequence; Antibodies; Breast Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Endothelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 5; Humans; Molecular Sequence Data; Mutation; Neovascularization, Pathologic; Peptides; Receptors, Urokinase Plasminogen Activator; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transfection; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.

Topics: Benzamides; Breast Neoplasms; Cell Line, Tumor; Dioxoles; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Smad Proteins; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Tumor growth factor-β (TGF-β) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-β signaling is modulated by the TGF-β co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-β-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-β signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

Topics: Animals; Antigens, CD; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Endoglin; Female; Gene Silencing; Humans; Lung; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Prognosis; Receptor, ErbB-2; Receptors, Cell Surface; Signal Transduction; Transforming Growth Factor beta

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

Topics: Antineoplastic Agents; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; Selective Estrogen Receptor Modulators; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

We have delineated TGFβ signaling pathways in the production of osteolytic factors interleukin-8 and interleukin-11 in breast cancer cells with different bone metastases potential. Bone seeking MDA-MB-231(hm) cells expressed higher levels of IL-11, but lower levels of IL-8 compared to MDA-MB-231 cells. MCF-7 cells (mainly osteoblastic) did not express IL-8 or IL-11; MDA-MB-468 cells (weakly metastatic) expressed IL-8, but not IL-11. The up-regulation of IL-11 and IL-8 was associated with the rapid activation of SMAD2/3 and p38 MAPK through the TGFβ/TGFβR system. Analysis of TGFβ receptors indicated that MCF-7 cells do not express TGFβRII, and MDA-MB-468 cells do not express SMAD4. Inactivation of SMAD4 or p38PMAPK gene via RNAi resulted in the inhibition of IL-11 and IL-8 production in MDA-MB-231(hm) cells; and over-expression of SMAD4 gene resulted in IL-11 production in MDA-MB-468 cells. TGFβ-1 induced SMAD3 translocation to the nuclei in MDA-MB-231, MDA-MB-231(hm) as well as in SMAD4 deficient MDA-MB-468, indicating that an alternate non-canonical pathway could be responsible for TGFβ-1 induced cytokine production in MDA-MB-468 cells. Thus, four breast cancer cell lines used in this study show differential expression and up-regulation of the osteolytic factors in response to TGFβ-1 that involves both SMAD pathway, a non-canonical SMAD pathway, as well as p38 MAPK pathways.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-11; Interleukin-8; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Recent studies indicate that a subset of cancer cells possessing stem cell properties, referred to as cancer-initiating or cancer stem cells (CSCs), have crucial roles in tumor initiation, metastasis and resistance to anticancer therapies. Transforming growth factor (TGF)-β and their family members have been implicated in both normal (embryonic and somatic) stem cells and CSCs. In this study, we observed that exposure to TGF-β increased the population of breast cancer (BC) cells that can form mammospheres in suspension, a feature endowed by stem cells. This was mediated by the micro (mi)RNA family miR-181, which was upregulated by TGF-β at the post-transcriptional level. Levels of the miR-181 family members were elevated in mammospheres grown in undifferentiating conditions, compared with cells grown in two-dimensional conditions. Ataxia telangiectasia mutated (ATM), a target gene of miR-181, exhibited reduced expression in mammospheres and upon TGF-β treatment. Overexpression of miR-181a/b, or depletion of ATM or its substrate CHK2, was sufficient to induce sphere formation in BC cells. Finally, knockdown of ATM enhanced in vivo tumorigenesis of the MDA361 BC cells. Our results elucidate a novel mechanism through which the TGF-β pathway regulates the CSC property by interfering with the tumor suppressor ATM, providing insights into the cellular and environmental factors regulating CSCs, which may guide future studies on therapeutic strategies targeting these cells.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Shape; Checkpoint Kinase 2; DNA-Binding Proteins; Female; Gene Knockdown Techniques; Humans; Mice; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Neoplastic Stem Cells; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Suppressor Proteins

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the ΔNp63α and β isoforms, leaving only ΔNp63γ, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the ΔNp63α isoform. We also show that ΔNp63γ expressed in a background where all the other ΔNp63 are knocked down causes EMT with an increase in TGFβ-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGFβ was identified. Inhibition of the TGFβ response with a specific inhibitor results in reversion of EMT in ΔNp63α- and β-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

Topics: Binding Sites; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasms, Glandular and Epithelial; Protein Isoforms; Smad Proteins; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins; Up-Regulation

Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-β and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-β signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-β receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-β1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Proteins; Cell Nucleus; Female; HCT116 Cells; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Neoplasm Proteins; Protein Structure, Tertiary; Proto-Oncogene Proteins c-mdm2; Receptors, Transforming Growth Factor beta; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction; Smad3 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Suppressor Protein p53

A significant amount of evidence shows that microenvironmental signals generated from extracellular matrix (ECM) molecules, soluble factors, and cell-cell adhesion complexes cooperate at the extra- and intracellular level. This synergetic action of microenvironmental cues is crucial for normal mammary gland development and breast malignancy. To explore how the microenvironmental genes coordinate in human breast cancer at the genome level, we have performed gene co-expression network analysis in three independent microarray datasets and identified two microenvironment networks in human breast cancer tissues. Network I represents crosstalk and cooperation of ECM microenvironment and soluble factors during breast malignancy. The correlated expression of cytokines, chemokines, and cell adhesion proteins in Network II implicates the coordinated action of these molecules in modulating the immune response in breast cancer tissues. These results suggest that microenvironmental cues are integrated with gene transcriptional networks to promote breast cancer development.

Topics: Breast Neoplasms; Cadherins; Cytokines; Extracellular Matrix; Extracellular Matrix Proteins; Female; Gene Expression Profiling; Gene Regulatory Networks; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Mammary Glands, Human; Oligonucleotide Array Sequence Analysis; Receptor, Platelet-Derived Growth Factor beta; Receptors, Cytokine; Transforming Growth Factor beta; Tumor Microenvironment

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Female; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Mice; Mice, Nude; Nuclear Proteins; Parathyroid Hormone-Related Protein; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta; Veratrum Alkaloids; Zinc Finger Protein Gli2

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cyclins; Female; Humans; Phenotype; Phosphorylation; Proteoglycans; Receptors, Transforming Growth Factor beta; Smad Proteins; Transforming Growth Factor beta

Increased stiffness represents a hallmark of breast cancer that has been attributed to the altered physicochemical properties of the extracellular matrix (ECM). However, the role of fibronectin (Fn) in modulating the composition and mechanical properties of the tumor-associated ECM remains unclear. We have utilized a combination of biochemical and physical science tools to evaluate whether paracrine signaling between breast cancer cells and adipose progenitor cells regulates Fn matrix assembly and stiffness enhancement in the tumor stroma. In particular, we utilized fluorescence resonance energy transfer imaging to map the molecular conformation and stiffness of Fn that has been assembled by 3T3-L1 preadipocytes in response to conditioned media from MDA-MB231 breast cancer cells. Our results reveal that soluble factors secreted by tumor cells promote Fn expression, unfolding, and stiffening by adipose progenitor cells and that transforming growth factor-β serves as a soluble cue underlying these changes. In vivo experiments using orthotopic co-transplantation of primary human adipose-derived stem cells and MDA-MB231 into SCID mice support the pathological relevance of our results. Insights gained by these studies advance our understanding of the role of Fn in mammary tumorigenesis and may ultimately lead to improved anti-cancer therapies.

Topics: 3T3 Cells; Adipose Tissue; Animals; Breast Neoplasms; Cell Line, Tumor; Cells, Cultured; Female; Fibronectins; Fluorescence Resonance Energy Transfer; Humans; Mice; Mice, SCID; Stem Cells; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-β (TGF-β) signaling to drive sustained ZEB expression. Furthermore, we show that prolonged autocrine TGF-β signaling induced reversible DNA methylation of the miR-200 loci with corresponding changes in miR-200 levels. Collectively, these findings demonstrate the existence of an autocrine TGF-β/ZEB/miR-200 signaling network that regulates plasticity between epithelial and mesenchymal states. We find a strong correlation between ZEBs and TGF-β and negative correlations between miR-200 and TGF-β and between miR-200 and ZEBs, in invasive ductal carcinomas, consistent with an autocrine TGF-β/ZEB/miR-200 signaling network being active in breast cancers.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line; Cofilin 2; DNA Methylation; Dogs; Epithelial-Mesenchymal Transition; Feedback, Physiological; Female; Homeodomain Proteins; Humans; MicroRNAs; Repressor Proteins; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

We showed previously that cruciferous vegetable constituent benzyl isothiocyanate (BITC) inhibits growth of cultured and xenografted human breast cancer cells and suppresses mammary cancer development in a transgenic mouse model. We now show, for the first time, that BITC inhibits epithelial-mesenchymal transition (EMT) in human breast cancer cells. Exposure of estrogen-independent MDA-MB-231 and estrogen-responsive MCF-7 human breast cancer cell lines and a pancreatic cancer cell line (PL-45) to BITC resulted in upregulation of epithelial markers (e.g., E-cadherin and/or occludin) with a concomitant decrease in protein levels of mesenchymal markers, including vimentin, fibronectin, snail, and/or c-Met. The BITC-mediated induction of E-cadherin protein was accompanied by an increase in its transcription, whereas BITC-treated MDA-MB-231 cells exhibited suppression of vimentin, snail, and slug mRNA levels. Experimental EMT induced by exposure to TGFβ and TNFα or Rb knockdown in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also partially reversed by BITC treatment. The TGFβ-/TNFα-induced migration of MCF-10A cells was inhibited in the presence of BITC, which was partially attenuated by RNA interference of E-cadherin. Inhibition of MDA-MB-231 xenograft growth in vivo in female athymic mice by BITC administration was associated with an increase in protein level of E-cadherin and suppression of vimentin and fibronectin protein expression. In conclusion, this study reports a novel anticancer effect of BITC involving inhibition of EMT, a process triggered during progression of cancer to invasive state.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cadherins; Cell Differentiation; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Isothiocyanates; Membrane Proteins; Mice; Occludin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vimentin

Latent Transforming Growth Factor beta (TGFβ) Binding Proteins (LTBPs) are chaperones and determinants of TGFβ isoform-specific secretion. They belong to the LTBP/Fibrillin family and form integral components of the fibronectin and microfibrillar extracellular matrix (ECM). LTBPs serve as master regulators of TGFβ bioavailability, functioning to incorporate and spatially pattern latent TGFβ at regular intervals within the ECM, and actively participate in integrin-mediated stretch activation of TGFβ in vivo. In so doing they create a highly patterned sensory system where local changes in ECM tension can be detected and transduced into focal signals. The physiological role of LTBPs in the mammary gland remains largely unstudied, however both loss and gain of LTBP expression is found in breast cancers and breast cancer cell lines. Importantly, elevated LTBP1 levels appear in two gene signatures predictive of enhanced metastatic behavior. LTBP may promote metastasis by providing the bridge between structural and signaling components of the epithelial to mesenchymal transition (EMT).

Topics: Animals; Breast Neoplasms; Female; Humans; Latent TGF-beta Binding Proteins; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Breast; Breast Neoplasms; Female; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Transforming Growth Factor beta

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Evidence supports a critical role for microRNAs (miRNAs) in regulation of tissue-specific differentiation and development. Signifying a disruption of these programs, expression profiling has revealed extensive miRNA dysregulation in tumors compared with healthy tissue. The miR-200 family has been established as a key regulator of epithelial phenotype and, as such, is deeply involved in epithelial to mesenchymal transition (EMT) processes in breast cancer. However, the effects of the miR-200 family on transformation of normal mammary epithelial cells have yet to be fully characterized. By examining a TGF-β driven model of transformation of normal mammary epithelium, we demonstrate that the class III histone deacetylase silent information regulator 1 (SIRT1), a proposed oncogene in breast cancer, is overexpressed upon EMT-like transformation and that epigenetic silencing of miR-200a contributes at least in part to the overexpression of SIRT1. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. We also observed SIRT1 and miR-200a participation in a negative feedback regulatory loop. Restoration of miR-200a or the knockdown of SIRT1 prevented transformation of normal mammary epithelial cells evidenced by decreased anchorage-independent growth and decreased cell migration. Finally, we observed SIRT1 overexpression in association with decreased miR-200a in breast cancer patient samples. These observations provide further evidence for a critical tumor suppressive role of the miR-200 family in breast epithelium in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 up-regulation in breast cancer.

Topics: Antigens, CD; Base Sequence; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Methylation; Epithelial-Mesenchymal Transition; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Gene Silencing; HEK293 Cells; Humans; Mammary Glands, Human; MicroRNAs; Promoter Regions, Genetic; Sirtuin 1; Transforming Growth Factor beta

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Line; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta

Two recent reports identify ZNF703 as an oncogene driving selection of frequent chromosome 8p12 amplifications in luminal B breast tumors. The estrogen-responsive ZNF703 gene encodes a transcriptional cofactor that, when overexpressed, induces cell proliferation and interferes with transforming growth factor beta signaling. In MCF7 cells, increased ZNF703 expression results in activation of genes involved in stem cell self-renewal - while in primary human mammary epithelial cells, ZNF703 increases the ratio of luminal to basal progenitors. Expression of the murine homolog of ZNF703 reduces cell adhesion and promotes metastasis. ZNF703 overexpression thus alters regulation of proliferation and differentiation in luminal B tumors.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 8; Female; Gene Amplification; Humans; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-β and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

Topics: Animals; Autocrine Communication; Bone Morphogenetic Proteins; Breast; Breast Neoplasms; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Mammary Glands, Animal; Mesoderm; Mice; Neoplastic Stem Cells; Paracrine Communication; Signal Transduction; Stem Cells; Transforming Growth Factor beta; Wnt Proteins

Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of prometastatic factors including matrix metalloproteinase (MMP) 9 and COX2. Suppression of TAK1 signaling by dominant-negative TAK1 (dn-TAK1) in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intracardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, parathyroid hormone-related protein (PTHrP) and interleukin 8 (IL-8), but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2, and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies show that the TAK1-TAB2-TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of proinvasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cyclooxygenase 2; Female; Humans; Interleukin-8; Male; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 9; Mice; Mice, SCID; Prostatic Neoplasms; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor β1 (TGFB1) T29C and TGF β receptor type 1 (TGFBR1) 6A/9A polymorphisms have been implicated in the modulation of risk for breast cancer in Caucasian women. We analyzed these polymorphisms and combinations of their genotypes, in pre menopausal breast cancer patients (N = 182) and healthy women (N = 236) from western India as well as in breast cancer patients and healthy women from the Parsi community (N = 48 & 171, respectively). Western Indian women were characterized by a higher frequency of TGFB1*C allele of the TGF β T29C polymorphism (0.48 vs 0.44) and a significantly lower frequency of TGFBR1*6A allele of the TGFBR1 6A/9A polymorphism (0.02 vs 0.068, p<0.01) as compared to healthy Parsi women. A strong protective effect of TGFB1*29C allele was seen in younger western Indian women (<40 yrs; OR = 0.45, 95% CI 0.25-0.81). Compared to healthy women, the strikingly higher frequencies of low or intermediate TGF β signalers in patients suggested a strong influence of the combination of these genotypes on the risk for breast cancer in Parsi women (for intermediate signalers, OR = 4.47 95%CI 1.01-19.69). The frequency of low signalers in Parsi healthy women, while comparable to that reported in Europeans and Americans, was three times higher than that in healthy women from western India (10.6% vs 3.3%, p<0.01). These observations, in conjunction with the low incidence rate of breast cancer in Indian women compared to White women, raise a possibility that the higher frequency of TGFB1*29C allele and lower frequency of TGFBR1*6A allele may represent important genetic determinants that together contribute to a lower risk of breast cancer in western Indian women.

Topics: Adult; Alleles; Breast Neoplasms; Case-Control Studies; Female; Humans; Polymorphism, Genetic; Risk Factors; Signal Transduction; Transforming Growth Factor beta; West Indies

Fermitin family member 1 (FERMT1, Kindlin-1) is an epithelial-specific regulator of integrin functions and is associated with Kindler syndrome, a genetic disorder characterized by skin blistering, atrophy, and photosensitivity. However, the possible role of kindlin-1 in cancer remains unknown.. Kindlin-1 expression was quantified in several human cancers using quantitative real-time polymerase chain reaction and published microarray datasets. The association between kindlin-1 expression and patient metastasis-free survival (N = 516) was assessed with Kaplan-Meier analyses. Effects of ectopic expression or silencing of kindlin-1 on cell signaling, migration, and invasion were assessed in human breast cancer cell lines using western blotting, immunofluorescence, wound healing assays, and invasion on Matrigel or type I collagen substrates. Breast tumor growth and lung metastasis were evaluated in 12-week-old female BALB/c mice (10 controls and six Kindlin-1-knockdown mice). All statistical tests were two-sided.. Kindlin-1 expression was consistently higher in tumors than in normal tissues in various cancer types metastasizing to the lungs, including colon and bladder cancer. Kindlin-1 expression was associated with metastasis-free survival in both breast and lung adenocarcinoma (breast cancer: hazard ratio of lung metastasis = 2.55, 95% confidence intervals [CI] = 1.39 to 4.69, P = .001; lung cancer: hazard ratio of metastasis = 1.96, 95% CI = 1.25 to 3.07, P = .001). Overexpression of kindlin-1 induced changes indicating epithelial-mesenchymal transition and transforming growth factor beta (TGFβ) signaling, constitutive activation of cell motility, and invasion (number of migrating cells, Kindlin-1 cells vs control, mean = 164.66 vs. 19.00, difference = 145.6, 95% CI = 79.1 to 212.2, P = .004; invasion rate, Kindlin-1-cells vs control = 9.65% vs. 1.92%, difference = 7.73%, 95% CI = 4.75 to 10.70, P < .001). Finally, Kindlin-1 depletion in an orthotopic mouse model statistically significantly inhibited breast tumor growth (P < .001) and lung metastasis (P = .003).. These results suggest a role for kindlin-1 in breast cancer lung metastasis and lung tumorigenesis and advance our understanding of kindlin-1 as a regulator of TGFβ signaling, offering new avenues for therapeutic intervention against cancer progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Proliferation; Female; Fluorescent Antibody Technique; Focal Adhesions; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Proportional Hazards Models; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell-cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24-30 h after exposure to TGF-β1.

Topics: Animals; Biosensing Techniques; Breast Neoplasms; Cell Differentiation; Cells, Cultured; Electric Impedance; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Mammary Glands, Animal; Mice; Microscopy, Fluorescence; Transforming Growth Factor beta

Protein L-isoaspartyl O-methyltransferase (PIMT) regulates cell adhesion in various cancer cell lines through activation of integrin αv and the PI3K pathway. The epithelial mesenchymal transition (EMT) enables epithelial cells to acquire the characteristics of mesenchymal cells, and to allow them to migrate for metastasis. Here, we examined the relationship between PIMT and EMT with attached or detached MDA-MB 231 cells.. Human breast cancer cell line MDA-MB-231 cells were maintained in a suspension on poly-HEMA in the presence or absence of PIMT siRNA or ERK inhibitor PD98059. The mRNAs and proteins were analyzed using RT-PCR and immunoblotting, respectively.. During cellular incubation under detached conditions, PIMT, integrin αv and EMT proteins, such as Snail, Slug and matrix metalloproteinase 2 (MMP-2), were significantly increased in correlation with the phosphorylation of ERK1/2. The ERK inhibitor PD98059 (25 μmol/L) strongly suppressed the expression of the proteins and PIMT. Interestingly, PIMT siRNA blocked the phosphorylation of ERK and the expression of the EMT proteins. Additionally, PIMT and ERK phosphorylation were both co-activated by treatment with TGF-β (10 ng/mL) and TNF-α (10 ng/mL).. A tight cross-regulation exists between ERK and PIMT in regards to their activation and expression during the EMT.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Organic Cation Transport Proteins; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Lysophosphatidic acid (LPA) is a multifunctional intercellular phospholipid mediator present in blood and other biological fluids. In cancer cells, LPA stimulates expression or activity of inflammatory cytokines, angiogenic factors, matrix metalloproteinases, and other oncogenic proteins. In this study, we showed that LPA upregulated expression of the cyclin-dependent kinase inhibitor p21(Waf1) in TGFβ-sensitive breast and ovarian cancer cells, but not in TGFβ-resistant ones. We examined the possibility that LPA-induced p21 might contribute to the cytostatic response to TGFβ. In serum-free conditions, TGFβ alone induced p21 expression weakly in TGFβ-sensitive cells. Serum or serum-borne LPA cooperated with TGFβ to elicit the maximal p21 induction. LPA stimulated p21 via LPA(1) and LPA(2) receptors and Erk-dependent activation of the CCAAT/enhancer binding protein beta transcription factor independent of p53. Loss or gain of p21 expression led to a shift between TGFβ-sensitive and -resistant phenotypes in breast and ovarian cancer cells, indicating that p21 is a key determinant of the growth inhibitory activity of TGFβ. Our results reveal a novel cross-talk between LPA and TGFβ that underlies TGFβ-sensitive and -resistant phenotypes of breast and ovarian cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Female; Humans; Immunohistochemistry; Lysophospholipids; Ovarian Neoplasms; Phosphorylation; Transforming Growth Factor beta

Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. Here we demonstrate that neutrophils accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H(2)O(2) and tumor secreted CCL2 is a critical mediator of optimal antimetastatic entrainment of G-CSF-stimulated neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, whereas tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site.

Topics: Animals; Bone Marrow Cells; Breast Neoplasms; Cell Line, Tumor; Chemokine CCL2; Cytotoxicity, Immunologic; Female; Granulocyte Colony-Stimulating Factor; Humans; Hydrogen Peroxide; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Invasiveness; Neutrophils; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta

Cyclin D1 is a well-characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in 'inhibitor of differentiation 1' (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer.. Protein and gene expression of ID1, CCND1 and EMT markers were determined in MDA-MB-231 and ZR75 cells by western blot and qPCR. Cell migration and promoter occupancy were monitored by transwell and ChIP assays, respectively. Gene expression was analysed from publicly available datasets.. The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1-independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1low/ID1high tumours displayed increased expression of EMT markers and were associated with reduced recurrence free survival. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like 'claudin-low' subtype of breast cancer than in other subtypes.. These results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cluster Analysis; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inhibitor of Differentiation Protein 1; Prognosis; Recurrence; Risk; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a normal developmental program that is considered to also play an important role in cancer metastasis. Ultimate inducers of EMT are transcriptional repressors that individually can induce experimental EMT, yet in many cells, particularly cancer cells, multiple inducers are expressed simultaneously. Why, and if, and how they interact to regulate EMT is unanswered. Using RNA interference technology to affect protein knockdown and avoid potential overexpression artifact coupled with transient TGFβ treatment to better mimic in vivo conditions we show, in both nontumorigenic and tumorigenic epithelial cancer cells, that Snail1 is uniquely required for EMT initiation, whereas Twist1 is required to maintain late EMT. Twist1, present in resting epithelial cells, is dispensable for EMT initiation. Mechanistically, in response to transient TGFβ treatment, transient Snail1 expression represses Twist1 transcription directly, which is subsequently upregulated, as Snail1 levels decrease, to sustain E-cadherin downregulation and growth arrest of EMT. Persistent Twist1 expression is associated with a p38 and extracellular signal-regulated kinase signal feedback loop that sustains growth-inhibitory signals characteristic of quiescent micrometastatic tumors. This Snail1-Twist1 temporal and spatial cooperation was also observed in vivo during human breast cancer progression to metastasis. Twist1 level, but not Snail1 level, and Twist1:Snail1 ratio in disseminated micrometastatic bone marrow tumor cells was found to correlate with survival and treatment resistance and is highly predictive of metastatic or recurrent disease.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Cell Dedifferentiation; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Nuclear Proteins; Prognosis; Promoter Regions, Genetic; Protein Binding; Recurrence; RNA Interference; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo.. TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo.. Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures.. These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.

Topics: Amino Acids; Animals; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Combined Modality Therapy; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, SCID; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Radiation-Sensitizing Agents; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1; Xanthenes; Xenograft Model Antitumor Assays

The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways.

Topics: Acetylation; Benzamides; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Dioxoles; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Genetic Loci; Histones; Humans; Intercellular Junctions; Mammary Glands, Human; Phenotype; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Snail Family Transcription Factors; Substrate Specificity; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

Biomarkers are lacking for identifying the switch of transforming growth factor-β (TGF-β) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-β to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-β. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-β-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-β-induced cell migration. Because TGF-β is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-β during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-β in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-β and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-β.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Female; Genes, p53; Humans; Male; Mice; Mice, Inbred C57BL; Mutation; Prostatic Neoplasms; Shc Signaling Adaptor Proteins; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Src Homology 2 Domain-Containing, Transforming Protein 1; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Transforming growth factor-β (TGF-β) promotes cancer invasion and metastasis and is therefore a potential drug target for cancer treatment. Fresolimumab, which neutralizes all mammalian active isoforms of TGF-β, was radiolabeled with (89)Zr for PET to analyze TGF-β expression, antibody tumor uptake, and organ distribution.. (89)Zr was conjugated to fresolimumab using the chelator N-succinyldesferrioxamine-B-tetrafluorphenol. (89)Zr-fresolimumab was analyzed for conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. (89)Zr-fresolimumab tumor uptake and organ distribution were assessed using 3 protein doses (10, 50, and 100 μg) and compared with (111)In-IgG in a human TGF-β-transfected Chinese hamster ovary xenograft model, human breast cancer MDA-MB-231 xenograft, and metastatic model. Latent and active TGF-β1 expression was analyzed in tissue homogenates with enzyme-linked immunosorbent assay.. (89)Zr was labeled to fresolimumab with high specific activity (>1 GBq/mg), high yield, and high purity. In vitro validation of (89)Zr-fresolimumab showed a fully preserved immunoreactivity and long (>1 wk) stability in solution and in human serum. In vivo validation showed an (89)Zr-fresolimumab distribution similar to IgG in most organs, except for a higher uptake in the liver in all mice and higher kidney uptake in the 10-μg group. (89)Zr-fresolimumab induced no toxicity in mice; it accumulated in primary tumors and metastases in a manner similar to IgG. Both latent and active TGF-β was detected in tumor homogenates, whereas only latent TGF-β could be detected in liver homogenates. Remarkably high (89)Zr-fresolimumab uptake was seen in sites of tumor ulceration and in scar tissue, processes in which TGF-β is known to be highly active.. Fresolimumab tumor uptake and organ distribution can be visualized and quantified with (89)Zr-fresolimumab PET. This technique will be used to guide further clinical development of fresolimumab and could possibly identify patients most likely to benefit.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Isotope Labeling; Liver; Male; Mice; Neoplasm Metastasis; Positron-Emission Tomography; Radioisotopes; Transfection; Transforming Growth Factor beta; Zirconium

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.

Topics: Animals; Antibodies; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Collagen; Female; Humans; Mice; Mice, Nude; Osteoblasts; Osteoclasts; Osteogenesis; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta

Advanced breast cancer cells acquire metastatic properties in response to TGFβ. We show here that the expression of c-Myb increases in TGFβ-treated ER (+) breast cancer cells by protein stabilization, transcription activation and release from miR200-dependent down-regulation. In particular, we mapped 2 sites for miR200b, miR200c and miR429 binding in the 3' UTR of the human c-myb gene. These microRNAs decreased the expression of c-Myb when transfected in MCF-7 cells. In addition, luciferase activity from a vector containing the 3' UTR of the c-myb gene was inhibited by miR200s through a binding-dependent mechanism. siRNA- and shRNA-mediated down-regulation was used to investigate the role of c-Myb for the effects induced by TGFβ in ER(+) breast cancer MCF-7 and ZR-75.1 cells. Transfection with c-Myb siRNAs blocked the increase of Slug (SNAI2) and Bcl-2 expression and reversed the decrease in E-cadherin expression induced by TGF-β treatment. Conversely, c-Myb down-regulation decreased invasion and anchorage-independent growth of breast cancer cells expressing a constitutively active TGFβ receptor I. Finally, apoptosis induced by etoposide increased in c-Myb-silenced TGFβ-treated ER(+) cell lines. In summary, exposure of ER(+) breast cancer cells to TGFβ induces an increase of c-Myb expression which is required for expression of EMT-associated markers, in vitro invasion and anchorage-independent growth. Furthermore, our findings suggest a potentially detrimental effect of TGFβ and c-Myb co-expression in breast cancer.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Etoposide; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, myb; Humans; Lentivirus; MicroRNAs; Mutagenesis, Site-Directed; Neoplasm Invasiveness; Protein Processing, Post-Translational; Protein Stability; Proto-Oncogene Proteins c-myb; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Transcriptional Activation; Transfection; Transforming Growth Factor beta

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage(1,2). Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes(5). In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways(6). To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Neoplasm Invasiveness; Spheroids, Cellular; Transforming Growth Factor beta

In contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.. MCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.. Runx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.. E2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Core Binding Factor Alpha 1 Subunit; Epithelial-Mesenchymal Transition; Estrogens; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Wnt Proteins

Topics: Animals; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Humans; MicroRNAs; Transforming Growth Factor beta

High mammographic density is associated with a increased risk of breast cancer. We hypothesized that specific pathways exist that are associated with increased mammographic density, and may therefore be used to identify potential targets for chemoprevention. Histologically confirmed normal breast tissue was collected from women undergoing breast surgery who had available demographic data and mammograms for review. Women with low versus high mammographic breast density were compared. Differentially expressed genes using Affymetrix HG U133Plus2 chips were identified in dense versus non-dense tissue. Immunohistochemical analysis (IHC) of estrogen receptor, progesterone receptor, Ki67, and COX2 expression was performed. About 66 women were identified, 28 (42%) had high, and 38 (58%) had low mammographic density. About 73 genes had differential expression between normal breast tissue with high and low mammographic density (P < 0.001, fold change > or = 1.5 with a low false discovery rate (<10%). Network and canonical pathway analysis indicated decreased TGFbeta signaling (TGFBR2, SOS, SMAD3, CD44 and TNFRSF11B) in dense breast tissue relative to non-dense breast. By IHC, only COX2 expression in the stroma was statistically significant on multivariate analysis. TGFbeta ligands are currently the only growth factors known to prevent mammary epithelial cell proliferation. TGFbeta signaling has been reported to be inhibited by COX-2, and these molecules are highly differentially expressed in individuals at high risk of developing breast cancer. These results strongly suggest that COX2 inhibition should be investigated for breast cancer prevention despite possible increase in cardiovascular risk.

Topics: Adult; Aged; Aged, 80 and over; Anticarcinogenic Agents; Breast Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Immunohistochemistry; Ligands; Logistic Models; Mammography; Middle Aged; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Risk Assessment; Risk Factors; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

To investigate a presumed crosstalk between estrogen receptor alpha (ERalpha) and the TGF-beta signaling pathway in breast cancer, we analyzed the TGF-beta-induced expression of the plasminogen activator inhibitor 1 (PAI-1) gene in ER-positive MCF-7 cells. After siRNA-mediated knock-down of endogenous ERalpha, the transcription level of PAI-1 was upregulated, pointing to an attenuation of TGF-beta signaling by the presence of ERalpha. We verified these findings by a vice versa approach using a primary ER-negative cell model transiently overexpressing either ERalpha or ERbeta. We found that ERalpha, but not ERbeta, led to a strong inhibition of the TGF-beta1 signal, monitored by TGF-beta reporter assays. This attenuation was completely independent of receptor stimulation by beta-estradiol (E2) or inhibition by the pure antagonist ICI 182.780 (ICI). Our results indicate a permanent repression of PAI-1 by ERalpha and suggest a ligand-independent crosstalk between ERalpha and TGF-beta signaling in breast cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Plasminogen Activator Inhibitor 1; Receptor Cross-Talk; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Transfection; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta signaling makes a significant contribution to the pathogenesis of breast cancer bone metastasis. In other tumor types, TGF-beta has been shown to promote tumor vascularity. Here, we report that inhibition of TGF-beta significantly reduces microvessel density in mammary tumor-induced bone lesions, mediated by decreased expression of both vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1, both known angiogenic factors. Cathepsin G upregulation at the tumor-bone interface has been linked to increased TGF-beta signaling, and we also report that inhibition of Cathepsin G reduced tumor vascularity, as well as VEGF and MCP-1 expression.

Topics: Animals; Antibodies; Bone Neoplasms; Breast Neoplasms; Cathepsin G; Cell Line, Tumor; Chemokine CCL2; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; RNA, Messenger; Serine Proteinase Inhibitors; Signal Transduction; Tosylphenylalanyl Chloromethyl Ketone; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

We were interested in developing oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we constructed mhTERTAd.sTbetaRFc, a 01/07-based adenoviral vector expressing the soluble form of transforming growth factor-beta (TGFbeta) receptor II fused with the human Fc IgG1 (sTGFbetaRIIFc) gene, in which viral replication is under the control of a modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTbetaRFc-mediated sTGFbetaRIIFc production targets the TGFbeta pathway known to contribute to the tumor progression of breast cancer metastasis. We chose to use the mhTERT promoter because it was found to be relatively more active (approximately 20 times) in breast cancer cells compared with normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 h with mhTERTAd.sTbetaRFc produced high levels of sTGFbetaRIIFc (greater than 1 microg ml(-1)) in the medium. Breast cancer cells produced nearly a 6000-fold increase in viral titers during the 48 h infection period. However, mhTERTAd.sTbetaRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication-deficient virus Ad(E1(-)).sTbetaRFc also produced high levels of sTGFbetaRIIFc, but under these conditions, no detectable viral replication was observed. Adenoviral-mediated production of sTGFbetaRIIFc was shown to bind with TGFbeta-1, and to abolish the effects of TGFbeta-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTbetaRFc intravenously into MDA-MB-231 human xenograft-bearing mice resulted in a significant inhibition of tumor growth and production of sTGFbetaRIIFc in the blood. Conversely, intravenous injection of Ad(E1(-)).sTbetaRFc did not show a significant inhibition of tumor growth, but resulted in sTGFbetaRIIFc in the blood, suggesting that viral replication along with sTGFbetaRIIFc protein production is critical in inducing the inhibition of tumor growth. These results warrant future investigation of mhTERTAd.sTbetaRFc as an antitumor agent in vivo.

Topics: Adenoviridae; Adenoviridae Infections; Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cytopathogenic Effect, Viral; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Immunoglobulin Fc Fragments; Mice; Mice, Nude; Oncolytic Virotherapy; Phosphorylation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Telomerase; Transforming Growth Factor beta; Virus Replication; Xenograft Model Antitumor Assays

It is well known that tumor and its microenvironment, or stroma, interact with each other and that this interaction plays a critical role in tumor initiation, growth, and metastasis. This interaction consists of complex relations between tumor cells, stromal cells such as fibroblasts, epithelial cells and immunocytes, the vascular system, the extracellular matrix, and cytokines secreted by the cells. Understanding these relationships may lead to new therapeutic approaches to cancer. In the present paper, we consider tumor-stroma crosstalk in a simple in vitro situation which involves interaction between tumor epithelial cells from breast cancer and a microenvironment consisting of just fibroblasts. The two populations of cells are separated by a semi-permeable membrane that allows only cytokines to cross over. We develop a mathematical model that includes two critical growth factors: TGF-beta, produced by the tumor cells, and EGF, secreted by the fibroblasts. The TGF-beta modifies the microenvironment by transforming fibroblasts into myofibroblasts. Myofibroblasts secrete higher concentrations of EGF than fibroblasts, thereby, increasing the proliferation of tumor cells. Thus already in this simple setup one sees a mutual interaction between tumor cells and their microenvironment. We conducted experiments which show good agreement with the model's simulations, hence confirming the model's ability to predict aspects of tumor cell behavior in response to signaling from fibroblasts.

Topics: Breast Neoplasms; Cell Communication; Cell Transformation, Neoplastic; Computer Simulation; Epidermal Growth Factor; Epithelial Cells; Female; Fibroblasts; Humans; Models, Immunological; Transforming Growth Factor beta

This paper is concerned with early development of transformed epithelial cells (TECs) in the presence of fibroblasts in the tumor micro-environment. These two types of cells interact by means of cytokines such as transforming growth factor (TGF-beta) and epidermal growth factor (EGF) secreted, respectively, by the TECs and the fibroblasts. As this interaction proceeds, TGF-beta induces fibroblasts to differentiate into myofibroblasts which secrete EGF at a larger rate than fibroblasts. We monitor the entire process in silico, in a setup which mimics experiments in a Tumor Chamber Invasion Assay, where a semi-permeable membrane coated by extracellular matrix (ECM) is placed between two chambers, one containing TECs and another containing fibroblasts. We develop a mathematical model, based on a system of PDEs, that includes the interaction between TECs, fibroblasts, myofibroblasts, TGF-beta, and EGF, and we show how model parameters affect tumor progression. The model is used to generate several hypotheses on how to slow tumor growth and invasion. In an Appendix, it is proved that the mathematical model has a unique global in-time solution.

Topics: Algorithms; Breast Neoplasms; Cell Communication; Cell Count; Cell Differentiation; Cell Proliferation; Chemotaxis; Computer Simulation; Diffusion; Epidermal Growth Factor; Extracellular Matrix; Female; Fibroblasts; Humans; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Models, Biological; Neoplasm Invasiveness; Neoplasms, Glandular and Epithelial; Permeability; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clusterin; Epithelial Cells; Extracellular Space; Female; Gene Expression Profiling; Humans; Immunoglobulin G; Lung Neoplasms; Mesoderm; Mice; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis; Phenotype; Reproducibility of Results; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

The transforming growth factor beta (TGF-beta) pathway can play either a tumor-suppressing or a tumor-promoting role in human breast carcinogenesis. In order to determine whether expression of TGF-beta signaling factors varies by age at onset and breast tumor characteristics that have prognostic significance, we undertook a study of 623 women with invasive breast carcinoma enrolled in a population-based case-control study conducted in Poland from 2000 to 2003. TGF-beta signaling factors were analyzed by immunohistochemistry in tumor tissue microarrays. We found that most tumors expressed extracellular-TGF-beta1 (78%), TGF-beta2 (91%), TGF-beta3 (93%), TGF-betaR2 (72%), and phospho-SMAD2 (61%), whereas intracellular-TGF-beta1 was expressed in 32% of tumors. Expression of TGF-beta ligands (beta1, beta2, and beta3) was associated with prognostically favorable pathological features including small size, and low grade, and these associations were similar for ER-positive and negative tumors. On the contrary, expression of the receptor TGF-betaR2 was primarily associated with small tumor size among ER-negative tumors, while expression of the transcription factor phospho-SMAD2 was associated with positive nodal status among ER-negative tumors. The greater frequency of expression of phospho-SMAD2 in cancers associated with lymph node metastases is consistent with a pro-progression role for TGF-beta. In addition, expression of extracellular-TGF-beta1 (P = 0.005), TGF-betaR2 (P = 8.2E-11), and phospho-SMAD2 (P = 1.3E-8) was strongly associated with earlier age at onset, independent of ER status. Our data provide evidence that TGF-beta signaling patterns vary by age and pathologic features of prognostic significance including ER expression. These results warrant analysis in studies of clinical outcomes accounting for age, ER status and treatment.

Topics: Adult; Age Distribution; Age of Onset; Aged; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Female; Humans; Incidence; Middle Aged; Poland; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Estrogen; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta can suppress and promote breast cancer progression. How TGF-beta elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-beta-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-beta-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-beta-induced vascular endothelial growth factor (VEGF) expression. TGF-beta induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-beta-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo.

Topics: Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Carcinogenicity Tests; Cell Line, Tumor; Humans; Mice; Mice, Knockout; Neoplasm Metastasis; Neoplasms, Second Primary; Neovascularization, Pathologic; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

The insulin-like growth factor (IGF) system mediates growth, differentiation and developmental processes; it is also involved in various metabolic activities. Deregulation of IGF system expression and action is linked to diverse pathologies, ranging from growth deficits to cancer development. Targeting of the IGF axis emerged in recent years as a promising therapeutic approach in cancer and other medical conditions. Rational use of IGF-I-induced gene signatures may help to identify patients who might benefit from IGF axis-directed therapeutic modalities. In the accompanying research article in BMC Medicine, Rajski et al. show that IGF-I-induced gene expression in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients.See the associated research paper by Rajski et al: http://www.biomedcentral.com/1741-7015/8/1.

Topics: Biomarkers, Pharmacological; Breast Neoplasms; Cell Line, Tumor; Computational Biology; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Oligonucleotide Array Sequence Analysis; Prognosis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Polarity; Disease Progression; Epithelial Cells; Female; Humans; Mesenchymal Stem Cells; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Antiestrogens are universally used to treat estrogen receptor--positive breast cancer, but relapses occur commonly due to the development of drug resistance. The ability of antiestrogen to induce transforming growth factor beta (TGFbeta) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFbeta on cancer progression but also at the level of its effects on the host immune system. To evaluate the potential role of tumor-derived, antiestrogen-induced TGFbeta as an immune suppressor, we established in vitro mixed lymphocyte tumor reactions (MLTR) using MCF-7 cells and peripheral blood mononuclear cells (PBMC), as well as tumor tissue and autologous tumor infiltrating lymphocytes (TIL) obtained from primary breast cancer biopsies. In allogeneic MLTR, antiestrogen-treated MCF-7 cells caused downregulation of the effector molecules granzyme B, perforin, and Fas ligand in CD8(+) T cells, and suppressed the generation of cytotoxic effector cells in a TGFbeta-dependent manner. Furthermore, we documented induction of regulatory T cells in CD4(+) T cells, based on Foxp3 expression and T-cell activation in cocultures. In autologous MLTR, antiestrogen treatment gave rise to enhanced Foxp3 expression of TIL/PBMC and decreased the number of apoptotic tumor cells. These effects were reversed by addition of a TGFbeta neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFbeta-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Female; Forkhead Transcription Factors; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Immune Tolerance; Tamoxifen; Transforming Growth Factor beta

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

Topics: Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Movement; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Profiling; Humans; Immunoprecipitation; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Plasminogen Activator Inhibitor 1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases

Topics: Animals; Antineoplastic Agents; Bone Density Conservation Agents; Bone Marrow; Bone Neoplasms; Breast Neoplasms; Chemokines; Diphosphonates; Female; Humans; Osteoblasts; Osteoclasts; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Chemokine; Signal Transduction; Transforming Growth Factor beta

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFbeta/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

Topics: Animals; Annexin A1; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Signal Transduction; Transforming Growth Factor beta

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-beta1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-beta appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-beta pathway.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Coculture Techniques; Culture Media, Conditioned; Female; Fibroblasts; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Transforming Growth Factor beta

As carcinomas progress, the tumors may lose epithelial morphology and acquire mesenchymal characteristics typically called epithelial-mesenchymal transition (EMT), which is commonly associated with increased cell migration, enables cells to dissociate from their original tissue and form metastasis in distant organs. In addition to molecular and morphologic changes, the EMT transformed cells also showed the change of sensitivity to chemotherapeutics. In order to detect the EMT transition in vivo clinically, we detected the change of metabolism of MCF-7 cells after being induced by TGF-beta to form EMT condition by MTT and 18F-FDG uptake.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Epithelial Cells; Female; Fluorodeoxyglucose F18; Glucose Transporter Type 1; Humans; Mesoderm; Radiopharmaceuticals; Transforming Growth Factor beta; Vimentin

Recent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFbeta is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models.. We report that chemotherapeutic drug doxorubicin activates TGFbeta signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFbeta type I receptor kinase inhibitor (TbetaRI-KI). We investigated the potential synergistic anti-tumor activity of TbetaR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TbetaRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TbetaR1-KI was administered in combination with doxorubicin.. These observations suggest that the adverse activation of TGFbeta pathway by chemotherapeutics in the cancer cells together with elevated TGFbeta levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFbeta inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Humans; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Neoplasms, Experimental; Transforming Growth Factor beta; Transplantation, Heterologous

Transforming Growth Factor beta (TGF-beta) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-beta antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-beta pathway antagonists (1D11, a mouse monoclonal pan-TGF-beta neutralizing antibody and LY2109761, a chemical inhibitor of TGF-beta type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR).. Both 1D11 and LY2109761 effectively blocked TGF-beta-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-beta stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-beta. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-beta antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.. In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Pyrazoles; Pyrroles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Chronic bacterial infection increased the risk of many solid malignancies and the underlying mechanism is usually ascribed to bacterial-caused inflammation. However, the direct interaction of infectious bacteria with cancer cells has been largely overlooked. We identified that highly metastatic breast cancer MDA-MB-231 cells expressed high level of Toll-like receptor 2 (TLR2) in contrast to poorly metastatic breast cancer cells and homogenous untransformed breast cells. TLR2 in MDA-MB-231 cells were actively triggered by peptidoglycan (PGN) from infectious bacterium Staphylococcus aureus (PGN-SA), resulting in the promoted invasiveness and adhesiveness of the cancer cells in vitro. PGN-SA induced phosphorylation of TAK1 and IkappaB in the TLR2-NF-kappaB pathway of the cancer cells and stimulated IL-6 and TGF-beta secretion in MDA-MB-231 cells. All these effects were abrogated by TLR2 blockade. Further investigation showed that the NF-kappaB, STAT3 and Smad3 activities were augmented sequentially in MDA-MB-231 cells after PGN-SA stimulation. Phosphorylation of NF-kappaBp65 was initially increased and then followed by phosphorylation of STAT3 and Smad3 in the delayed 4 or 6 hours. NF-kappaB inhibition attenuated STAT3 and Smad3 activities whereas PGN-SA-stimulated cell culture supernatants reversed these inhibitory effects. Our study indicated that TLR2 activation by infectious bacterial PGN played an important role in breast cancer cell invasiveness and illustrated a new link between infectious bacteria and the cancer cells, suggesting the importance of antibiotic therapy to treat cancer with bacterial infection.

Topics: Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Neoplasm Invasiveness; NF-kappa B; Peptidoglycan; Signal Transduction; Smad3 Protein; Staphylococcus aureus; STAT3 Transcription Factor; Toll-Like Receptor 2; Transforming Growth Factor beta

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].

Topics: Actins; Autophagy; Biomarkers, Tumor; Breast Neoplasms; Calcium-Binding Proteins; Calponins; Carcinoma, Intraductal, Noninfiltrating; Caveolin 1; Cell Line, Tumor; Chloroquine; Coculture Techniques; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Microfilament Proteins; Phenotype; Prognosis; Smad2 Protein; Transforming Growth Factor beta; Vimentin

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-beta. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Interleukin-17; Matrix Metalloproteinases; Mice; Mice, Nude; Neoplasm Metastasis; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

TMEPAI is a transforming growth factor-beta (TGF-beta)-induced transmembrane protein that is overexpressed in several cancers. How TMEPAI expression relates to malignancy is unknown. Here, we report high expression of TMEPAI in estrogen receptor/progesterone receptor-negative and human epidermal growth factor receptor-2-negative breast cancer cell lines and primary breast cancers that was further increased by TGF-beta treatment. Basal and TGF-beta-induced expression of TMEPAI were inhibited by the TGF-beta receptor antagonist SB431542 and overexpression of Smad7 or a dominant-negative mutant of Alk-5. TMEPAI knockdown attenuated TGF-beta-induced growth and motility in breast cancer cells, suggesting a role for TMEPAI in growth promotion and invasiveness. Further, TMEPAI knockdown decreased breast tumor mass in a mouse xenograft model in a manner associated with increased expression of phosphatase and tensin homologue (PTEN) and diminished phosphorylation of Akt. Consistent with the effects through the phosphatidylinositol 3-kinase pathway, tumors with TMEPAI knockdown exhibited elevated levels of the cell cycle inhibitor p27kip1 and attenuated levels of DNA replication and expression of hypoxia-inducible fator 1alpha and vascular endothelial growth factor. Together, these results suggest that TMEPAI functions in breast cancer as a molecular switch that converts TGF-beta from a tumor suppressor to a tumor promoter.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Growth Processes; Cell Movement; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Membrane Proteins; Mice; Mice, Nude; RNA, Small Interfering; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-β in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-β was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-β increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with TβR-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-β stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-β in aggressive and invasive forms of breast cancer.

Topics: Animals; Breast Neoplasms; Cadherins; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Indoles; Mammary Neoplasms, Experimental; Mice; p38 Mitogen-Activated Protein Kinases; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Sulfonamides; Transforming Growth Factor beta

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous 7-amino acid peptide hormone of the renin-angiotensin system that has antiproliferative properties. In this study, Ang-(1-7) inhibited the growth of cancer-associated fibroblasts (CAF) and reduced fibrosis in the tumor microenvironment. A marked decrease in tumor volume and weight was observed in orthotopic human breast tumors positive for the estrogen receptor (BT-474 or ZR-75-1) and HER2 (BT-474) following Ang-(1-7) administration to athymic mice. Ang-(1-7) concomitantly reduced interstitial fibrosis in association with a significant decrease in collagen I deposition, along with a similar reduction in perivascular fibrosis. In CAFs isolated from orthotopic breast tumors, the heptapeptide markedly attenuated in vitro growth as well as reduced fibronectin, transforming growth factor-β (TGF-β), and extracellular signal-regulated kinase 1/2 kinase activity. An associated increase in the mitogen-activated protein kinase (MAPK) phosphatase DUSP1 following treatment with Ang-(1-7) suggested a potential mechanism by which the heptapeptide reduced MAPK signaling. Consistent with these in vitro observations, immunohistochemical analysis of Ang-(1-7)-treated orthotopic breast tumors revealed reduced TGF-β and increased DUSP1. Together, our findings indicate that Ang-(1-7) targets the tumor microenvironment to inhibit CAF growth and tumor fibrosis.

Topics: Angiotensin I; Animals; Antihypertensive Agents; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Dual Specificity Phosphatase 1; Female; Fibronectins; Fibrosis; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lung Diseases, Interstitial; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 3; Peptide Fragments; Phosphorylation; Transforming Growth Factor beta; Tumor Cells, Cultured

The present analyses were done to define the role of fetuin-A (Fet) in mammary tumorigenesis using the polyoma middle T antigen (PyMT) transgenic mouse model. We crossed Fet-null mice in the C57BL/6 background with PyMT mice in the same background and after a controlled breeding protocol obtained PyMT/Fet+/+, PyMT/Fet+/-, and PyMT/Fet-/- mice that were placed in control and experimental groups. Whereas the control group (PyMT/Fet+/+) formed mammary tumors 90 days after birth, tumor latency was prolonged in the PyMT/Fet-/- and PyMT/Fet+/- mice. The majority of the PyMT/Fet-/- mice were tumor-free at the end of the study, at approximately 40 weeks. The pathology of the mammary tumors in the Fet-null mice showed extensive fibrosis, necrosis, and squamous metaplasia. The preneoplastic mammary tissues of the PyMT/Fet-/- mice showed intense phopho-Smad2/3 staining relative to control tissues, indicating that transforming growth factor-β signaling is enhanced in these tissues in the absence of Fet. Likewise, p19ARF and p53 were highly expressed in tumor tissues of PyMT/Fet-/- mice relative to the controls in the absence of Fet. The phosphatidylinositol 3-kinase/Akt signaling pathway that we previously showed to be activated by Fet, on the other hand, was unaffected by the absence of Fet. The data indicate that Fet is a powerful modulator of breast tumorigenesis in this model system and has the potential to modulate breast cancer progression in humans.

Topics: alpha-2-HS-Glycoprotein; Animals; Antigens, Polyomavirus Transforming; Blood Proteins; Breast Neoplasms; Disease Progression; Female; Humans; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phosphatidylinositol 3-Kinase; Signal Transduction; Transforming Growth Factor beta

The activating transcription factor 3 (ATF3) gene is induced by a variety of signals, including many of those encountered by cancer cells. We present evidence that ATF3 is induced by TGFβ in the MCF10CA1a breast cancer cells and plays an integral role for TGFβ to upregulate its target genes snail, slug and twist, and to enhance cell motility. Furthermore, ATF3 upregulates the expression of the TGFb gene itself, forming a positive-feedback loop for TGFβ signaling. Functionally, ectopic expression of ATF3 leads to morphological changes and alterations of markers consistent with epithelial-to-mesenchymal transition (EMT). It also leads to features associated with breast-cancer-initiating cells: increased CD24(low)-CD44(high) population of cells, mammosphere formation and tumorigenesis. Conversely, knockdown of ATF3 reduces EMT, CD24(low)-CD44(high) cells and mammosphere formation. Importantly, knocking down twist, a downstream target, reduces the ability of ATF3 to enhance mammosphere formation, indicating the functional significance of twist in ATF3 action. To our knowledge, this is the first report demonstrating the ability of ATF3 to enhance breast cancer-initiating cell features and to feedback on TGFβ. Because ATF3 is an adaptive-response gene and is induced by various stromal signals, these findings have significant implications for how the tumor microenvironment might affect cancer development.

Topics: Activating Transcription Factor 3; Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Chromatin Immunoprecipitation; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Immunoblotting; Immunoprecipitation; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

The oncoprotein c-Ski has been implicated in the negative regulation of transforming growth factor-β (TGF-β) signaling owing to its ability to repress Smad transcriptional activity via recruitment of a transcriptional corepressor complex containing histone deacetylases. However, c-Ski has also been shown to localize to the cytoplasm, raising the interesting possibility that it might disable TGF-β signaling through alternative mechanisms. Here, we provide evidence that c-Ski can restrict TGF-β signaling by interacting directly with the activated TGF-β type I receptor (TβRI). We explored the physiologic relevance of the c-Ski/TβRI interaction and found that it can culminate in a constitutive association of TβRI with a nonfunctional R-Smad/Smad4 complex. Based on these findings, we hypothesize that the interaction between c-Ski and TβRI might interfere with nuclear translocation of the R-Smad/Smad4 complex, thereby attenuating TGF-β signaling. Such a mechanism may play a crucial role in tumor progression, because many tumors that express high levels of c-Ski also display impaired nuclear accumulation of Smads.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Female; Humans; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Smad Proteins, Receptor-Regulated; Smad2 Protein; Smad3 Protein; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Cell Differentiation; Chemokine CXCL12; Female; Humans; Mammary Glands, Human; Mice; Myofibroblasts; Neoplasm Invasiveness; Receptors, CXCR4; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The TGF-β, a tumor suppressive cytokine in normal cells, is abused in cancer to promote the malignancy. In this study, we reported that TGF-β downregulated the mutS homolog 2 (MSH2), a central component of the DNA mismatch repair (MMR) system, in HER2-transformed MCF10A mammary epithelial cells and in breast cancer (BC) cells. This was mediated by a TGF-β-induced micro RNA (miRNA), miR-21, which targeted the 3' untranslated region of MSH2 mRNA and downregulated its expression. A negative correlation between the expression of TGF-β1 and MSH2 was also detected in primary breast tumors. In contrast, TGF-β upregulated MSH2 in nontransformed cells through Smad-mediated, p53-dependent promoter activation, which was absent in BC cells with impaired p53 function. Although this upregulating mechanism also existed in MCF10A/HER2 and p53-proficient BC cells, both basal and TGF-β-induced MSH2 promoter activities were significantly lower than those in MCF10A. Moreover, the basal and TGF-β-induced miR-21 levels were markedly higher in transformed cells, suggesting that the preset levels of miR-21 and MSH2 promoter activity, which is affected by the p53 status, determine the outputs of the bidirectional regulation of MSH2 by TGF-β in a certain cellular context. We further found that by downregulating MSH2, TGF-β contributed to resistance to DNA-damaging chemotherapy agents in cancer cells. Our results indicated a regulatory antagonism between promoter activation and miRNA-mediated posttranscriptional inhibition underlying a dual effect of TGF-β on the DNA repair machinery, which may influence the genomic stability in a context-dependent manner and contribute to chemoresistance in cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; DNA Damage; DNA Mismatch Repair; Down-Regulation; Drug Resistance, Neoplasm; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; MicroRNAs; MutS Homolog 2 Protein; Promoter Regions, Genetic; Smad Proteins; Transfection; Transforming Growth Factor beta; Up-Regulation

The Six1 homeodomain protein is a developmental transcription factor that has been implicated in tumor onset and progression. Our recent work shows that Six1 overexpression in human breast cancer cell lines is sufficient to induce epithelial-to-mesenchymal transition (EMT) and metastasis. Importantly, Six1-induced EMT and metastasis are dependent on TGF-β signaling. The TGF-β pathway plays a dual role in cancer, acting as a tumor suppressor in early lesions but enhancing metastatic spread in more advanced tumors. Our previous work indicated that Six1 may be a critical mediator of the switch in TGF-β signaling from tumor suppressive to tumor promotional. However, the mechanism by which Six1 impinges on the TGF-β pathway was, until now, unclear. In this work, we identify the TGF-β type I receptor (TβRI) as a target of Six1 and a critical effector of Six1-induced TGF-β signaling and EMT. We show that Six1-induced upregulation of TβRI is both necessary and sufficient to activate TGF-β signaling and induce properties of EMT. Interestingly, increased TβRI expression is not sufficient to induce experimental metastasis, providing in vivo evidence that Six1 overexpression is required to switch TGF-β signaling to the prometastatic phenotype and showing that induction of EMT is not sufficient to induce experimental metastasis. Together, these results show a novel mechanism for the activation of TGF-β signaling, identify TβRI as a new target of Six1, and implicate Six1 as a determinant of TGF-β function in breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Metastatic tumour cells are characterised by acquisition of migratory and invasive properties; properties shared by cells, which have undergone epithelial-to-mesenchymal transition (EMT). Disabled-2 (Dab2) is a putative tumour suppressor whose expression has been shown to be downregulated in various cancer types including breast cancer; however, its exact function in suppressing tumour initiation or progression is unclear.. Disabled-2 isoform expression was determined by RT-PCR analysis in human normal and breast tumour samples. Using shRNA-mediated technology, Dab2 was stably downregulated in two cell model systems representing nontumourigenic human mammary epithelial cells. These cells were characterised for expression of EMT markers by RT-PCR and western blot analysis.. Decreased expression of the p96 and p67 isoforms of Dab2 is observed in human breast tumour samples in comparison to normal human breast tissue. Decreased Dab2 expression in normal mammary epithelial cells leads to the appearance of a constitutive EMT phenotype. Disabled-2 downregulation leads to increased Ras/MAPK signalling, which facilitates the establishment of an autocrine transforming growth factor β (TGFβ) signalling loop, concomitant with increased expression of the TGFβ2 isoform.. Loss of Dab2 expression, commonly observed in breast cancer, may facilitate TGFβ-stimulated EMT, and therefore increase the propensity for metastasis.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Humans; MAP Kinase Signaling System; ras Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone.

Topics: Algorithms; Bone Neoplasms; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Neoplasm Metastasis; Osteoclasts; Osteolysis; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; Transforming Growth Factor beta; Tumor Microenvironment

Transforming Growth Factor-b (TGFb) is a major driving force of the Epithelial-to-Mesenchymal (EMT) genetic program, which becomes overactive in the pathophysiology of many age-related human diseases.  TGFb-driven EMT is sufficient to generate migrating cancer stem cells by directly linking the acquisition of cellular motility with the maintenance of tumor-initiating (stemness) capacity.  Chronic diseases exhibiting excessive fibrosis can be caused by repeated and sustained infliction of TGFb-driven EMT, which increases collagen and extracellular matrix synthesis.  Pharmacological prevention and/or reversal of TGFb-induced EMT may therefore have important clinical applications in the management of cancer metastasis as well as in the prevention and/or treatment of end-state organ failures.  Earlier studies from our group have revealed that clinically-relevant concentrations of the biguanide derivative metformin, the most widely used oral agent to lower blood glucose concentration in patients with type 2 diabetes and metabolic syndrome, notably decreased both the self-renewal and the proliferation of trastuzumab-refractory breast cancer stem cell populations.  Given that: a.) tumor-initiating cancer stem cells display a significant enrichment in the expression of basal/mesenchymal or myoepithelial markers, including an increased secretion of TGFb; b.) metformin treatment impedes the ontogeny of generating the stem cell phenotype by transcriptionally repressing key drivers of the EMT genetic program (e.g. ZEB1, TWIST1, SNAIL2 [Slug], TGFbs), we recently hypothesized that prevention of TGFb-induced EMT might represent a common molecular mechanism underlying the anti-cancer stem cells and anti-fibrotic actions of metformin.  Remarkably, metformin exposure not only impedes TGFb-promoted loss of the epithelial marker E-cadherin in MCF-7 breast cancer cells but it prevents further TGF-induced cell scattering and accumulation of the mesenchymal marker vimentin in Madin-Darby canine kidney (MDCK) cells.  We now propose that metformin, by weakening the ability of TGFb signaling to fully induce mesenchymal cell states in a variety of pathological processes including fibrosis (e.g. chronic renal disease, non-alcoholic steatohepatitis, heart failure or sclerosis) and malignant progression (and likely by reducing TGFb-regulated inflammation and immune responses -inflamm-aging-), molecularly behaves as a bona fide anti-aging modality.

Topics: Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cellular Senescence; Dogs; Epithelial-Mesenchymal Transition; Female; Fibrosis; Humans; Hypoglycemic Agents; Metformin; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Tumor progression requires ablation of suppressor functions mediated by transforming growth factor β (TGFβ) signaling and by oncogene-induced senescence (OIS), but how these functions are canceled in specific subtypes of breast cancer remains unknown. In this study, we show that HER2-overexpressing breast cancer cells avert TGFβ- and OIS-mediated tumor suppression by switching expression of 2 functionally distinct isoforms of the transcription factor C/EBPβ, which has been implicated previously in breast cancer development. HER2 signaling activates the translational regulatory factor CUGBP1, which favors the production of the transcriptionally inhibitory isoform LIP over that of the active isoform LAP. LIP overexpression prevents the assembly of LAP/Smad transcriptional repressor complexes on the MYC promoter in response to TGFβ, and interferes with activation of OIS responses. Treatment of HER2-transformed mammary epithelial cells with the HER2 antibody trastuzumab reduces LIP levels, restoring these suppressor responses. Our findings reveal a novel mechanism through which HER2 silences tumor suppression in a concerted manner, contributing to the potency of this oncogene in breast cancer.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Line; Cell Line, Tumor; Cellular Senescence; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Mice; Mice, Nude; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Biosynthesis; Protein Isoforms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transforming Growth Factor beta; Trastuzumab

Current attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies.. We successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers.. SNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r² = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, P(trend) = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, P(trend) = 0.018).. This study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Female; Gene Expression; Genes, BRCA2; Genetic Predisposition to Disease; Genome-Wide Association Study; Humans; Linkage Disequilibrium; Middle Aged; Mutation; Polymorphism, Single Nucleotide; Risk Factors; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

American women have a nearly 25% lifetime risk of developing breast cancer, with 20% to 40% of these patients developing life-threatening metastases. More than 70% of patients presenting with metastases have skeletal involvement, which signals progression to an incurable stage. Tumor-stroma cell interactions are only superficially understood, specifically regarding the ability of stromal cells to affect metastasis. In vivo models show that exogenously supplied human bone marrow-derived stem cells (hBMSC) migrate to breast cancer tumors, but no reports have shown endogenous hBMSC migration from the bone to primary tumors. Here, we present a model of in vivo hBMSC migration from a physiologic human bone environment to human breast tumors. Furthermore, hBMSCs alter tumor growth and bone metastasis frequency. These may home to certain breast tumors based on tumor-derived TGF-β1. Moreover, at the primary tumor level, interleukin 17B (IL-17B)/IL-17BR signaling may mediate interactions between hBMSCs and breast cancer cells.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Communication; Cell Line, Tumor; Female; Gene Expression Profiling; Humans; Interleukin-17; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Receptors, Interleukin-17; Reverse Transcriptase Polymerase Chain Reaction; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous

The histological manifestation of growth-regulating and differentiation-inducing signals in cancer cells is considered as a key component for clinical outcome prediction and commonly defined as tumor differentiation grade. However, the molecular and functional framework underlying this clinical parameter remains poorly understood. Our correlative data display a significant association (P>0.001) between mitochondrial uncoupling protein 2 (UCP2) and tumor grade in primary breast cancer (n=234). Through mechanistic analyses, we show a synergistic link between UCP2 and established cellular pathways in conferring grade-associated functional phenotypes. Here, the application of well to moderately differentiated primary tumor cell lines has enabled direct observation of SMAD recruitment to the UCP2 promoter underlying repression of gene transcription. In contrast, poorly differentiated tumor cells, known to be TGFβ resistant, displayed aberrant UCP2 regulation, and consequently, gene overexpression, which reduced mitochondrial calcium and facilitated the maintenance of mitochondrial membrane potential, thereby significantly decreasing oxidative stress and inhibiting cell death. Conversely, UCP2 silencing in such cells rapidly led to the induction of apoptosis and cell differentiation, concurrent with reduced cell survival and proliferation, confirming gene-specific effects. Demonstration of a biologically driven role for UCP2 dysregulation in promoting multiple characteristics of tumor aggressiveness strongly endorses assessment of gene expression at clinical presentation to augment therapeutic decision-making and improve patient outcome through personalized targeting approaches.

Topics: Apoptosis; Breast Neoplasms; Calcium; Cell Differentiation; Female; Gene Expression Regulation, Neoplastic; Humans; Ion Channels; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Neoplasm Staging; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Uncoupling Protein 2

The transforming growth factor beta-1 gene (TGFB1) is a plausible candidate for breast cancer susceptibility. The L10P variant of TGFB1 is associated with higher circulating levels and secretion of TGF-beta, and recent large-scale studies suggest strongly that this variant is associated with breast cancer risk in the general population.. To evaluate whether TGFB1 L10P also modifies the risk of breast cancer in BRCA1 or BRCA2 mutation carriers, we undertook a multi-center study of 3,442 BRCA1 and 2,095 BRCA2 mutation carriers.. We found no evidence of association between TGFB1 L10P and breast cancer risk in either BRCA1 or BRCA2 mutation carriers. The per-allele HR for the L10P variant was 1.01 (95%CI: 0.92-1.11) in BRCA1 carriers and 0.92 (95%CI: 0.81-1.04) in BRCA2 mutation carriers.. These results do not support the hypothesis that TGFB1 L10P genotypes modify the risk of breast cancer in BRCA1 or BRCA2 mutation carriers.

Topics: Adult; Alleles; Breast Neoplasms; Cohort Studies; Female; Genes, BRCA1; Genes, BRCA2; Genetic Predisposition to Disease; Genotype; Heterozygote; Humans; Mutation; Risk; Transforming Growth Factor beta

During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.

Topics: Activins; Animals; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Dioxoles; Humans; Mice; Phosphorylation; Protein Binding; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad5 Protein; Transforming Growth Factor beta

MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB-231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-kappaB and induced phosphorylation of TAK1 and IkappaBalpha in the TLR2/NF-kappaB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-beta, VEGF and MMP9 secretion, which are associated with TLR2-NF-kappaB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-kappaB signaling activity and highly invasive capacity of MDA-MB-231 cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Humans; Interleukin-6; Matrix Metalloproteinase 9; Neoplasm Invasiveness; NF-kappa B; Toll-Like Receptor 2; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

It has been proposed that cross talk between integrin and growth factor receptor signaling such as ErbB2 (HER2) is required for activation of downstream effectors and ErbB2-mediated mammary tumorigenesis. Here we show that transforming growth factor beta (TGF-beta) induced focal adhesion kinase (FAK)-dependent clustering of HER2 and integrins alpha(6), beta(1), and beta(4) in HER2-overexpressing mammary epithelial cells without altering the total and surface levels of HER2 receptors. This effect was mediated by ligand-induced epidermal growth factor receptor (EGFR) activation and the subsequent phosphorylation of Src and FAK. We have previously reported that TGF-beta up-regulates EGFR ligand shedding through a mechanism involving the phosphorylation of tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17). Knockdown of TACE, FAK, or integrin alpha(6) by siRNA or inhibition of EGFR or Src by specific inhibitors abrogated TGF-beta-induced receptor clustering and signaling to phosphatidylinositol 3-kinase-Akt. Finally, inhibition of Src-FAK reversed TGF-beta-induced resistance to the therapeutic HER2 inhibitor trastuzumab in HER2-overexpressing breast cancer cells. Taken together, these data suggest that, by activating Src-FAK, TGF-beta integrates ErbB receptor and integrin signaling to induce cell migration and survival during breast cancer progression.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cytoskeleton; Enzyme Activation; ErbB Receptors; Focal Adhesion Kinase 1; Humans; Integrins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor Cross-Talk; Receptor, ErbB-2; src-Family Kinases; Transforming Growth Factor beta; Trastuzumab

The bone-tumor microenvironment encompasses unique interactions between the normal cells of the bone and marrow cavity and the malignant cells from a primary or metastasized cancer. A multitude of paracrine factors within this microenvironment such as the growth factor, TGF-beta, and the chemokine, MCP-1, are secreted by many of these cell types. These factors can act in concert to modulate normal and malignant cell proliferation, malignant cell migration and invasion and, often, mediate bone cancer pain. Although many valuable in vitro and in vivo models exist, identifying the relevant paracrine factors and deciphering their interactions is still a challenge. The aim of our study is to test an ex vivo coculture model that will allow monitoring of the expression, release and regulation of paracrine factors during interactions of an intact femur explant and tumor cells.. Intact or marrow-depleted neonatal mouse femurs and select murine and human sarcoma or carcinoma cell lines were incubated singly or in coculture in specialized well plates. Viability of the bone and cells was determined by immunohistochemical stains, microscopy and marrow cytopreps. Secretion and mRNA expression of paracrine factors was quantitated by ELISA and real-time RT-PCR.. Compartments of the bone were optimally viable for up to 48 h in culture and tumor cells for up to 4 days. Bone was the major contributor of TGF-beta and MMP2 whereas both bone and sarcoma cells secreted the chemokine MCP-1 in cocultures. Synergistic interaction between the femur and sarcoma resulted in enhanced MCP-1 secretion and expression in cocultures and was dependent on the presence of the hematopoietic component of the bone as well as other bone cells. In contrast, coculturing with breast carcinoma cells resulted in reduction of TGF-beta and MCP-1 secretion from the bone.. These studies illustrate the feasibility of this model to examine paracrine interactions between intact bone and tumor cells. Further study of unique regulation of MCP-1 secretion and signaling between these cell types in different types of cancer will be possible using this simulated microenvironment.

Topics: Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Chemokine CCL2; Coculture Techniques; Female; Gene Expression; Humans; Male; Mice; Models, Biological; Paracrine Communication; Protein Transport; Signal Transduction; Transforming Growth Factor beta

CD44, a transmembrane receptor for hyaluronic acid, is implicated in various adhesion-dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Recent studies demonstrated that CD44 expressed in cancer cells can be proteolytically cleaved at the ectodomain by membrane type 1-matrix metalloproteinase (MT1-MMP) to form soluble CD44 and that CD44 cleavage plays a critical role in cancer cell migration. Here, we show that transforming growth factor-beta (TGF-beta), a multifunctional cytokine involved in cell proliferation, differentiation, migration and pathological processes, induces MT1-MMP expression in MDA-MB-435s cells. TGF-beta-induced MT1-MMP expression was blocked by the specific extracellular regulated kinase-1/2 (ERK1/2) inhibitor PD98059 and the specific phosphoinositide 3-OH kinase (PI3K) inhibitor LY294002. In addition, treatment with SP600125, an inhibitor for c-Jun NH(2)-terminal kinase (JNK), resulted in a significant inhibition of MT1-MMP production. These data suggest that ERK1/2, PI3K, and JNK likely play a role in TGF-beta-induced MT1-MMP expression. Interestingly, treatment of MDA-MB-435s cells with TGF-beta resulted in a colocalization of MT1-MMP and CD44 in the cell membrane and in an increased level of soluble CD44. Using an electric cell-substrate impedance sensing cell-electrode system, we demonstrated that TGF-beta treatment promotes MDA-MB-435s cell migration, involving MT1-MMP-mediated CD44 cleavage. MT1-MMP siRNA transfection-inhibited TGF-beta-induced cancer cell transendothelial migration. Thus, this study contributes to our understanding of molecular mechanisms that play a critical role in tumor cell invasion and metastasis.

Topics: Breast Neoplasms; Cell Movement; Cells, Cultured; Humans; Hyaluronan Receptors; Matrix Metalloproteinase 14; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Extracellular matrix (ECM) molecules modify gene expression through attachment-dependent (focal adhesion-related) integrin receptor signaling. It was previously unknown whether the same molecules acting as soluble peptides could generate signal cascades without the associated mechanical anchoring, a condition that may be encountered during matrix remodeling and degradation and relevant to invasion and metastatic processes. In the current study, the role of ECM ligand-regulated gene expression through this attachment-independent process was examined. It was observed that fibronectin, laminin, and collagen type I and II induce Smad2 activation in MCF-10A and MCF-7 cells. This activation is not caused by transforming growth factor (TGF)-beta ligand contamination or autocrine TGF involvement and is 3- to 5-fold less robust than the TGF-beta1 ligand. The resulting nuclear translocation of Smad4 in response to ECM ligand indicates downstream transcriptional responses occurring. Coimmunoprecipitation experiments determined that collagen type II and laminin act through interaction with integrin alpha(2)beta(1) receptor complex. The ECM ligand-induced Smad activation (termed signaling crosstalk) resulted in cell type and ligand-specific transcriptional changes, which are distinct from the TGF-beta ligand-induced responses. These findings show that cell-matrix communication is more complex than previously thought. Soluble ECM peptides drive transcriptional regulation through corresponding adhesion and non-attachment-related processes. The resultant gene expressional patterns correlate with pathway activity and not by the extent of Smad activation. These results extend the complexity and the existing paradigms of ECM-cell communication to ECM ligand regulation without the necessity of mechanical coupling.

Topics: Breast Neoplasms; Cell Line, Tumor; Collagen Type I; Collagen Type II; Epithelial Cells; Extracellular Matrix; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha2beta1; Laminin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor beta (TGFbeta) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA-MB-231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFbeta-inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFbeta signaling in metastases was monitored by microPET imaging of HSV1-TK/GFP expression with [(18)F]FEAU and by a more sensitive and cost-effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFbeta signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFbeta signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFbeta signaling.

Topics: Animals; Base Sequence; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; DNA, Complementary; Female; Genes, Reporter; Green Fluorescent Proteins; Humans; Luciferases, Firefly; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Positron-Emission Tomography; Recombinant Proteins; Signal Transduction; Tomography, X-Ray Computed; Transforming Growth Factor beta; Transplantation, Heterologous

Transforming growth factor-beta (TGF-beta) increases or decreases nuclear factor kappa B (NFkappaB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-beta receptor (TbetaRIII) is a TGF-beta superfamily co-receptor with emerging roles in both mediating and regulating TGF-beta superfamily signaling. We have previously reported a novel interaction of TbetaRIII with the scaffolding protein, beta-arrestin2, which results in TbetaRIII internalization and downregulation of TGF-beta signaling. beta-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFkappaB-signaling pathways. Here, we demonstrate that TbetaRIII, through its interaction with beta-arrestin2, negatively regulates NFkappaB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TbetaRIII expression reduced NFkappaB-mediated transcriptional activation and IkappaBalpha degradation, whereas a TbetaRIII mutant unable to interact with beta-arrestin2, TbetaRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TbetaRIII expression or beta-arrestin2 expression increased NFkappaB-mediated transcriptional activation and IkappaBalpha degradation. Functionally, TbetaRIII-mediated repression of NFkappaB signaling is important for TbetaRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TbetaRIII regulates NFkappaB signaling and expand the roles of this TGF-beta superfamily co-receptor in regulating epithelial cell homeostasis.

Topics: Arrestins; beta-Arrestins; Blotting, Western; Breast Neoplasms; Cell Movement; Cross-Linking Reagents; Female; Humans; Luciferases; NF-kappa B; Proteoglycans; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Humans; Mice; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; ras Proteins; Smad Proteins; Specific Pathogen-Free Organisms; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

During rodent mammary gland involution there is a dramatic increase in the expression of the transforming growth factor-beta isoform, TGF-beta3. The TGF-betas are multifunctional cytokines which play important roles in wound healing and in carcinogenesis. The responses that are activated in the remodeling of the gland during involution have many similarities with the wound healing process and have been postulated to generate a mammary stroma that provides a microenvironment favoring tumor progression. In this review we will discuss the putative role of TGF-beta during involution, as well as its effects on the mammary microenvironment and possible implications for pregnancy-associated tumorigenesis.

Topics: Animals; Apoptosis; Breast Neoplasms; Cicatrix; Disease Progression; Female; Gene Expression Profiling; Humans; Inflammation; Lactation; Mammary Glands, Animal; Mice; Parity; Pregnancy; Pregnancy Complications, Neoplastic; Prognosis; Protein Isoforms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Transforming Growth Factor beta3; Treatment Outcome; Wound Healing

In human breast cancer, loss of carcinoma cell-specific response to TGF-beta signaling has been linked to poor patient prognosis. However, the mechanisms through which TGF-beta regulates these processes remain largely unknown. In an effort to address this issue, we have now identified gene expression signatures associated with the TGF-beta signaling pathway in human mammary carcinoma cells. The results strongly suggest that TGF-beta signaling mediates intrinsic, stromal-epithelial, and host-tumor interactions during breast cancer progression, at least in part, by regulating basal and oncostatin M-induced CXCL1, CXCL5, and CCL20 chemokine expression. To determine the clinical relevance of our results, we queried our TGF-beta-associated gene expression signatures in 4 human breast cancer data sets containing a total of 1,319 gene expression profiles and associated clinical outcome data. The signature representing complete abrogation of TGF-beta signaling correlated with reduced relapse-free survival in all patients; however, the strongest association was observed in patients with estrogen receptor-positive (ER-positive) tumors, specifically within the luminal A subtype. Together, the results suggest that assessment of TGF-beta signaling pathway status may further stratify the prognosis of ER-positive patients and provide novel therapeutic approaches in the management of breast cancer.

Topics: Antigens, Viral, Tumor; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Chemokines; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Polyomavirus; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Breast cancer is known to be a hormone-dependent disease, and estrogens through an interaction with estrogen receptor (ER) enhance the proliferative and metastatic activity of breast tumor cells. Here we show a critical role of transactivation of BIG3, brefeldin A-inhibited guanine nucleotide-exchange protein 3, in activation of the estrogen/ER signaling in breast cancer cells. Knocking-down of BIG3 expression with small-interfering RNA (siRNA) drastically suppressed the growth of breast cancer cells. Subsequent coimmunoprecipitation and immunoblotting assays revealed an interaction of BIG3 with prohibitin 2/repressor of estrogen receptor activity (PHB2/REA). When BIG3 was absent, stimulation of estradiol caused the translocation of PHB2/REA to the nucleus, enhanced the interaction of PHB2/REA and ERalpha, and resulted in suppression of the ERalpha transcriptional activity. On the other hand, when BIG3 was present, BIG3 trapped PHB2/REA in the cytoplasm and inhibited its nuclear translocation, and caused enhancement of ERalpha transcriptional activity. Our results imply that BIG3 overexpression is one of the important mechanisms causing the activation of the estrogen/ERalpha signaling pathway in the hormone-related growth of breast cancer cells.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Estrogens; Extracellular Matrix Proteins; Female; Fluorescent Dyes; Gene Silencing; Green Fluorescent Proteins; Humans; Immunohistochemistry; Immunoprecipitation; Mass Spectrometry; Prohibitins; Receptors, Estrogen; Repressor Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection; Transforming Growth Factor beta

SMAD7 and GREM1 are signaling components on the transforming growth factor-beta pathway, which regulates normal mammary gland development and has been implicated in breast tumor invasion and metastasis. Three variants within SMAD7 and two variants in CRAC1 (a colorectal cancer-associated region on chromosome 15 in which GREM1 is located) have been associated with colorectal cancer risks [odds ratios (OR), 0.85-1.26; all P < 10(-7)]. We genotyped these five variants in a series of 1,267 bilateral breast cancer cases and 900 controls to determine whether they are associated with breast as well as colorectal cancer risk. None of these single nucleotide polymorphisms were associated with breast cancer risk in our study and the 95% confidence limits of our data, pooled with data from the Cancer Genetic Markers of Susceptibility study, exclude per allele ORs of <0.94 or >1.08. One or more of these variants may be associated with a very small OR for breast cancer, but our data suggest that the effects of these alleles are cancer site-specific.

Topics: Adult; Aged; Breast Neoplasms; Calcium Channels; Colorectal Neoplasms; Female; Genetic Predisposition to Disease; Genotype; Humans; Intercellular Signaling Peptides and Proteins; Middle Aged; ORAI1 Protein; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Risk Factors; Smad7 Protein; Transforming Growth Factor beta

In a recent issue of Breast Cancer Research, investigators from the Serra laboratory describe a novel mechanism of transforming growth factor (TGF)-beta tumor suppression. Previously, the authors discovered that stromal TGF-beta signaled through Wnt5a to restrain pubertal ductal elongation and branching. Here, they show that inhibition of stromal TGF-beta signaling or Wnt5a loss leads to increased beta-catenin transcriptional activity and reduced latency in mammary tumor models, with tumors displaying a higher proportion of progenitor cell markers. These findings reveal a novel intersection of two tumor suppressors with a potent oncogenic pathway and highlight the need for further study on the role played by canonical Wnt signaling in breast cancer susceptibility and subtype.

Topics: Animals; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Medical Oncology; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins; Wnt-5a Protein

In breast carcinomas, prolonged signalling through the TGF-beta receptor promotes latent tumour progression, metastasis and the epithelial-to-mesenchymal transition of tumour cells. Previously, it has been found that the 154 amino acid RING finger protein, RNF11, was overexpressed in high-grade breast tumours and was capable of modulating TGF-beta signalling.. Utilizing cellular and biochemical assays, key interactions and molecular roles for the RNF11 protein in the TGF-beta pathway were explored.. It is shown that RNF11 is required for TGF-beta signalling and is capable of enhancing the Smad-TGF-beta signalling pathway directly. Further, that endogenous RNF11 and Smad4 proteins associate and co-localize in a TGF-beta-enhanced manner. This study indicates that RNF11 induces an increase in Smad4 protein levels. In functional assays, it is observed that RNF11 enhances Smad4-dependant TGF-beta signalling and that RNF11 alone can recapitulate Smad4-dependant apoptosis in cellular assays.. RNF11 acts directly on Smad4 to enhance Smad4 function, and plays a role in prolonged TGF-beta signalling and possibly in latent tumour progression.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Carcinoma, Hepatocellular; Carrier Proteins; Cells, Cultured; DNA-Binding Proteins; Fluorescent Antibody Technique; Humans; Immunoblotting; Immunoprecipitation; Kidney; Liver Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin

The precise sequence of events that enable mammary tumorigenesis to convert transforming growth factor-beta (TGF-beta) from a tumor suppressor to a tumor promoter remains incompletely understood. We show here that X-linked inhibitor of apoptosis protein (xIAP) is essential for the ability of TGF-beta to stimulate nuclear factor-kappaB (NF-kappaB) in metastatic 4T1 breast cancer cells. Indeed whereas TGF-beta suppressed NF-kappaB activity in normal mammary epithelial cells, those engineered to overexpress xIAP demonstrated activation of NF-kappaB when stimulated with TGF-beta. Additionally up-regulated xIAP expression also potentiated the basal and TGF-beta-stimulated transcriptional activities of Smad2/3 and NF-kappaB. Mechanistically xIAP (i) interacted physically with the TGF-beta type I receptor, (ii) mediated the ubiquitination of TGF-beta-activated kinase 1 (TAK1), and (iii) facilitated the formation of complexes between TAK1-binding protein 1 (TAB1) and IkappaB kinase beta that enabled TGF-beta to activate p65/RelA and to induce the expression of prometastatic (i.e. cyclooxygenase-2 and plasminogen activator inhibitor-1) and prosurvival (i.e. survivin) genes. We further observed that inhibiting the E3 ubiquitin ligase function of xIAP or expressing a mutant ubiquitin protein (i.e. K63R-ubiquitin) was capable of blocking xIAP- and TGF-beta-mediated activation of NF-kappaB. Functionally xIAP deficiency dramatically reduced the coupling of TGF-beta to Smad2/3 in NMuMG cells as well as inhibited their expression of mesenchymal markers in response to TGF-beta. More importantly, xIAP deficiency also abrogated the formation of TAB1.IkappaB kinase beta complexes in 4T1 breast cancer cells, thereby diminishing their activation of NF-kappaB, their expression of prosurvival/metastatic genes, their invasion through synthetic basement membranes, and their growth in soft agar. Collectively our findings have defined a novel role for xIAP in mediating oncogenic signaling by TGF-beta in breast cancer cells.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Models, Biological; Neoplasm Metastasis; NF-kappa B; Transforming Growth Factor beta; Ubiquitin; Ubiquitin-Protein Ligases; X-Linked Inhibitor of Apoptosis Protein

Recent large-scale tumor resequencing studies have identified a number of mutations that might be involved in tumorigenesis. Analysis of the frequency of specific mutations across different tumors has been able to identify some, but not all of the mutated genes that contribute to tumor initiation and progression. One reason for this is that other functionally important genes are likely to be mutated more rarely and only in specific contexts. Thus, for example, mutation in one member of a collection of functionally related genes may result in the same net effect, and/or mutations in certain genes may be observed less frequently if they play functional roles in later stages of tumor development, such as metastasis. We modified and applied a network reconstruction and coexpression module identification-based approach to identify functionally related gene modules targeted by somatic mutations in cancer. This method was applied to available breast cancer, colorectal cancer, and glioblastoma sequence data, and identified Wnt/TGF-beta cross-talk, Wnt/VEGF signaling, and MAPK/focal adhesion kinase pathways as targets of rare driver mutations in breast, colorectal cancer, and glioblastoma, respectively. These mutations do not appear to alter genes that play a central role in these pathways, but rather contribute to a more refined shaping or "tuning" of the functioning of these pathways in such a way as to result in the inhibition of their tumor-suppressive signaling arms, and thereby conserve or enhance tumor-promoting processes.

Topics: Algorithms; Breast Neoplasms; Colorectal Neoplasms; Computational Biology; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Glioblastoma; Humans; Mitogen-Activated Protein Kinase Kinases; Mutation; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wnt Proteins

Although the transforming growth factor-beta (TGF-beta) pathway has been implicated in breast cancer metastasis, its in vivo dynamics and temporal-spatial involvement in organ-specific metastasis have not been investigated. Here we engineered a xenograft model system with a conditional control of the TGF-beta-SMAD signaling pathway and a dual-luciferase reporter system for tracing both metastatic burden and TGF-beta signaling activity in vivo. Strong TGF-beta signaling in osteolytic bone lesions is suppressed directly by genetic and pharmacological disruption of the TGF-beta-SMAD pathway and indirectly by inhibition of osteoclast function with bisphosphonates. Notably, disruption of TGF-beta signaling early in metastasis can substantially reduce metastasis burden but becomes less effective when bone lesions are well established. Our in vivo system for real-time manipulation and detection of TGF-beta signaling provides a proof of principle for using similar strategies to analyze the in vivo dynamics of other metastasis-associated signaling pathways and will expedite the development and characterization of therapeutic agents.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Diagnostic Imaging; Female; Genetic Vectors; Humans; Mice; Mice, Nude; Models, Biological; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Treatment Outcome; Tumor Cells, Cultured; Validation Studies as Topic; Xenograft Model Antitumor Assays

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFbeta, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFbeta-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell-like features.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelium; Female; Gene Expression; Homeodomain Proteins; Humans; Male; Mammary Neoplasms, Experimental; Mesoderm; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-beta signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-beta signaling. TGF-beta signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-beta signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-beta signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-beta signaling and EMT, is a powerful and global promoter of cancer metastasis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelium; Female; Homeodomain Proteins; Humans; Mammary Neoplasms, Experimental; Mesoderm; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Prognosis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transplantation, Heterologous

Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- beta. We asked whether hypoxia (via HIF-1alpha) and TGF-beta signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.. We analyzed interactions between HIF-1alpha and TGF-beta pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-beta and hypoxia, with effects on the proximal promoters. We inhibited HIF-1alpha and TGF-beta pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.. Hypoxia and TGF-beta signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1alpha and TGF-beta may improve treatment of bone metastases and increase survival.

Topics: Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Neoplasm Metastasis; Receptors, CXCR4; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

ErbB2, a metastasis-promoting oncoprotein, is overexpressed in approximately 25% of invasive/metastatic breast cancers, but in 50%-60% of noninvasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3zeta in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3zeta overexpression, respectively, increased cell migration and decreased cell adhesion, two prerequisites of tumor cell invasion. 14-3-3zeta overexpression reduced cell adhesion by activating the TGF-beta/Smads pathway that led to ZFHX1B/SIP-1 upregulation, E-cadherin loss, and epithelial-mesenchymal transition. Importantly, patients whose breast tumors overexpressed both ErbB2 and 14-3-3zeta had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one.

Topics: 14-3-3 Proteins; Breast Neoplasms; Cadherins; Carcinoma, Ductal, Breast; Cell Adhesion; Cell Movement; Disease Progression; Epithelium; Female; Genes, erbB-2; Homeodomain Proteins; Humans; Mesoderm; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Nerve Tissue Proteins; Risk Factors; RNA-Binding Proteins; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

Mammary tumorigenesis is associated with the increased expression of several proteins in the focal adhesion complex, including focal adhesion kinase (FAK) and various integrins. Aberrant expression of these molecules occurs concomitant with the conversion of TGF-beta function from a tumor suppressor to a tumor promoter. We previously showed that interaction between beta3 integrin and TbetaR-II facilitates TGF-beta-mediated oncogenic signaling, epithelial-mesenchymal transition (EMT), and metastasis. However, the molecular mechanisms by which the focal adhesion complex contributes to beta3 integrin:TbetaR-II signaling and the oncogenic conversion of TGF-beta remain poorly understood.. FAK expression and activity were inhibited in normal and malignant mammary epithelial cells (MECs) either genetically by using lentiviral-mediated delivery of shRNAs against FAK, or pharmacologically through in vitro and in vivo use of the FAK inhibitors, PF-562271 and PF-573228. Altered Smad2/3 and p38 MAPK activation, migration, EMT, and invasion in response to TGF-beta1 were monitored in FAK-manipulated cells. TbetaR-II expression was increased in metastatic breast cancer cells by retroviral transduction, and the metastasis of FAK- and TbetaR-II-manipulated tumors was monitored by using bioluminescent imaging.. TGF-beta stimulation of MECs stabilized and activated FAK in a beta3 integrin- and Src-dependent manner. Furthermore, by using the human MCF10A breast cancer progression model, we showed that increased FAK expression in metastatic breast cancer cells mirrored the acquisition of enhanced activation of p38 MAPK by TGF-beta. Administering FAK inhibitors or rendering metastatic breast cancer cells FAK deficient abrogated the interaction between beta3 integrin and TbetaR-II, thereby preventing TGF-beta from (a) activating p38 MAPK; (b) stimulating MEC invasion, migration, and EMT; and (c) inducing early primary tumor dissemination to the lungs. Finally, in contrast to FAK depletion, adjuvant FAK chemotherapy of mammary tumors decreased their growth in part by diminished macrophage tumor infiltration.. Our studies identify an essential function for FAK in mediating the interaction between beta3 integrin and TbetaR-II, and thus in facilitating the oncogenic conversion of TGF-beta required for mammary tumor metastasis. Furthermore, this study establishes chemotherapeutic targeting of FAK as an effective, two-pronged approach in preventing tumor progression both by decreasing innate immune cell infiltration, and by inhibiting early TGF-beta-dependent metastasis.

Topics: Animals; Breast Neoplasms; Cell Movement; Drug Delivery Systems; Focal Adhesion Protein-Tyrosine Kinases; Humans; Indoles; Integrin beta3; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Quinolones; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Sulfonamides; Sulfones; Transforming Growth Factor beta

During breast cancer progression, transforming growth factor-beta (TGF-beta) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-beta function depends upon fundamental changes in the TGF-beta signaling system. We show here that these changes in TGF-beta signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-beta1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-beta signaling evoked (i) resistance to TGF-beta-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-beta-stimulated Smad2/3 activity, which restored TGF-beta-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TbetaR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TbetaR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-beta signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-beta. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-beta signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-beta during breast cancer progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Crk-Associated Substrate Protein; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Here we use intravital imaging to demonstrate a reversible transition to a motile state as breast cancer cells spread. Imaging primary tumours revealed heterogeneity in cell morphology and motility. Two distinct modes of motility were observed: collective and single-celled. By monitoring the localization of Smad2 and the activity of a TGFbeta-dependent reporter gene during breast cancer cell dissemination, we demonstrate that TGFbeta signalling is transiently and locally activated in motile single cells. TGFbeta1 switches cells from cohesive to single cell motility through a transcriptional program involving Smad4, EGFR, Nedd9, M-RIP, FARP and RhoC. Blockade of TGFbeta signalling prevented cells moving singly in vivo but did not inhibit cells moving collectively. Cells restricted to collective invasion were capable of lymphatic invasion but not blood-borne metastasis. Constitutive TGFbeta signalling promoted single cell motility and intravasation but reduced subsequent growth in the lungs. Thus, transient TGFbeta signalling is essential for blood-borne metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Female; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN-gamma ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu-specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN-alpha and reduced transforming growth factor (TGF)beta1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFbeta1, reduced IFN-alpha, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis.

Topics: Bone Marrow; Breast; Breast Neoplasms; Case-Control Studies; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interferon-gamma; Middle Aged; Mucin-1; Prognosis; Survival Rate; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, differentiation, and apoptosis of various types of cells. Autophagy is emerging as a critical response of normal and cancer cells to environmental changes, but the relationship between TGF-beta signaling and autophagy has been poorly understood. Here, we showed that TGF-beta activates autophagy in human hepatocellular carcinoma cell lines. TGF-beta induced accumulation of autophagosomes and conversion of microtubule-associated protein 1 light chain 3 and enhanced the degradation rate of long-lived proteins. TGF-beta increased the mRNA expression levels of BECLIN1, ATG5, ATG7, and death-associated protein kinase (DAPK). Knockdown of Smad2/3, Smad4, or DAPK, or inhibition of c-Jun NH(2)-terminal kinase, attenuated TGF-beta-induced autophagy, indicating the involvement of both Smad and non-Smad pathway(s). TGF-beta activated autophagy earlier than execution of apoptosis (6-12 versus 48 h), and reduction of autophagy genes by small interfering RNA attenuated TGF-beta-mediated growth inhibition and induction of proapoptotic genes Bim and Bmf, suggesting the contribution of autophagy pathway to the growth-inhibitory effect of TGF-beta. Additionally, TGF-beta also induced autophagy in some mammary carcinoma cells, including MDA-MB-231 cells. These findings show that TGF-beta signaling pathway activates autophagy in certain human cancer cells and that induction of autophagy is a novel aspect of biological functions of TGF-beta.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Bcl-2-Like Protein 11; Beclin-1; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Proto-Oncogene Proteins; Receptors, Transforming Growth Factor beta; Smad Proteins; Transfection; Transforming Growth Factor beta; Ubiquitin-Activating Enzymes

Colony-stimulating factor-1 (CSF-1) and its receptor (CSF-1R) have been implicated in the pathogenesis and progression of various types of cancer, including breast cancer. This is based on high levels of circulating CSF-1 in patient sera with aggressive disease and increased CSF-1R staining in the tumor tissues. However, there have been no direct in vivo studies to determine whether a CSF-1 autocrine signaling loop functions in human breast cancer cells in vivo and whether it contributes to invasion. Recently, in mouse and rat models, it has been shown that invasion and metastasis are driven by an epidermal growth factor (EGF)/CSF-1 paracrine loop between tumor cells and host macrophages. In this macrophage-dependent invasion, tumor cells secrete CSF-1 and sense EGF, whereas the macrophages secrete EGF and sense CSF-1. Here, we test the hypothesis that in human breast tumors, the expression of both the CSF-1 ligand and its receptor in tumor cells leads to a CSF-1/CSF-1R autocrine loop which contributes to the aggressive phenotype of human breast tumors. Using MDA-MB-231 cell-derived mammary tumors in severe combined immunodeficiency mice, we show here for the first time in vivo that invasion in a human mammary tumor model is dependent on both paracrine signaling with host macrophages as well as autocrine signaling involving the tumor cells themselves. In particular, we show that the autocrine contribution to invasion is specifically amplified in vivo through a tumor microenvironment-induced upregulation of CSF-1R expression via the transforming growth factor-beta1.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Humans; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, SCID; Neoplasm Invasiveness; Rats; Receptor, Macrophage Colony-Stimulating Factor; Transforming Growth Factor beta; Transplantation, Heterologous

TGF-beta resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-beta regulates ERalpha-induced gene transcription and potential mechanisms of frequent TGF-beta resistance in breast cancer.. Effect of TGF-beta on ERalpha-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERalpha-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays.. A complex of Smad3/4 mediates TGF-beta inhibition of ERalpha-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-beta from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERalpha-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma.. These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-beta suppression of ERalpha transactivity in breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Estrogen Receptor alpha; Estrogens; Female; Humans; Response Elements; Smad3 Protein; Smad4 Protein; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Imaging, Three-Dimensional; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Cells of the monocyte/macrophage lineage are important for tumour cell migration, invasion and metastasis. Fusion between macrophages and cancer cells in animal models in vitro and in vivo causes hybrids with increased metastatic potential. Primary breast cancer cells were characterized for macrophage antigens to test if phenotypic resemblance to macrophages is related to early distant recurrence. Immunostaining for CD163, MAC387 and CD68 was performed in a breast cancer tissue micro array from 127 patients consequently followed up for a median of 13 years. Tumour-associated macrophages expressed all 3 antigens. The breast cancers expressed CD163 to 48%, MAC387 to 14% while CD68 was not expressed. TGF-beta staining intensity was positively related to both CD163 and MAC387 expression. Expression of CD163 in the cancer cells was compared to their DNA ploidy, Nottingham Histological Grade, TNM-stage, node state, presence of estrogen receptors and occurrence of distant metastases and survival. Cancers of a more advanced histological grade expressed CD163 to a higher extent. Cells expressing MAC387 were more common in cancers with a high proportion of CD163 positive cells. Multivariate analysis showed that expression of the macrophage antigen CD163 in breast cancer cells has a prognostic impact on the occurrence of distant metastases and reduced patient survival time.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Breast Neoplasms; Cell Line, Tumor; Disease-Free Survival; Female; Humans; Immunohistochemistry; Interleukin-10; Lymphatic Metastasis; Macrophages; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Ploidies; Proportional Hazards Models; Receptors, Cell Surface; Transforming Growth Factor beta

MMPs are zinc-dependent endopeptidases that are involved in proteolysis of extracellular matrix in both physiological and pathological processes including cancer. MMPs are involved at all stages of tumor progression, including tumor growth, angiogenesis, and metastasis. We recently showed that overexpression of Lefty in cancer cells restrains tumor growth. Here, we show that small forms of Lefty are generated by MMP2 (gelatinase A) mediated cleavage. In turn, these forms of Lefty strongly inhibit the autocatalytic, gelatinolytic and caseinolytic activities of MMP2 in vitro. We show that a short synthesized form of Lefty peptide (CASDGALVP) inhibits gelatinolytic and caseinolytic activities of MMP2 in vitro and inhibits tumor growth in vivo. Together, these findings show that lefty peptides are a new class of gelatinase A inhibitors that restrain tumor growth.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Female; Gelatinases; Humans; Left-Right Determination Factors; Matrix Metalloproteinase 2; Mice; Neoplasms; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA and protein synthesis. Aberrant miRNA expression leads to developmental abnormalities and diseases, such as cardiovascular disorders and cancer; however, the stimuli and processes regulating miRNA biogenesis are largely unknown. The transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) family of growth factors orchestrates fundamental biological processes in development and in the homeostasis of adult tissues, including the vasculature. Here we show that induction of a contractile phenotype in human vascular smooth muscle cells by TGF-beta and BMPs is mediated by miR-21. miR-21 downregulates PDCD4 (programmed cell death 4), which in turn acts as a negative regulator of smooth muscle contractile genes. Surprisingly, TGF-beta and BMP signalling promotes a rapid increase in expression of mature miR-21 through a post-transcriptional step, promoting the processing of primary transcripts of miR-21 (pri-miR-21) into precursor miR-21 (pre-miR-21) by the DROSHA (also known as RNASEN) complex. TGF-beta- and BMP-specific SMAD signal transducers are recruited to pri-miR-21 in a complex with the RNA helicase p68 (also known as DDX5), a component of the DROSHA microprocessor complex. The shared cofactor SMAD4 is not required for this process. Thus, regulation of miRNA biogenesis by ligand-specific SMAD proteins is critical for control of the vascular smooth muscle cell phenotype and potentially for SMAD4-independent responses mediated by the TGF-beta and BMP signalling pathways.

Topics: Animals; Apoptosis Regulatory Proteins; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line; Chlorocebus aethiops; DEAD-box RNA Helicases; Gene Expression Regulation; Humans; Ligands; Mice; MicroRNAs; Muscle, Smooth; Phenotype; Protein Binding; Ribonuclease III; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The retinoblastoma tumor suppressor protein (Rb) is mutated or expressed at very low levels in several tumor types, including retinoblastoma and osteosarcoma, as well as small cell lung, colon, prostate, bladder, and breast carcinomas. Loss or reduction of Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb function and a less-differentiated state, increased proliferation, and high metastatic potential. In this study, we found that knockdown of Rb by small interfering RNA in MCF7 breast cancer cells disrupts cell-cell adhesion and induces a mesenchymal-like phenotype. The epithelial-to-mesenchymal transition (EMT), a key event in embryonic morphogenesis, is implicated in the metastasis of primary tumors. Additionally, Rb is decreased during growth factor- and cytokine-induced EMT and overexpression of Rb inhibits the EMT in MCF10A human mammary epithelial cells. Ectopic expression and knockdown of Rb resulted in increased or reduced expression of E-cadherin, which is specifically involved in epithelial cell-cell adhesion. Other EMT-related transcriptional factors, including Slug and Zeb-1, are also induced by Rb depletion. Furthermore, we confirmed that Rb binds to an E-cadherin promoter sequence in association with the transcription factor activator protein-2alpha. Finally, in breast cancer specimens, we observed a concurrent down-regulation of Rb and E-cadherin expression in mesenchymal-like invasive cancers. These findings suggest that Rb inactivation contributes to tumor progression due to not only loss of cell proliferation control but also conversion to an invasive phenotype and that the inhibition of EMT is a novel tumor suppressor function of Rb.

Topics: Breast Neoplasms; Cadherins; Cell Adhesion; Cell Movement; Disease Progression; Embryonic Development; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Neoplasm Invasiveness; Phenotype; Retinoblastoma Protein; rho GTP-Binding Proteins; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The ZNF217 oncoprotein is a constituent of a core transcriptional complex that includes CoREST, histone deacetylase 1/2, lysine demethylase 1, and the C-terminal binding protein 1/2. We have combined genome-wide expression profiling and chromatin immunoprecipitation with directed selection and ligation (ChIP-DSL) to identify a subset of genes directly regulated by ZNF217. Our results establish p15(ink4b) as a direct target of the ZNF217 complex. Downregulation of ZNF217 in MCF-7 breast cancer cells resulted in a dramatic increase in p15(ink4b) expression and coincided with increases in dimethylation of H3-K4 and, surprisingly, a decrease in K9/K14-H3 acetylation. Stimulation of HaCaT cells with transforming growth factor beta (TGF-beta) resulted in a release of ZNF217 and a concomitant binding of SMAD2 to the proximal promoter, which preceded increases in ink4b protein expression. Furthermore, the changes in chromatin marks at the p15(ink4b) promoter following TGF-beta stimulation were similar to those observed following ZNF217 downregulation. Collectively, these results establish the ZNF217 complex as a novel negative regulator of the p15(ink4b) gene and may constitute an important link between amplification of ZNF217 and the loss of TGF-beta responsiveness in breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Co-Repressor Proteins; Cyclin-Dependent Kinase Inhibitor p15; DNA-Binding Proteins; Genomics; Humans; Nerve Tissue Proteins; Promoter Regions, Genetic; Repressor Proteins; Trans-Activators; Transforming Growth Factor beta

In HER2-overexpressing mammary epithelial cells, transforming growth factor beta (TGF-beta) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-beta or expression of an activated TGF-beta type I receptor (Alk5 with the mutation T204D [Alk5(T204D)]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-alpha, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-beta-induced activation of Akt and cell invasiveness. Treatment with TGF-beta or expression of Alk5(T204D) in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5(T204D) expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-beta may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.

Topics: ADAM Proteins; ADAM17 Protein; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cells, Cultured; Enzyme Activation; Female; Gene Expression Profiling; Humans; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; RNA Interference; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Trastuzumab; Treatment Outcome

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Disease-Free Survival; Humans; Radiation Pneumonitis; Radiation Tolerance; Radiodermatitis; Tamoxifen; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a normal physiological process that regulates tissue development, remodeling and repair; however, aberrant EMT also elicits disease development in humans, including lung fibrosis, rheumatoid arthritis and cancer cell metastasis. Transforming growth factor-beta (TGF-beta) is a master regulator of EMT in normal mammary epithelial cells (MECs), wherein this pleiotropic cytokine also functions as a potent suppressor of mammary tumorigenesis. In contrast, malignant MECs typically evolve resistance to TGF-beta-mediated cytostasis and develop the ability to proliferate, invade and metastasize when stimulated by TGF-beta. It therefore stands to reason that establishing how TGF-beta promotes EMT may offer new insights into targeting the oncogenic activities of TGF-beta in human breast cancers. By monitoring alterations in the actin cytoskeleton and various markers of EMT, we show here that the TGF-beta gene target, fibulin-5 (FBLN5), initiates EMT and enhances that induced by TGF-beta. Whereas normal MECs contain few FBLN5 transcripts, those induced to undergo EMT by TGF-beta show significant upregulation of FBLN5 messenger RNA, suggesting that EMT and the dedifferentiation of MECs override the repression of FBLN5 expression in polarized MECs. We also show that FBLN5 stimulated matrix metalloproteinase expression and activity, leading to MEC invasion and EMT, to elevated Twist expression and to reduced E-cadherin expression. Finally, FBLN5 promoted anchorage-independent growth in normal and malignant MECs, as well as enhanced the growth of 4T1 tumors in mice. Taken together, these findings identify a novel EMT and tumor-promoting function for FBLN5 in developing and progressing breast cancers.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Extracellular Matrix Proteins; Female; Immunohistochemistry; Mammary Glands, Animal; Metalloproteases; Mice; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNrho0-8 and MCFrho0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNrho0-8 and MCFrho0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro. In addition, the induction of vimentin and the repression of E-cadherin expression in rho0 cells indicate that they are mesenchymal cells. Since LNrho0-8 and MCFrho0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the rho0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-beta) and type I TGF-beta receptor (TGF-betaRI). EMT requires cooperation of TGF-beta signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; DNA, Mitochondrial; Epithelial Cells; Female; Humans; In Vitro Techniques; Male; Mesoderm; Pancreatic Neoplasms; Phenotype; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Vimentin

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

Topics: Breast Neoplasms; Cell Line, Tumor; Enzyme Activation; Estradiol; Estrogen Antagonists; Estrogens; Female; Fulvestrant; Gene Silencing; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-beta-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-beta-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-beta induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-beta-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3' untranslated region largely reversed the phenotype induced by miR-155 and TGF-beta. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Line; Cell Movement; Epithelial Cells; Female; Gene Expression Profiling; Humans; Mice; Microarray Analysis; MicroRNAs; Molecular Sequence Data; Neoplasm Invasiveness; rhoA GTP-Binding Protein; Sequence Alignment; Signal Transduction; Smad4 Protein; Transcription, Genetic; Transforming Growth Factor beta

The active acquisition of epigenetic changes is a poorly understood but important process in development, differentiation, and disease. Our work has shown that repression of the p16/pRb pathway in human epithelial cells, a condition common to stem cells and many tumor cells, induces dynamic epigenetic remodeling resulting in the targeted methylation of a selected group of CpG islands. We hypothesized that cells in this epigenetically plastic state could be programmed by the microenvironment to acquire epigenetic changes associated with tumorigenesis. Here, we describe an in vitro model system where epigenetically plastic cells were placed in an environment that induced epithelial to mesenchymal transition (EMT) and led to a program of acquired de novo DNA methylation at targeted sites. In this model, we found that repression of E-cadherin transcription preceded the subsequent acquisition of methylated CpG sites. Furthermore, the induction of EMT was accompanied by de novo methylation of several other gene promoters, including those of the estrogen receptor and Twist. These data demonstrate that signals from the microenvironment can induce phenotypic and gene expression changes associated with targeted de novo epigenetic alterations important in tumor progression, and that these alterations occur through a deterministic, rather than stochastic, mechanism. Given the dynamic epigenetic reprogramming that occurs in these cells, DNA methylation profiles observed in human tumors may reflect the history of environmental exposures during the genesis of a tumor.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA Methylation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Mesoderm; Promoter Regions, Genetic; Serum; Smad2 Protein; Transforming Growth Factor beta

Runx2, required for bone formation, is ectopically expressed in breast cancer cells. To address the mechanism by which Runx2 contributes to the osteolytic disease induced by MDA-MB-231 cells, we investigated the effect of Runx2 on key components of the "vicious cycle" of transforming growth factor beta (TGFbeta)-mediated tumor growth and osteolysis. We find that Runx2 directly up-regulates Indian Hedgehog (IHH) and colocalizes with Gli2, a Hedgehog signaling molecule. These events further activate parathyroid hormone-related protein (PTHrP). Furthermore, Runx2 directly regulates the TGFbeta-induced PTHrP levels. A subnuclear targeting deficient mutant Runx2, which disrupts TGFbeta-induced Runx2-Smad interactions, failed to induce IHH and downstream events. In addition, Runx2 knockdown in MDA-MB-231 inhibited IHH and PTHrP expression in the presence of TGFbeta. In vivo blockade of the Runx2-IHH pathway in MDA-MB-231 cells by Runx2 short hairpin RNA inhibition prevented the osteolytic disease. Thus, our studies define a novel role of Runx2 in up-regulating the vicious cycle of metastatic bone disease, in addition to Runx2 regulation of genes related to progression of tumor metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Core Binding Factor Alpha 1 Subunit; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Mice; Mice, SCID; Models, Biological; Nuclear Proteins; Osteoclasts; Parathyroid Hormone-Related Protein; RNA, Small Interfering; Signal Transduction; Tissue Distribution; Transcriptional Activation; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Zinc Finger Protein Gli2

Transforming growth factor beta regulates many biological processes including cell motility and invasion. Podosomes are specialized F-actin rich structures found in normal cells, such as osteoclasts and macrophages. Tumor cells often form related structures called invadopodia that are thought to promote invasion and metastasis. Here we show that human breast cancer cells organize F-actin rich structures in response to transforming growth factor beta that colocalize with areas of extracellular matrix degradation. We further show that organizing the complex of proteins needed to form these structures requires signaling through phosphatidylinositide 3-kinase and Src kinase, while activating the proteases involved in degradation of extracellular matrix requires extracellular signal-regulated kinase signaling, and that each of these pathways is activated by transforming growth factor beta in CA1D human breast cancer cells.

Topics: Actins; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Extracellular Matrix; Female; Gelatin; Humans; Matrix Metalloproteinase 9; Mice; Microscopy, Fluorescence; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Ubiquitin-dependent protein degradation is involved in various biological processes, and accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are E3 ubiquitin ligases, which suppress transforming growth factor-beta (TGF-beta) family signaling through degradation of Smads and receptors for TGF-beta and bone morphogenetic proteins. In addition, Smurf1 has been reported to promote RhoA ubiquitination and degradation and regulate cell motility, suggesting the involvement of Smurf1 in cancer progression. However, the regulation and biological function of Smurf1 and Smurf2 in cancer development remain to be elucidated. In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. Of note, knockdown of Smurf1, but not of Smurf2, enhanced TGF-beta signaling in MDA-MB-231 cells, suggesting that increased an protein level of Smurf1 offsets the effect of Smurf2 knockdown on TGF-beta signaling. These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Knockdown Techniques; Humans; Neoplasm Metastasis; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination

mTOR, the mammalian target of rapamycin, is a critical target of survival signals in many human cancers. In the absence of serum, rapamycin induces apoptosis in MDA-MB-231 human breast cancer cells. However, in the presence of serum, rapamycin induces G(1) cell cycle arrest-indicating that a factor(s) in serum suppresses rapamycin-induced apoptosis. We report here that transforming growth factor-beta (TGF-beta) suppresses rapamycin-induced apoptosis in serum-deprived MDA-MB-231 cells in a protein kinase Cdelta (PKCdelta)-dependent manner. Importantly, if TGF-beta signaling or PKCdelta was suppressed, rapamycin induced apoptosis rather than G(1) arrest in the presence of serum. And, if cells were allowed to progress into S phase, rapamycin induced apoptosis in the presence of serum. BT-549 and MDA-MB-468 breast, and SW-480 colon cancer cells have defects in TGF-beta signaling and rapamycin induced apoptosis in these cells in the presence of either serum or TGF-beta. Thus, in the absence of TGF-beta signaling, rapamycin becomes cytotoxic rather than cytostatic. Importantly, this study provides evidence indicating that tumors with defective TGF-beta signaling--common in colon and pancreatic cancers--will be selectively sensitive to rapamycin or other strategies that target mTOR.

Topics: Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Signal Transduction; Sirolimus; Transforming Growth Factor beta

Transforming growth factor beta1 (TGF-beta1) suppresses tumor development at early stages of cancer, but enhances tumor invasion and formation of metastasis. TGF-beta1-mediated tumor invasion is associated with epithelial to mesenchymal transition (EMT) and matrix proteolysis. The mechanisms of these TGF-beta1 responses in normal and tumor cells are not well understood. Recently, we have reported that TGF-beta1 increases expression of high-molecular weight tropomyosins (HMW-tropomyosins) and formation of actin stress fibers in normal epithelial cells. The present study investigated the role of tropomyosin in TGF-beta1-mediated cell motility and invasion. We found that TGF-beta1 restricts motility of normal epithelial cells although it promotes EMT and formation of actin stress fibers and focal adhesions. Cell motility was enhanced by siRNA-mediated suppression of HMW-tropomyosins. TGF-beta1 stimulated migration and matrix proteolysis in breast cancer MDA-MB-231 cells that express low levels of HMW-tropomyosins. Tet-Off-regulated expression of HMW-tropomyosin inhibited cell migration and matrix proteolysis without affecting expression of matrix metalloproteinases. Tropomyosin increased cell adhesion to matrix by enhancing actin fibers and focal adhesions. Finally, tropomyosin impaired the ability of tumor cells to form lung metastases in SCID mice. Thus, these results suggest that HMW-tropomyosins are important for TGF-beta-mediated control of cell motility and acquisition of the metastatic potential.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Movement; Collagen; Drug Combinations; Humans; Immunoblotting; Laminin; Lung Neoplasms; Mammary Glands, Animal; Matrix Metalloproteinases; Mice; Mice, SCID; Molecular Weight; Neoplasm Invasiveness; Proteoglycans; Rats; Transforming Growth Factor beta; Tropomyosin; Tumor Cells, Cultured; Wound Healing

We recently showed that bone morphogenetic protein 7 (BMP7) is overexpressed in primary breast tumors. Here we explored the clinical significance of BMP7 expression in breast cancer.. This study included 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas, and 40 local recurrences. BMP7 protein expression was determined using immunohistochemistry.. BMP7 was expressed in 47% of the primary tumor samples and 13% of the local recurrences. The primary tumors expressed BMP7 more often than the corresponding local recurrences (P = 0.004). BMP7 expression was dependent on the tumor subtype; 57% of the lobular carcinomas but only 37% of the ductal carcinomas were BMP7 positive (P = 0.0001). BMP7 expression was associated with accelerated bone metastasis formation (P = 0.040), especially in ductal carcinomas (P = 0.033), and multivariate analysis confirmed that BMP7 is an independent prognostic indicator for early bone metastasis development (P = 0.032).. BMP7 is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis. This is the first time that bone inducing factor BMP7 has been linked to the bone metastasis process in breast cancer.

Topics: Adult; Aged; Analysis of Variance; Biomarkers, Tumor; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cohort Studies; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Proportional Hazards Models; Retrospective Studies; Risk Assessment; Survival Analysis; Time Factors; Transforming Growth Factor beta

We and others previously observed immunosurveillance against transplantable tumors in mice, and enhancement thereof by blockade of negative regulation by T reg cells or the NKT-IL-13-myeloid cell-TGF-beta regulatory circuit. However, it was unknown whether natural immunosurveillance inhibits growth of completely spontaneous autochthonous tumors, and whether it can be improved by inhibition of negative regulation.. To examine the existence of T cell-mediated immunosurveillance against spontaneous tumors, BALB-neuT mice were treated with anti-CD4 and/or anti-CD8. A role for IL-13 in the suppression of immunosurveillance was investigated by treating mice with IL-13 inhibitor.. We show that even spontaneous autochthonous breast carcinomas arising in Her-2/neu transgenic mice appear more quickly when the mice are depleted of T cells, evidence for T-cell mediated immunosurveillance slowing tumor growth. This immunosurveillance could be further enhanced by blockade of IL-13 (but not IL-4) which slowed the appearance of these autologous tumors compared to control antibody-treated mice.. Thus, even completely spontaneous, autochthonous breast cancers can be controlled in part by natural immunosurveillance, and blockade of negative regulation can improve this control.

Topics: Animals; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Gene Expression Regulation, Neoplastic; Interleukin-13; Interleukin-4; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Transgenic; Monitoring, Immunologic; Neoplasm Transplantation; Rats; Transforming Growth Factor beta

The breast cancer incidence has been increasing in the south Indian women. A case (n=250)-control (n=500) study was undertaken to investigate the role of Single Nucleotide Polymorphisms (SNP's) in GSTM1 (Present/Null); GSTP1 (Ile105Val), p53 (Arg72Pro), TGFbeta1 (Leu10Pro), c-erbB2 (Ile655Val), and GSTT1 (Null/Present) in breast cancer. In addition, the value of the SNP's in predicting primary tumor's pathologic response following neo-adjuvant chemo-radiotherapy was assessed. Genotyping was done using PCR (GSTM1, GSTT1), Taqman Allelic discrimination assay (GSTP1, c-erbB2) and PCR-CTPP (p53 and TGFbeta1). None of the gene SNP's studied were associated with a statistically significant increased risk for the breast cancer. However, combined analysis of the SNP's showed that p53 (Arg/Arg and Arg/Pro) with TGFbeta1 (Pro/Pro and Leu/Pro) were associated with greater than 2 fold increased risk for breast cancer in Univariate (P=0.01) and Multivariate (P=0.003) analysis. There was no statistically significant association for the GST family members with the breast cancer risk. TGFbeta1 (Pro/Pro) allele was found to predict complete pathologic response in the primary tumour following neo-adjuvant chemo-radiotherapy (OR=6.53 and 10.53 in Univariate and Multivariate analysis respectively) (P=0.004) and was independent of stage. This study suggests that SNP's can help predict breast cancer risk in south Indian women and that TGFbeta1 (Pro/Pro) allele is associated with a better pCR in the primary tumour.

Topics: Antineoplastic Agents; Breast Neoplasms; Case-Control Studies; Chemotherapy, Adjuvant; DNA, Neoplasm; Extracellular Matrix Proteins; Female; Genetic Predisposition to Disease; Genetic Testing; Glutathione S-Transferase pi; Glutathione Transferase; Humans; India; Mastectomy; Neoadjuvant Therapy; Odds Ratio; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Radiotherapy, Adjuvant; Risk Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Development of acquired resistance to tamoxifen is a major clinical problem during endocrine treatment in estrogen receptor positive breast cancer. Transforming growth factor-beta1 (TGF-beta) has been implicated in tamoxifen-induced cellular signaling in breast cancer, and increased Akt activation is associated with tamoxifen-resistant cell types. We hypothesized that the relationship between TGF-beta and Akt signaling may be involved in the development and progression of tamoxifen resistance. Tamoxifen-resistant (Tam-R) cells were established from parental MCF-7 cells by continuously exposing them to 4-hydroxytamoxifen (4-OHT). Tam-R cells were associated with a decrease in TGF-beta1 secretion, TGF-beta-mediated transcriptional response, and growth inhibitory effects of 4-OHT. Tam-R cells expressed significantly higher levels of phosphorylated Akt and lower levels of phosphorylated Smad 3 in both the absence and presence of 4-OHT when compared to MCF-7 cells treated with 4-OHT. Ectopic expression of constitutively active Akt (Myc-Akt(Myr)) rendered MCF-7 cells resistant to activation by TGF-beta and the growth inhibitory effects of 4-OHT, while over-expression of kinase-dead Akt (Myc-Akt(K179M)) or LY294002 treatment of Tam-R cells enhanced TGF-beta activation and blocked cell growth. These results suggest that suppression of TGF-beta signaling by activated Akt is correlated with the development of tamoxifen resistance in breast cancer.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Proto-Oncogene Proteins c-akt; Signal Transduction; Tamoxifen; Transforming Growth Factor beta

Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.

Topics: Animals; Breast Neoplasms; CD11b Antigen; Cell Line, Tumor; Female; Gene Deletion; Humans; Matrix Metalloproteinases; Mice; Models, Biological; Myeloid Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

We describe here the regulation of ABCG2 expression and side population (SP) abundance in MCF7 human breast cancer cells. The level of ABCG2 mRNA and protein were increased in purified MCF7 SP relative to non-SP cells, and incubation with an ABCG2-specific inhibitor or ABCG2 short interfering RNA eliminated the MCF7 SP. The purified MCF7 SP could generate a heterogeneous population containing both SP and non-SP cells in culture. In vivo tumorigenicity experiments showed that the purified MCF7 SP has an increased ability to colonize the mouse mammary gland. Importantly, the MCF7 SP was depleted by a transforming growth factor-beta (TGFbeta)-directed epithelial-mesenchymal transition (EMT), and this effect was associated with a strong down-regulation of ABCG2 gene expression, and an increased sensitivity to mitoxantrone. ABCG2 expression and SP abundance were restored upon the removal of transforming growth factor-beta and reversion of the cells to an epithelial phenotype. Knock-down of E-cadherin also reduced SP abundance, but this effect was not accompanied by the loss of ABCG2 mRNA or protein. We conclude that ABCG2 expression in MCF7 cells is regulated during an EMT, and that the EMT effect reflects posttranslational regulation of ABCG2 function by E-cadherin as well as transcriptional repression of the ABCG2 gene.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cadherins; Cell Growth Processes; Cell Line, Tumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Mice; Mice, Inbred NOD; Mice, SCID; Mitoxantrone; Neoplasm Proteins; Protein Processing, Post-Translational; Recombinant Proteins; RNA, Messenger; RNA, Small Interfering; Transfection; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) is an important event during carcinoma progression and leads to increased tumor cell malignancy. Here, we show that vascular endothelial (VE)-cadherin is induced during EMT in mammary tumor cells and is aberrantly expressed in invasive human breast carcinomas. VE-cadherin enhanced the capacity of fibroblastoid tumor cells to proliferate, form cord-like invasive structures, and adhere to endothelial cells, characteristics that are key contributors to their increased malignancy and metastatic potential. Consistently, VE-cadherin expression in malignant fibroblastoid tumor cells promoted the growth of experimental mammary carcinomas in vivo. Analysis of the signaling mechanisms involved revealed that VE-cadherin expression influences the levels of Smad2 phosphorylation and expression of target genes of transforming growth factor-beta (TGF-beta), a major mediator of advanced tumor progression and malignant tumor cell proliferation. VE-cadherin might thus promote tumor progression not only by contributing to tumor angiogenesis but also by enhancing tumor cell proliferation via the TGF-beta signaling pathway. This article provides evidence for a novel function of VE-cadherin in tumor progression and reveals a previously unknown molecular link between VE-cadherin expression and TGF-beta signaling. Our findings may have important implications for the clinical application of anti-VE-cadherin strategies.

Topics: Animals; Antigens, CD; Breast Neoplasms; Cadherins; Cell Line, Tumor; Disease Progression; Gene Expression Profiling; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta

Invasive lobular carcinoma (ILC) and lobular carcinoma in situ characteristically show loss of E-cadherin expression and so immunohistochemistry for E-cadherin is being increasingly used as a tool to differentiate between lobular and ductal lesions in challenging situations. However, misinterpretation of "aberrant" positive staining may lead some to exclude a diagnosis of lobular carcinoma. E-cadherin and beta-catenin immunohistochemistry was analyzed in 25 ILCs. E-cadherin "positive" ILCs were subjected to molecular analysis including comparative genomic hybridization. Different morphologic components of case 25, showing heterogenous E-cadherin expression, were analyzed by E-cadherin gene sequencing, methylation, and DASL gene expression profiling. Four ILCs were positive for E-cadherin, but each also had neoplastic cells with aberrant staining. Two of these ILCs were positive for beta-catenin, again with some aberrantly stained neoplastic cells, and 2 were negative. The solid component of case 25 was positive for E-cadherin whereas the classic and alveolar areas were negative. All components harbored an in-frame deletion in exon 7 (867del24) of the E-cadherin gene and loss of the wild type allele. Comparative genomic hybridization demonstrated evidence of clonal evolution from E-cadherin-positive to E-cadherin-negative components. E-cadherin down-regulation seems to be through transcriptional repression via activation of transforming growth factor-beta/SMAD2 rather than methylation. Positive staining for E-cadherin should not preclude a diagnosis of lobular in favor of ductal carcinoma. Molecular evidence suggests that even when E-cadherin is expressed, the cadherin-catenin complex maybe nonfunctional. Misclassification of tumors may lead to mismanagement of patients in clinical practice, particularly in the context of in situ disease at margins.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cadherins; Carcinoma in Situ; Carcinoma, Ductal; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Diagnosis, Differential; DNA Methylation; DNA, Neoplasm; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Loss of Heterozygosity; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Neoplasm; Sequence Analysis, DNA; Smad2 Protein; Transforming Growth Factor beta

The microenvironment of a tumor is known to influence tumor progression and spread to distant sites (metastasis). Padua et al. (2008) now show that transient exposure of breast cancer cells to the signaling molecule transforming growth factor beta (TGFbeta) promotes their extravasation from blood vessels and entry into the lung by upregulation of the adipokine angiopoietin-like 4 (ANGPTL4). Their work shows that the later stages of metastasis can be influenced by transient signals produced in the primary tumor microenvironment.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Breast Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Endothelial Cells; Female; Gene Expression Profiling; Humans; Intercellular Junctions; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neoplasms, Experimental; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

The current hypothesis of tumorigenesis in humans suggests that cancer cells acquire their hallmarks of malignancy through the accumulation of advantageous gene activation and inactivation events over long periods of time. For breast cancer development, this multistep process may manifest itself as a sequence of pathologically defined stages. It is widely held that breast cancer originates at the premalignant stage of atypical ductal hyperplasia, progresses to the preinvasive stage of ductal carcinoma in situ, and culminates in the potentially lethal stage of invasive ductal carcinoma. Tumor grade has been a highly valuable prognostic factor for breast cancer, and high-grade ductal carcinoma in situ lesions are associated with poor clinical outcome. The aim of this work was to investigate the BigH3 protein expression changes associated with various stages of breast cancer progression in comparison to benign specimens using tissue microarray technology. Pathological characteristics of breast tissues ranged from benign lesions to breast cancers either of lobular or ductal carcinomas in origin, and included in situ ductal carcinomas, lobular carcinomas, infiltrating ductal carcinomas, carcinomas, scirrhous carcinomas, adenocarcinomas and infiltrating colloid carcinomas. BigH3 protein expression was analyzed by immunohistochemistry in 192 cases of breast tumors. Results indicated a decrease in BigH3 protein expression from benign tissues to in situ ductal carcinoma, lobular carcinoma, infiltrating ductal carcinomas, carcinomas, scirrhous carcinoma, adenocarcinomas to infiltrating colloid carcinomas. We observed that the benign tissue had a 23-fold increase in BigH3 protein expression compared to the infiltrating colloid carcinoma which was the most malignant tissue analyzed. In summary, these studies confirmed the suppressor effect of the BigH3 gene expressed as protein expression in those processes related to the progression of breast tumorigenesis. We conclude that this protein can be used as a marker for breast cancer progression.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Extracellular Matrix Proteins; Female; Humans; Mammary Glands, Human; Microarray Analysis; Middle Aged; Transforming Growth Factor beta

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Adhesion; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The development of osteolytic breast cancer bone metastases relies on the ability of tumor cells to stimulate the formation of bone-resorbing osteoclasts. We have studied the effects of soluble factors produced by MDA-MB-231 breast carcinoma cells on osteoclast formation from human monocytic precursors and RAW 264.7 monocytic cells. Although factors produced by breast cancer cells were ineffective in inducing osteoclast formation from monocytes, priming with RANKL for 1-3 days dramatically increased receptiveness of osteoclast precursors to cancer-derived factors, which enhanced osteoclast formation 2-3 fold in the absence of supporting cell types. Osteoclasts formed by exposure to cancer factors expressed proteases critical for bone resorption, cathepsin K and matrix metalloproteinase 9, and were capable of resorbing calcified matrices. Expression of key osteoclastogenic transcription factor NFATc1 in osteoclast precursors was dramatically increased by short treatment with RANKL. NFATc1 was localized in the nuclei of primed osteoclast precursors when RANKL was present; however removal of RANKL led to rapid nuclear export of NFATc1. Cancer-derived factors were able to substitute for RANKL in supporting nuclear localization of NFATc1. Using neutralizing antibodies against TGFbeta, and a kinase inhibitor targeting the TGFbeta type I receptor, we identified TGFbeta as a permissive factor, required for the effects of breast cancer cells on NFATc1 nuclear accumulation and osteoclast formation. Our data suggest that, during differentiation, osteoclast precursors acquire the competency to respond to factors secreted by breast cancer cells, which may serve to promote tumor growth at skeletal sites undergoing active bone turnover.

Topics: Animals; Biomarkers; Bone Resorption; Breast Neoplasms; Cell Differentiation; Cell Line; Cell Nucleus; Humans; Mice; NFATC Transcription Factors; Osteoclasts; Protein Transport; RANK Ligand; Solubility; Stem Cells; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) regulate diverse cellular processes, such as proliferation, differentiation, and apoptosis. The BMPs have been studied in several cancers, but thus far contradictory results have been obtained and, especially in breast cancer, information on BMPs is still limited. We performed a systematic expression survey of BMPs and their receptors in breast cancer. mRNA expression was studied of seven BMP ligands (BMP2-BMP8) and six receptors (ACVR1, BMPR1A, BMPR1B, BMPR2, ACVR2A, and ACVR2B) that specifically mediate BMP signals. Expression levels were determined in 22 breast cancer cell lines, 39 primary breast tumors, normal human mammary epithelial cell line, and normal mammary gland using semiquantitative RT-PCR. The expression frequencies and expression levels of different BMPs varied considerably in breast cancer with BMP4 and BMP7 being most frequently expressed and showing highest expression levels. The BMP specific receptors were more uniformly expressed and indicated that breast cancer is fully capable of transmitting BMP signals. Expression frequencies and levels for both the ligands and the receptors were in good concordance between the breast cancer cell lines and primary tumors. We can conclude that breast cancers possess functional BMP signaling machinery on the cell surface with distinct differences in the expression of various BMP ligands. Our survey focuses the attention particularly toward BMP4 and BMP7 and suggests their importance in breast cancer. Breast cancer cell lines and the data generated here serve as a good resource for further studies on BMP function in breast cancer.

Topics: Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Breast; Breast Neoplasms; Female; Humans; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Increased breast cancer risks have been reported among women with gross cystic breast disease (GCBD), although the mechanism for this increase remains unexplained. Relationships between GCBD characteristics, breast cancer risk factors, and the biochemical composition and growth properties of 142 breast cyst fluid (BCF) samples were studied among 93 women with GCBD. Concentrations of melatonin, estrogen (17-beta-estradiol), dehydroepiandrosterone-sulfate (DHEA-S), epidermal growth factor (EGF), transforming growth factor beta (TGF-B1 and TGF-B2), sodium (Na), and potassium (K) were quantified in BCF samples, and human breast cancer cells (MCF-7) were treated with BCF in vitro. Patients were grouped according to BCF Na:K ratios previously linked with increased breast cancer risks (Na:K 3, Type 2) and mixed cyst groups. Women with larger and more frequently occurring cysts had higher BCF estrogen and DHEA-S, and lower TGF-B1 levels. Women with Type 1 cysts had elevated BCF melatonin, estrogen, DHEA-S, and EGF, and lower concentrations of TGF-B2 compared to women with Type 2 cysts. BCF generally inhibited cell growth relative to serum-treated controls, consistent with previous studies. Melatonin and estrogen in BCF independently predicted growth inhibition and stimulation, respectively. Biological monitoring of BCF may help identify women with GCBD at greatest risk for breast cancer development.

Topics: Adult; Breast Cyst; Breast Neoplasms; Cell Line, Tumor; Cyst Fluid; Dehydroepiandrosterone Sulfate; Epidermal Growth Factor; Estradiol; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Melatonin; Middle Aged; Transforming Growth Factor beta

SnoN is an important negative regulator of transforming growth factor beta signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed pro-oncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both pro-tumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.

Topics: Animals; Breast Neoplasms; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Glandular and Epithelial; Proto-Oncogene Proteins; rhoA GTP-Binding Protein; Transforming Growth Factor beta

The anti-estrogen tamoxifen and vitamin A-related compound, all-trans retinoic acid (RA), in combination act synergistically to inhibit the growth of MCF-7 human breast cancer cells. In the present study, we applied two-dimensional gel electrophoresis based proteomic approach to globally analyze this synergistic effect of RA and tamoxifen. Proteomic study revealed that multiple clusters of proteins were involved in RA and tamoxifen-induced apoptosis in MCF-7 breast cancer cells, including post-transcriptional and splicing factors, proteins related to cellular proliferation or differentiation, and proteins related to energy production and internal degradation systems. The negative growth factor-transforming growth factor beta (TGFbeta) was secreted by RA and/or tamoxifen treatment and was studies as a potential mediator of the synergistic effects of RA and tamoxifen in apoptosis. By comparing protein alterations in treatments of RA and tamoxifen alone or in combination to those of TGFbeta treatment, or co-treatment with TGFbeta inhibitor SB 431542, proteomic results showed that a number of proteins were involved in TGFbeta signaling pathway. These results provide valuable insights into the mechanisms of RA and tamoxifen-induced TGFbeta signaling pathway in breast cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Estrogen Antagonists; Humans; Neoplasm Proteins; Protein Biosynthesis; Proteomics; Tamoxifen; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.

Topics: Antigens, CD; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; CD24 Antigen; Cell Lineage; Cells, Cultured; Endothelial Protein C Receptor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Mammary Glands, Human; Neoplastic Stem Cells; Pregnancy; Receptors, Cell Surface; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Radiotherapy and chemotherapy often induce DNA double-strand breaks in both normal and malignant cells. The proteins involved in the repair of such lesions are central to cancer prognosis and treatment, as they can be overexpressed in many cancers, accelerating malignant transformation and increasing repair capacity, potentially leading to cellular resistance. If malignant cells can be selectively targeted repair proteins could also be candidates for targeted therapy. In this study, two keyplayers in eukaryotic DNA double-strand break repair, Rad51 and DNA-dependent protein kinase catalytic subunit, were analysed in noncancerous human breast cells (MCF12A) and the breast cancer cell lines (MDA MB 231 and MCF7) in response to treatment with doxorubicin. A cell cycle-independent increase in Rad51 protein levels (a recombinase involved in homologous recombination repair) was observed 24 and 48 h after treatment in MDA MB 231 and MCF12A when exposed to low levels of doxorubicin, whereas MCF7 cells displayed a continuous decrease in Rad51 protein with increasing drug concentration. DNA-dependent protein kinase catalytic subunit, which is involved in nonhomologous end joining of DNA lesions, remained unaltered under all conditions tested. Topoisomerase II-alpha protein, the primary target of doxorubicin, was upregulated at low concentrations of doxorubicin in all cell lines tested. Here we show that Rad51 protein levels can be differentially regulated in normal and malignant breast cell lines in response to doxorubicin, independent of cell cycle state. These observations have direct relevance to chemosensitivity and add an additional prognostic factor that could be taken into account when designing targeted therapeutic regimes.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Doxorubicin; Female; Flow Cytometry; Genes, p53; Humans; Rad51 Recombinase; Receptors, Estrogen; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) can modulate the activity of various MAP kinases. However, how this pathway may mediate TGFbeta-induced malignant phenotypes remains elusive. We investigated the role of autocrine TGFbeta signaling through MAP kinases in the regulation of cell survival in breast carcinoma MCF-7 cells and untransformed human mammary epithelial cells (HMECs). Our results show that abrogation of autocrine TGFbeta signaling with the expression of a dominant negative type II TGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptor inhibitor significantly increased apoptosis in MCF-7 cell, but not in HMEC. The expression of DNRII markedly decreased activated/phosphorylated Erk, whereas increased activated/phosphorylated p38 in MCF-7 cells. In contrast, there was no or little change of phosphorylated Erk and p38 in HMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7 control cell induced apoptosis whereas restoration of Erk activity in MCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activity also inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbeta signaling can enhance the survival of MCF-7 cells by maintaining the level of active Erk high and the level of active p38 low. Furthermore, the survival properties of TGFbeta pathway appear related to transformation supporting the notion that it may be a potential target for cancer therapy.

Topics: Apoptosis; Autocrine Communication; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Survival; Female; Flavonoids; Humans; Imidazoles; Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Inhibition of platelet-derived growth factor receptor (PDGFR) signaling restricts the growth of human breast cancer in the bone of nude mice. We hypothesized that osteoblast-secreted substances may alter the response capacity of breast cancer cells to the PDGFRs tyrosine kinase inhibitor imatinib mesylate. We found that osteoblast-conditioned medium (OCM) increases the proliferation rate of the estrogen receptor negative (ER-) MDA-MB-231 and of the ER+ MCF-7 human breast cancer cell lines and the growth-promoting effect on ER+ cells is independent from estrogen. OCM significantly improved the dose- and the time-dependent sensitivity of the tumor cells to the anti-proliferative effect of imatinib. We also found that MDA-MB-231 and MCF-7 cells express the two PDGFRs subtypes, PDGFR-alpha and PDGFR-beta, and OCM treatment increases the expression of the PDGFRs. Furthermore, imatinib inhibited the phosphorylation rate of its target tyrosine kinase receptors. We conclude that bone microenvironment, through osteoblast-secreted substances may cause estrogen-independent proliferation of breast cancer cells by a mechanism mediated by the induction of PDGFRs expression. The enhanced sensitivity of OCM-treated breast cancer cells to imatinib would justify investigation on the efficacy of imatinib in bone breast cancer metastasis.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Humans; Imatinib Mesylate; Osteoblasts; Phosphorylation; Piperazines; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Estrogen; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 x 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-beta-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cluster Analysis; Dose Fractionation, Radiation; Female; Gene Expression; Gene Expression Profiling; Genes, p53; Glioma; Humans; Interferons; Male; Mice; Mice, Nude; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Estrogen Replacement Therapy; Estrogens; Female; Forkhead Transcription Factors; Gene Expression Regulation, Enzymologic; Humans; Mice; Osteoporosis, Postmenopausal; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Resveratrol; Risk Factors; src-Family Kinases; Stilbenes; Transforming Growth Factor beta

Transforming growth factor beta (TGF-ss) is able to inhibit proliferation of epithelial cells and is involved in the carcinogenesis of human mammary tumours. Three latent transforming growth factor-beta binding proteins (LTBP-1, -3 and -4) are involved in TGF-beta function. The aim of the study was to analyze the expression profiles of TGF-beta 1 and 2 and LTBP-4 in human mammary carcinoma cell lines as well as in human mammary tumours. Expression analysis was performed at the transcription and protein level under in vivo and in vitro conditions. LTBP-4 expression was quantitatively analysed in human carcinomas of the mammary gland and in healthy mammary tissues of the same patients. Downregulation of LTBP-4 in all investigated human mammary tumours compared to normal tissues could be demonstrated. Results also revealed that protein levels of TGF-beta 1 are downregulated and of TGF-beta 2 are upregulated in human mammary carcinoma cell lines compared to primary (normal) human mammary epithelial cells. LTBP-4 reduction in neoplasms leads to a possible decrease of TGF-beta 1 extracellular deposition with reduced TGF-beta 1 bioavailability. TGF-beta 2 was upregulated, which indicates a possible compensatory mechanism. This study demonstrated a possible functional role of LTBP-4 for TGF-beta bioavailability with respect to carcinogenesis of human mammary tumours in vivo and in vitro.

Topics: Adenocarcinoma; Breast Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Latent TGF-beta Binding Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

The activity of T regulatory cells (Tregs) is known to be closely associated with the expression of forkhead/winged helix transcription factor, FOXP3. To determine, whether accumulation and activation of intratumoral Tregs help in the progression of breast carcinoma, we have analyzed the intratumoral expression of FOXP3 in invasive breast carcinoma and compared it with its level in ductal carcinoma in situ (DCIS) and adjacent normal tissue with the main aim of using this factor as marker of tumor progression. Intratumoral FOXP3 levels were correlated with the levels of transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF, an invasogenic and angiogenic growth factor) and intratumoral microvessel density (IMD, a prognostic marker for angiogenesis). We also analyzed whether FOXP3 gene expression correlated with other clinicopathological variables like age, tumor stage, histological grade and lymph node metastasis. Infiltrating cancers had higher FOXP3 transcription (7.43+/-3.44) than did ductal carcinoma in situ (4.27+/-1.97, p<0.05) and normal tissues (3.51+/-1.22, p<0.001). Intratumoral FOXP3 expression was significantly higher in patients with stage III disease (TNM classification) compared to patients who had stage II disease (p=0.037). In infiltrating carcinoma, a significant positive correlation between FOXP3 expression and TGF-beta1 expression was noted (p<0.001). Furthermore, a positive correlation between FOXP3 expression with VEGF expression and IMD values was also detected, however, statistically that was non-significant. A linear association of intratumoral FOXP3 expression with invasion, size and vascularity suggests a utility of FOXP3, an indicator of Treg activity as a marker of tumor progression and metastasis in breast carcinoma.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Disease Progression; Female; Forkhead Transcription Factors; Gene Expression; Humans; Mastectomy; Middle Aged; Neoplasm Staging; Pilot Projects; T-Lymphocyte Subsets; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Breast tumorigenesis and breast cancer progression involves the deregulation or hyperactivation of intracellular signaling proteins that leads to uncontrolled cellular proliferation, invasion and metastasis. For example, the expression and cellular responses to estogen receptor (ER) and transforming growth factor beta (TGFbeta) signaling pathways change during breast tumorigenesis and breast cancer progression. While the expression and activity of ER and TGFbeta maybe significant in the development of breast cancer, alterations in the cross-talk between these pathways may be equally important. Autocrine and paracrine effects of TGFbeta on breast cancer cell growth have been known for some time, but only recently have direct interactions between ER and TGFbeta been described. The purpose of this article was to further characterize the cross-talk between ER and TGFbeta, by examining ER interaction with Smad3, a downstream mediator of TGFbeta signaling. Transient transfection of Cos1 cells with p3TP-lux, demonstrate that ERalpha and ERbeta(1) repress Smad3 transcriptional activity in an estradiol-dependent manner and that this effect is inhibited by antiestrogen treatment. The ERbeta variants, ERbeta(2) and ERbeta(5), did not have any effect on Smad3 transcriptional activity. Further experiments attempted to characterize the molecular mechanism by which activated ER inhibits Smad3 transcriptional activity. Results indicate that ligand-bound ER does not affect Smad3 protein expression levels and that ER does not form direct protein interactions with Smad3. Transient transfection of Cos1 cells with the Ap-1 transcription factor c-Jun but not c-Fos was able to rescue the inhibitory effect of estrogen on Smad3 transcriptional activity. Based on these results, a model is proposed whereby c-Jun is limiting in its ability to act as a Smad3 co-activator in the presence of E(2)-bound ER, possibly due to ER sequestering c-Jun away from the Smad3 responsive promoter.

Topics: Animals; Blotting, Western; Breast Neoplasms; Chlorocebus aethiops; COS Cells; Estrogen Receptor alpha; Estrogen Receptor beta; Gene Expression Regulation; Humans; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Response Elements; Smad3 Protein; Trans-Activators; Transcription Factor AP-1; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFbeta signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFbeta pathways in human breast cancer.

Topics: Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Movement; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Nuclear Proteins; Protein Processing, Post-Translational; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The estrogen receptor (ER)-alpha (ESR1) is a key regulatory molecule in mammary epithelial cell development. Loss of ER-alpha in breast cancer is correlated with poor prognosis, increased recurrence after treatment, and an elevated incidence of metastasis. A proposed molecular pathway by which ER-alpha acts to constrain invasive growth in breast cancer cells involves direct, ER-alpha-dependent expression of metastasis-associated protein 3, a cell-type-specific component of the Mi-2/NuRD chromatin remodeling complex. MTA3 in turn represses expression of Snail, a transcription factor linked to epithelial to mesenchymal transition and cancer metastasis. To elucidate its role(s) in epithelial to mesenchymal transition (EMT), we expressed Snail in the noninvasive, ER-alpha-positive MCF-7 cell line. Snail expression led to decreased cell-cell adhesion and increased cell invasiveness. Furthermore, we observed loss of ER-alpha expression at both the RNA and protein level that was accompanied by direct interaction of Snail with regulatory DNA sequences at the ESR1 locus. A consequence of loss of ER-alpha function in this system was the increased abundance of key components of the TGF-beta signaling pathway. Thus, cross-talk among ER-alpha, Snail, and the TGF-beta pathway appears to control critical phenotypic properties of breast cancer cells.

Topics: Acetylation; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Epithelial Cells; Estrogen Receptor alpha; Histones; Humans; Introns; Mesenchymal Stem Cells; Microarray Analysis; Phenotype; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

The anticancer agent prodigiosin has been shown to act as an efficient immunosuppressant, eliciting cell cycle arrest at non-cytotoxic concentrations, and potent proapoptotic and antimetastatic effects at higher concentrations. Gene expression profiling of MCF-7 cells after treatment with a non-cytotoxic concentration of prodigiosin showed that expression of the p21WAF1/CIP1 gene, a negative cell cycle regulator was induced. In this study, we show that prodigiosin induces p21 expression leading to cell cycle blockade. Subsequently, we attempted to elucidate the molecular mechanisms involved in prodigiosin-mediated p21 gene expression. We demonstrate that prodigiosin induces p21 in a p53-independent manner as prodigiosin induced p21 in cells with both mutated and dominant negative p53. Conversely, the transforming growth factor-beta (TGF-beta) pathway has been found to be necessary for p21 induction. Prodigiosin-mediated p21 expression was blocked by SB431542, a TGF-beta receptor inhibitor. Nevertheless, this pathway alone is not enough to induce p21 expression. The TGF-beta family member (nonsteroidal anti-inflammatory drug)-activated gene 1/growth differentiation factor 15 (NAG-1) may activate this pathway, as it has previously been suggested to signal through the TGF-beta pathway and is overexpressed in response to prodigiosin treatment. We show that NAG-1 colocalizes with TGF-beta receptor type I, suggesting a possible interaction between them. Taken together, these results suggest the TGF-beta pathway is required for induction of p21 expression after prodigiosin treatment of MCF-7 cells.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dioxoles; Female; Humans; Immunohistochemistry; Prodigiosin; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Profiling; Humans; Inhibitor of Differentiation Protein 1; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Epithelial Cells; Female; Humans; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Nude; Retrospective Studies; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

To investigate the association of cytokine gene polymorphism with the development of breast cancer.. The study was carried out in Uludag University Medical School, Bursa, Turkey. The study included 38 patients with breast cancer admitted to the Medical Oncology outpatient clinic, and 24 healthy controls, age and sex matched, from the Internal Medicine Department between 2004 and 2005. All genotyping of tumor necrosis factor-alpha (TNF-alpha), tumor growth factor-beta1 (TGF-beta1), interleukin (IL)-10, IL-6, and interferon-gamma (IFN-gamma) experiments were performed using polymerase chain reaction sequence-specific primers.. The frequencies of IL-6-174GC genotype and IL-10 (-1082, -819, -592) GCC/ATA haplotype were significantly higher in the patient group (p=0.0008) when compared with controls (p=0.020). Significantly lower frequencies of IL-10 (-1082, -819, -592) ACC/ATA haplotype were observed in the patient group in comparison to the controls (p=0.026). The distribution of IFN-gamma +874, TNF-alpha 308, and TGF-beta1 codon 10-25 genotypes failed to show any statistical significant association with the development of breast cancer.. Our data suggest that IL-10 (-1082, -819, -592) GCC/ATA haplotype and IL-6-174 GC genotype seem to be potential risk factors for the development of breast cancer. The presence of IL-10ACC/ATA haplotype may be protective for the oncogenesis of breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Codon; Cytokines; Female; Genotype; Haplotypes; Humans; Interleukin-10; Interleukin-6; Middle Aged; Polymorphism, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Turkey

The high frequency and mortality associated with breast cancer metastasis to bone has motivated efforts to elucidate tumor-stroma interactions in the bone microenvironment contributing to invasion and proliferation of metastatic cells. The development of engineered tissues has prompted the integration of engineered bone scaffolds into animal models as potential targets for metastatic spread. Silk scaffolds were coupled with bone morphogenetic protein-2 (BMP-2), seeded with bone marrow stromal cells (BMSC), and maintained in culture for 7 weeks, 4 weeks, and 1 day before s.c. implant in a mouse model of human breast cancer metastasis from the orthotopic site. Following injection of SUM1315 cells into mouse mammary fat pads, tumor burden of implanted tissues was observed only in 1-day scaffolds. Scaffold development and implantation was then reinitiated to identify the elements of the engineered bone that contribute to metastatic spread. Untreated scaffolds were compared with BMP-2-coupled, BMSC-seeded, or BMP-2/BMSC-combined treatment. Migration of SUM1315 cells was detected in four of four mice bearing scaffolds with BMP-2 treatment and with BMSC treatment, respectively, whereas only one of six mice of the BMP-2/BMSC combination showed evidence of metastatic spread. Histology confirmed active matrix modeling and stromal cell/fibroblast infiltration in scaffolds positive for the presence of metastasis. These results show the first successful integration of engineered tissues in a model system of human breast cancer metastasis. This novel platform now can be used in continued investigation of the bone environment and stem cell contributions to the process of breast cancer metastasis.

Topics: Animals; Bombyx; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Mice; Mice, SCID; Stromal Cells; Tissue Engineering; Transforming Growth Factor beta

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-beta signals that block cell cycle progression in G(1) are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-beta signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467-sites on Smads that get phosphorylated by the TGF-beta receptor and positively regulate TGF-beta signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-beta. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255-sites that are phosphorylated by MAP kinase and negatively regulate TGF-beta signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-beta. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-beta signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-beta signals that promote G(1) arrest.

Topics: Breast Neoplasms; Cell Line, Tumor; G1 Phase; Humans; Pancreatic Neoplasms; Phospholipase D; Phosphorylation; Signal Transduction; Transforming Growth Factor beta

Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Chromatin; DNA Methylation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Angiogenesis is an important event during the neoplastic process and is induced by the secretion of numerous growth factors from endothelial cells. Vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (FGF2), and transforming growth factor-beta1, beta2, beta3 (TGF-beta1, 2, 3) and cognate receptors (TGF-betaRI, II, III) mRNA expression pattern was evaluated by RT-PCR in 25 breast cancer tissue samples and adjacent normal tissues, and correlated to clinicopathological features. Western blot analysis was performed to evaluate VEGF and TGF-beta1 protein levels. TGF-beta1 and TGF-beta3 mRNA levels were significantly different in breast cancer specimens of differing histology (ductal, lobular, other) (P=0.020 and P=0.043). No statistically significant difference was observed at the mRNA level of VEGF between normal and tumor tissues while elevated VEGF protein levels in tumors were associated with patients' menopausal status. A strong hormonal influence of ER and PR on TGF-beta mRNA expression was established. FGF2 transcript levels were substantially decreased in cancer compared to adjacent normal specimens (P=0.031). A disruption of mRNA co-expression patterns was observed in malignant breast tissues compared to controls. Western blot analysis revealed differences between VEGF and TGFbeta1 mRNA and their corresponding protein levels. A substantial negative correlation of TGF-beta1 protein and TGF-beta1 mRNA levels (P=0.016) was demonstrated by breast tissue-pair analysis. Summarizing, our findings suggest that transcript levels of the examined markers in breast cancer are associated with menopausal and hormonal status, while their co-expression pattern is altered in malignant tissues compared to controls. In addition the difference between VEGF and TGF-beta1 mRNA and protein levels observed, indicates that post-transcriptional mechanisms may regulate expression of these molecules in breast cancer.

Topics: Adult; Aged; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Invasiveness; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Vascular Endothelial Growth Factor A

LIM-only protein 4 (LMO4) plays critical roles in mammalian development, and has been proposed to play roles in epithelial oncogenesis, including breast cancer. As LMO4 is highly expressed in the epithelial compartments at locations of active mesenchymal-epithelial interactions, we reasoned that LMO4 might act by modulating signaling pathways involved in mesenchymal-epithelial signaling. One such candidate signal is the transforming growth factor-beta (TGFbeta) cytokine pathway, which plays important roles both in development and cancer. We show here that the transcriptional response to TGFbeta in epithelial cells is sensitive to LMO4 levels; both up- and downregulation of LMO4 can enhance TGFbeta signaling as assessed by a TGFbeta-responsive reporter gene. Furthermore, LMO4 can interact with the MH1 and linker domains of receptor-mediated Smad proteins, and associate with the endogenous TGFbeta-responsive Plasminogen Activator Inhibitor-1 gene promoter in a TGFbeta-dependent manner, suggesting that such interactions may mediate the effects of LMO4 on TGFbeta signaling. When introduced into mammary epithelial cells, LMO4 potentiated the growth-inhibitory effects of TGFbeta in those cells. These results define a new function for LMO4 as a coactivator in TGFbeta signaling, and provide a potential novel mechanism for LMO4-mediated regulation in development and oncogenesis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Epithelial Cells; Homeodomain Proteins; Humans; Immunoprecipitation; Kidney; LIM Domain Proteins; Mice; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta

Constitutive nuclear factor-kappaB (NF-kappaB) activity plays a crucial role in the development and progression of lymphoma, leukemia, and some epithelial cancers. Given the contribution of NF-kappaB in carcinogenesis, a novel approach that interferes with its activity might have therapeutic potential against cancers that respond poorly to conventional treatments. Here, we have shown that a new IkappaB kinase beta inhibitor, IMD-0354, suppressed the growth of human breast cancer cells, MDA-MB-231, HMC1-8, and MCF-7, by arresting cell cycle and inducing apoptosis. In an electrophoretic mobility shift assay and a reporter assay, IMD-0354 abolished the NF-kappaB activity in MDA-MB-231 cells in a dose-dependent manner. In the cells incubated with IMD-0354, cell cycle arrested at the G0-G1 phase and apoptotic cells were increased. The expression of some cell cycle regulatory molecules and antiapoptotic molecules was suppressed in cells treated with IMD-0354. On the other hand, cyclin-dependent kinase suppressor p27Kip1 was up-regulated by the addition of IMD-0354. Daily administration of IMD-0354 inhibited tumor expansion in immunodeficient mice into which MDA-MB-231 cells were transplanted. These results indicate that NF-kappaB may contribute to cell proliferation through up-regulation of cell cycle progression; accordingly, inhibition of NF-kappaB activity might have a therapeutic ability in the treatment of human breast cancers.

Topics: Animals; Benzamides; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Disease Progression; Female; Humans; I-kappa B Kinase; Mice; Mice, Inbred BALB C; Mice, Nude; NF-kappa B; Protein Kinase Inhibitors; RNA, Messenger; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor-beta (TGFbeta)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFbeta1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFbeta1, TGFbeta1 protein level has been measured by enzyme-immunoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFbeta1 with a range of 0-684 pg mg(-1) protein. In the overall population, an increase of tumoral TGFbeta1 was observed in premenopausal patients when compared to postmenopausal subgroup (P=0.0006). When patients were subdivided according to nodal status, TGFbeta1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P=0.040). Multivariate analysis revealed that, after lymph node status (P=0.0002) and urokinase-type plasminogen activator (P=0.004), TGFbeta1 was an independent prognostic marker for DFS (P=0.005) in the overall population. In the node-negative population, TGFbeta1 was the prominent prognostic factor (P=0.010). In the same population, Kaplan-Meier curves demonstrated that high TGFbeta1 level was correlated with a shorter disease-free survival (P=0.020). These data suggest that the measurement of tumoral TGFbeta1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

Topics: Adult; Age Factors; Aged; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Enzyme-Linked Immunosorbent Assay; Female; Humans; Middle Aged; Prognosis; Survival Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Halofuginone inhibits fibrosis by decreasing type I collagen synthesis and tumor growth through an anti-angiogenic mechanism. In vitro data suggested that halofuginone inhibits angiogenesis through upregulating thrombospondin-1 (TSP-1) expression and by inhibiting cell proliferation. To determine whether thrombospondin-1 (TSP-1) is necessary for inhibition of tumor growth and angiogenesis by halofuginone, we tested the effect of halofuginone on mammary tumor growth in polyoma middle T antigen, TSP-1 null (TSP-1-/-PyT) transgenic mice. After 30 days of treatment, we found a significant decrease in tumor weight in these mice and the extent of tumor growth inhibition was comparable to that found in TSP-1 expressing PyT mice (TSP-1+/+PyT). However, no significant difference in tumor weight was observed after 60 days of halofuginone treatment between control and treated mice in both genotypes. Interestingly, type I collagen level was lower in the halofuginone treated TSP-1+/+PyT tumors at 30 days, but this was not observed in the TSP-1-/-PyT mice. Levels of type I collagen did not correlate with blood vessel number as a decrease in the number of vessels was observed in the halofuginone treated tumors from both the TSP-1+/+PyT and TSP-1-/-PyT mice as compared to control tumors. Because halofuginone has been shown to inhibit type I collagen synthesis by inhibiting the TGF-beta signaling pathway, we measured Smad 2/3 phosphorylation levels and found that halofuginone inhibited Smad 2/3 phosphorylation in cells derived from TSP-1+/+PyT tumors. We also found that it inhibited Smad 2/3 phosphorylation in cells treated with the TGF-beta activating sequence of TSP-1, TSR2+RFK. Our data demonstrate that halofuginone inhibits mammary tumor growth in a transgenic mouse model via a TSP-1 independent pathway, by decreasing tumor angiogenesis and by inhibiting TGF-beta signaling.

Topics: Animals; Antigens, Viral, Tumor; Antineoplastic Agents; Breast Neoplasms; Collagen Type I; Mice; Mice, Transgenic; Neovascularization, Pathologic; Piperidines; Polyomavirus; Quinazolines; Quinazolinones; Smad2 Protein; Smad3 Protein; Thrombospondin 1; Transforming Growth Factor beta; Tumor Burden

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme of prostaglandin synthesis, has been implicated in invasiveness and distant metastases of cancer. Bone is one of the most common target sites of cancer metastasis. However, the role of COX-2 in bone metastasis is unclear. We examined the surgical specimens of bone metastases from patients with various types of cancers by using immunohistochemistry and observed evident COX-2 expression in these bone metastases. In a nude mouse model of bone metastasis, the MDA-MB-231 human breast cancer cells showed no COX-2 expression at orthotopic sites, whereas these cells, when metastasized to bone, intensely expressed COX-2, suggesting that the bone microenvironment induced COX-2 expression. Consistent with this notion, inhibition of bone resorption by the bisphosphonate ibandronate reduced COX-2 expression in MDA-MB-231 cells in bone. Transforming growth factor-beta (TGFbeta), one of the most abundant growth factors stored in bone, increased COX-2 expression and prostaglandin E2 production in MDA-MB-231 cells in culture. MDA-MB-231 cells overexpressing dominant-negative TGFbeta type II receptors showed decreased bone metastases and reduced osteoclastic bone resorption with impaired COX-2 expression. The COX-2 inhibitors, NS-398 and nimesulide, significantly suppressed bone metastases with decreased osteoclast number and increased apoptosis in MDA-MB-231 cells. These results suggest that bone-derived TGFbeta up-regulates COX-2 expression in breast cancer cells, thereby increasing prostaglandin E2 production, which in turn, stimulates osteoclastic bone destruction, leading to the progression of bone metastases. Our results also suggest that COX-2 is a potential therapeutic target for bone metastases in breast cancer.

Topics: Animals; Apoptosis; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Female; Heart Ventricles; Humans; Mice; Neoplasm Transplantation; Nitrobenzenes; Osteoclasts; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Sulfonamides; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation

Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.

Topics: Animals; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Disease Progression; Epithelial Cells; Female; Gene Silencing; Humans; Interleukin-11; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Nude; RNA Interference; RNA, Small Interfering; Smad4 Protein; Transforming Growth Factor beta

Using genome-wide expression profiling of a panel of 27 human mammary cell lines with different mechanisms of E-cadherin inactivation, we evaluated the relationship between E-cadherin status and gene expression levels. Expression profiles of cell lines with E-cadherin (CDH1) promoter methylation were significantly different from those with CDH1 expression or, surprisingly, those with CDH1 truncating mutations. Furthermore, we found no significant differentially expressed genes between cell lines with wild-type and mutated CDH1. The expression profile complied with the fibroblastic morphology of the cell lines with promoter methylation, suggestive of epithelial-mesenchymal transition (EMT). All other lines, also the cases with CDH1 mutations, had epithelial features. Three non-tumorigenic mammary cell lines derived from normal breast epithelium also showed CDH1 promoter methylation, a fibroblastic phenotype and expression profile. We suggest that CDH1 promoter methylation, but not mutational inactivation, is part of an entire programme, resulting in EMT and increased invasiveness in breast cancer. The molecular events that are part of this programme can be inferred from the differentially expressed genes and include genes from the TGFbeta pathway, transcription factors involved in CDH1 regulation (i.e. ZFHX1B, SNAI2, but not SNAI1, TWIST), annexins, AP1/2 transcription factors and members of the actin and intermediate filament cytoskeleton organisation.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA Methylation; DNA Mutational Analysis; Down-Regulation; Epithelial Cells; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Neoplasm Invasiveness; Polymerase Chain Reaction; Promoter Regions, Genetic; Transcription, Genetic; Transforming Growth Factor beta

In recent years, adenoviruses that selectively replicate in tumor cells have been developed. However, there is a tremendous need to improve their anticancer efficacy. We wish to investigate whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of soluble form of transforming growth factor-beta type II receptor (sTGFbetaRII) offers a therapeutic advantage. We chose to target TGF-betas because they play a pivotal role in late-stage tumorigenesis by enhancing tumor invasion and metastasis. A sTGFbetaRII cDNA was cloned in conditionally replicating adenoviral vector rAd-sTRII and in a replication-deficient adenovirus Ad-sTRII. Infection of MDA-MB-231 breast cancer cells with rAd-sTRII or Ad-sTRII followed by Western blot analysis indicated the expression of diffused glycosylated forms of sTGFbetaRII that were also secreted into the extracellular medium. The secreted proteins were shown to bind with TGF-beta and antagonize TGF-beta-induced p38 mitogen-activated protein kinase activity. However, marked differences in the replication potential of rAd-sTRII and Ad-sTRII were observed in breast tumor cells. Infection of MDA-MB-231 cells with rAd-sTRII resulted in cytotoxicity and significant increase in the adenoviral titers that were comparable with a wild-type adenovirus dl309. However, Ad-sTRII was much less toxic to the tumor cells, and the viral titers of Ad-sTRII remained relatively unchanged. These results suggest that the infection of breast tumor cells with conditionally replicating adenoviral vector rAd-sTRII produced sTGFbetaRII that can abrogate TGF-beta signaling while maintaining the replication potential of the virus, indicating that rAd-sTRII could be a potential anticancer agent.

Topics: Adenoviridae; Breast Neoplasms; Humans; Oncolytic Virotherapy; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured; Virus Replication

A comparative study of the products of the cell cycle control genes p53 (mutated form), p21, Rb (nonphosphorylated and phosphorylated form) and TGFbeta was performed by immunohistochemistry and Western blot, in benign breast disorders and breast cancer (in situ and infiltrating tumors). For the five proteins studied, the relative numbers of positively stained cells were higher in in situ carcinoma than in benign breast diseases. In infiltrating breast tumors, the relative numbers of positively stained cells were even higher than in in situ tumors except for the percentage of pRb immunostained cells, which decreased slightly in infiltrative tumors. For the other four proteins, the percentages of positively stained cases were similar to those found in in situ tumors. In the three groups of patients, TGFbeta immunoreaction appeared in the cytoplasm while immunoreactions to p53, p21, Rb, and pRb were found always in the nucleus except for p21 in in situ tumors, which showed cytoplasmic immunoreaction. Present results suggest that accumulation of mutated p53, cytoplasmic p21, and pRb in breast gland epithelium might be a crucial point in the development of in situ adenocarcinoma. In the infiltrating tumors, the expression of p21 in the nuclei and the decrease in pRb expression suggest an insufficient attempt to hinder cell proliferation.

Topics: Adult; Aged; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Immunohistochemistry; Middle Aged; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

We investigated the regulatory effect of tamoxifen (TAM) on fibronectin (FN) expression in estrogen-dependent MCF-7 breast carcinoma cells both in vitro and in vivo. in vitro, MCF-7 cells were cultured with 17beta-estradiol (E2) and/or TAM. In the animal experiment in vivo, MCF-7 tumors were grown in ovariectomized athymic mice by implanting a sustained release E2 pellet. The E2 pellets were removed after 3 weeks of E2 treatment. Animals were then divided into four groups: 1) an E2 (0.72 mg/pellet) pellet [E2(+)]; 2) an E2 and a TAM (5 mg/pellet) pellet [E2(+)TAM]; 3) no treatment [E2(-)] and 4) a TAM pellet [E2(-)TAM]. Following each treatment for 4 weeks, all animals were sacrificed and tumors were removed. Specimens, cells (in vitro) or tumors (in vivo), were homogenized and assayed for FN by Western blots. In the in vitro experiment, FN expression in MCF-7 cells decreased by incubating with 10(-9) M E2 and increased with 10(-6) M TAM. The effect of TAM increasing FN expression was inhibited by incubation accompanied with 10(-9) M E2 or 1 microg/ml transforming growth factor-beta (TGF-beta) neutralizing antibody. In the in vivo animal experiment FN expression in the tumors of E2(+) mice was lower than that of E2(-) mice. However, TAM increased FN expression in the tumors regardless of E2 pellet. These results suggest that TAM increases FN expression of MCF-7 breast carcinoma cells and that these regulatory effects of TAM on FN expression are partly mediated by TAM-induced TGF-beta.

Topics: Animals; Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Estrogens; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured

Cancer develops through a series of genetic changes leading to malignant transformation. Numerous gene and pathways involved in stages of progression to frank malignancy have been elucidated. These genetic changes result in aberrations in fundamental cellular processes controlling proliferation, apoptosis, differentiation and genomic stability. Metastasis is the hallmark of malignancy. The process of metastasis is extremely complex and involves steps including dissemination of tumor cells from the primary tumor through the vascular and lymphatic system and growth selectively in distant tissues and organs. Transforming growth factor-beta which is a growth suppressive cytokine in many normal situations becomes an active and important participant in malignant disease including angiogenesis, extracellular matrix deposition, immuno-suppression and metastasis growth promotion. Transforming growth factor-beta and its receptors are targets for antibody therapeutics and small molecule kinase inhibitors.

Topics: Breast Neoplasms; Female; Humans; Male; Prostatic Neoplasms; Transforming Growth Factor beta

The role of circulating TGF-beta(1) in prognosis of breast cancer (BC) was investigated with an intention to define TGF-beta(1)-dependent high risk and low risk subsets of patients.. Fifty three BC patients of all clinical stages and 37 healthy donors (HD) were analyzed for plasma TGF-beta(1) by the TbetaRII receptor-based Quantikine TGF-beta(1) ELISA kit.. The plasma TGF-beta(1) level of Stage I/II disease (median: 0.94 ng/ml; n=10)) remained close to HD (median: 1.30 ng/ml; n=37; p>0.1). In contrast, Stage III/IV disease (median: 2.34 ng/ml; n=43) exhibited highly significant TGF-beta(1) elevation (p<0.001) relative to HD. Further analysis revealed that TGF-beta(1) increase was predominantly attributed to Stage IV, metastatic disease patients (Q3=4.23 ng/ml) rather than to the group Stage III/IV (Q3=3.58 ng/ml). Using the plasma TGF-beta(1) concentration of 3.00 ng/ml as the cut-off value, two subgroups of patients were formed. Overall 2-year survival of the first subgroup, having elevated plasma TGF-beta(1) (>3.00 ng/ml; n=10), was 10%. This was significantly decreased (p<0.05) compared to 52% survival observed for the second subgroup of patients with plasma TGFbeta(1) values close to HD (<3.00 ng/ml, n=19).. We have performed a pilot study to determine the relationship between overall survival and TGF-beta(1) concentration in the blood of metastatic breast cancer patients. The survival was significantly reduced in the patients with elevated plasma TGF-beta(1) levels compared to that of the patients with plasma TGF-beta(1) levels close to normal. We propose that plasma TGF-beta(1) concentration may be a new tumour marker attributed to the presence of metastatic BC cells that may be used in selection of metastatic BC patients with poor prognosis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Gene silencing by CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. It has been shown previously that Betaig-h3 gene, which encodes an extracellular matrix protein involved in cell adhesion and tumorigenesis, is down-regulated or silenced in a variety of human cancer cell lines. To unravel the underlying molecular mechanism(s) for this phenomenon, DNA methylation patterns of Betaig-h3 CpG island were examined in normal, immortalized, and cancer cell lines derived from lung, prostate, mammary, and kidney. A good correlation was observed between promoter hypermethylation and lost expression of Betaig-h3 gene, which was supported by the data that demethylation of promoter by 5-aza-2'-deoxycytidine reactivated Betaig-h3 and restored its expression in Betaig-h3-silenced tumor cell lines. This result was further substantiated by a luciferase reporter assay, showing the restoration of promoter activities and increased response to transforming growth factor-beta treatment in Betaig-h3-negative 293T cells when transfected with unmethylated Betaig-h3 promoter. In contrast, activity of Betaig-h3 promoter was completely inactivated by in vitro methylation. Furthermore, CpG methylation of Betaig-h3 promoter was also shown in primary lung tumors that expressed decreased level of Betaig-h3 protein. These results suggest that promoter methylation plays a critical role in promoter silencing of the Betaig-h3 gene in human tumor cells.

Topics: Azacitidine; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Epigenesis, Genetic; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Male; Neoplasms; Promoter Regions, Genetic; Prostatic Neoplasms; Transforming Growth Factor beta

We investigated gene expression changes induced by a novel Gemini Vitamin D(3) analog, RO-438-3582 (1alpha,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 (Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D(3) analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-beta systems, differently in the two cell lines. The Gemini Vitamin D(3) analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D(3) analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Calcitriol; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Epithelial Cells; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Molecular Structure; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor beta; Up-Regulation

Metastasis is the major cause of cancer morbidity, but strategies for direct interference with invasion processes are lacking. Dedifferentiated, late-stage tumor cells secrete multiple factors that represent attractive targets for therapeutic intervention. Here we show that metastatic potential of oncogenic mammary epithelial cells requires an autocrine PDGF/PDGFR loop, which is established as a consequence of TGF-beta-induced epithelial-mesenchymal transition (EMT), a faithful in vitro correlate of metastasis. The cooperation of autocrine PDGFR signaling with oncogenic Ras hyperactivates PI3K and is required for survival during EMT. Autocrine PDGFR signaling also contributes to maintenance of EMT, possibly through activation of STAT1 and other distinct pathways. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Consequently, overexpression of a dominant-negative PDGFR or application of the established cancer drug STI571 interfered with experimental metastasis in mice. Similarly, in mouse mammary tumor virus-Neu (MMTV-Neu) transgenic mice, TGF-beta enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. Finally, expression of PDGFRalpha and -beta correlated with invasive behavior in human mammary carcinomas. Thus, autocrine PDGFR signaling plays an essential role during cancer progression, suggesting a novel application of STI571 to therapeutically interfere with metastasis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autocrine Communication; Benzamides; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Enzyme Activation; Epithelial Cells; Female; Humans; Imatinib Mesylate; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mesoderm; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Piperazines; Protein Kinase Inhibitors; Pyrimidines; ras Proteins; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Transforming Growth Factor beta

Topics: Activin Receptors, Type I; Aged; Aged, 80 and over; Alleles; Breast Neoplasms; Case-Control Studies; Female; Genotype; Humans; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Postmenopause; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the relation between expression of angiogenesis-related factors, namely vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGFbeta(1)), and microvessel count (MVC) in invasive breast cancer and analyze its clinical implications.. VEGF, TGFbeta (1) and CD34 expressions in 62 surgical specimens of invasive breast cancer and 12 normal breast specimens were examined by immunohistochemistry and HE staining. MVC was calculated according to the quantification of positive CD34 expression. Clinicopathological characteristics of the patients including age, tumor size, histological type and auxiliary lymph node metastasis were recorded and compared with the results of MVC VEGF and TGFbeta1 expression and detection.. MVC and of VEGF and expressions TGFbeta (1) in invasive breast cancer group (55.62-/+11.07, 51.61%, 56.45%, respectively) were greater than those in the normal control group (12.65-/+5.73, 16.67%, 16.67%, respectively, P<0.05). MVC and the positivity rates of VEGF and TGFbeta (1) expressions were 65.53-/+20.36, 68.75% and 78.13%, respectively, in invasive breast cancer patients with axillary lymph node metastasis, significantly higher than those without metastasis (P<0.05). MVC was correlated with VEGF and TGFbeta (1) expressions in that MVC was significantly higher in patients positive for VEGF and TGFbeta (1) (62.82-/+16.31 and 59.35-/+12.76) than in those negative for their expressions (51.16-/+12.53 and 50.80-/+15.62, P<0.05). Significant correlation was also found between VEGF and TGFbeta (1) expressions (P<0.05).. The interaction between VEGF and TGFbeta (1) mediates angiogenesis, and MVC and VEGF and TGFbeta (1) expressions are correlated to lymph node metastasis, which may provide reference for prognostic evaluation of invasive breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Invasiveness; Neovascularization, Pathologic; Prognosis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.

Topics: Activin Receptors, Type I; Animals; Bone Neoplasms; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Enzyme Inhibitors; Female; Humans; Lung Neoplasms; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor (TGF)-beta suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-beta, thus enabling TGF-beta to promote the acquisition and development of metastatic phenotypes. The molecular mechanisms underlying this conversion of TGF-beta function remain poorly understood but may involve signaling inputs from integrins.. beta3 Integrin expression or function in MECs was manipulated by retroviral transduction of active or inactive beta3 integrins, or by transient transfection of small interfering RNA (siRNA) against beta3 integrin. Altered proliferation, invasion, and epithelial-mesenchymal transition (EMT) stimulated by TGF-beta in control and beta3 integrin manipulated MECs was determined. Src involvement in beta3 integrin mediated alterations in TGF-beta signaling was assessed by performing Src protein kinase assays, and by interdicting Src function pharmacologically and genetically.. TGF-beta stimulation induced alphavbeta3 integrin expression in a manner that coincided with EMT in MECs. Introduction of siRNA against beta3 integrin blocked its induction by TGF-beta and prevented TGF-beta stimulation of EMT in MECs. beta3 integrin interacted physically with the TGF-beta receptor (TbetaR) type II, thereby enhancing TGF-beta stimulation of mitogen-activated protein kinases (MAPKs), and of Smad2/3-mediated gene transcription in MECs. Formation of beta3 integrin:TbetaR-II complexes blocked TGF-beta mediated growth arrest and increased TGF-beta mediated invasion and EMT. Dual beta3 integrin:TbetaR-II activation induced tyrosine phosphorylation of TbetaR-II, a phosphotransferase reaction mediated by Src in vitro. Inhibiting Src activity in MECs prevented the ability of beta3 integrin to induce TbetaR-II tyrosine phosphorylation, MAPK activation, and EMT stimulated by TGF-beta. Lastly, wild-type and D119A beta3 integrin expression enhanced and abolished, respectively, TGF-beta stimulation of invasion in human breast cancer cells.. We show that beta3 integrin alters TGF-beta signaling in MECs via Src-mediated TbetaR-II tyrosine phosphorylation, which significantly enhanced the ability of TGF-beta to induce EMT and invasion. Our findings suggest that beta3 integrin interdiction strategies may represent an innovative approach to re-establishing TGF-beta mediated tumor suppression in progressing human breast cancers.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cells; Female; Gene Transfer Techniques; Humans; Integrin beta3; Mammary Glands, Animal; Mesenchymal Stem Cells; Mice; Neoplasm Invasiveness; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, plays an important role in breast cancer. There is increasing evidence that enhanced expression of TGF-beta promotes breast cancer progression contributing to metastasis and invasiveness of the tumor. We identified a functional polymorphism in the TGFB2 promoter, a 4-bp insertion at position -246 relative to the transcriptional start site (-246ins). Transient transfection experiments showed that the -246ins polymorphism significantly increased TGFB2 promoter activity in breast cancer cells. Electrophoretic mobility shift assays revealed binding of the transcription factor Sp1 to the -246ins allele. Overexpression of Sp1 enhanced promoter activity of the -246ins allele, demonstrating that Sp1 mediates transcriptional activation. Furthermore, the -246ins allele was associated with enhanced TGF-beta(2) expression in breast cancer tissue (P = 0.0005). To evaluate the role of the polymorphism in breast cancer, frequency of the -246ins allele was determined in breast cancer patients (n = 78) and healthy female controls (n = 143). No significant differences were found. However, the presence of the -246ins allele was associated with lymph node metastasis (P = 0.003). The -246ins allele was a significant predictor for lymph node metastasis independent of estrogen and progesterone receptor status in a multivariate logistic regression analysis (P = 0.0118, odds ratio, 5.18; 95% confidence interval, 1.44-18.62). We provide evidence that the TGFB2 -246ins polymorphism leads to enhanced TGF-beta(2) expression levels in vivo and might thereby contribute to tumor progression and development of metastases.

Topics: Alleles; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Genetic Predisposition to Disease; Humans; Polymorphism, Genetic; Promoter Regions, Genetic; Protein Binding; Sp1 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta2

Breast cancers may evade the growth-inhibitory action of TGFbeta by accumulating defects of unknown nature that selectively eliminate cytostatic gene responses. We found the transcription factor C/EBPbeta to be essential for TGFbeta induction of the cell cycle inhibitor p15INK4b by a FoxO-Smad complex and repression of c-MYC by an E2F4/5-Smad complex in human epithelial cells. These cytostatic responses are selectively missing in metastatic breast cancer cells from half of the patients that we tested. The basis for this loss was traced to an excess of the C/EBPbeta inhibitory isoform LIP. We suggest that C/EBPbeta plays a key role in the coordination of TGFbeta cytostatic gene responses, and its malfunction may trigger evasion of these responses in breast cancer.

Topics: Animals; Base Sequence; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p15; Humans; Mice; Molecular Sequence Data; Neoplasm Metastasis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.

Topics: Autocrine Communication; Blotting, Northern; Breast Neoplasms; Cell Membrane; Cell Proliferation; Collagen; Drug Combinations; Female; Genes, Dominant; Humans; Immunoblotting; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Paracrine Communication; Plasminogen Activator Inhibitor 1; Proteoglycans; Receptors, Transforming Growth Factor beta; RNA Stability; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Chemically Modified Tetracyclines (CMTs) are antiproteolytic agents that have been shown to inhibit tumor invasiveness and metastasis. CMT 300 has shown promise in phase I clinical trials in patients with Kaposi's Sarcoma, which is characterized by over-production of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF). In this study, we report a novel activity of CMT 308, a 9-amino derivative of CMT 300, on reducing levels of VEGF secreted by breast cancer cell lines. CMT 308, at sub-cytotoxic concentrations, reduced basal levels of secreted VEGF in the poorly invasive MCF-7 cell line as well as the more aggressively invasive MDA-MB-435s cell line in a dose-dependent manner. In addition, CMT 308 also reduced transforming growth factor beta (TGFbeta)-induced VEGF secretion in both cell lines. While VEGF could be detected in the conditioned media of untreated MCF-7 cells within 4h, levels of secreted VEGF in CMT 308-treated cells remained undetectable up to 8h. CMT 308 diminished secretion of VEGF from MCF-7 cells up to 8h regardless of previous time in culture. CMT 308 did not reduce the levels of basal VEGF mRNA in either cell line, but did reduce pools of total intracellular VEGF protein. Although TGFbeta stimulated an increase in VEGF levels in the conditioned media as well as in the cytoplasm, TGFbeta treatment did not increase VEGF mRNA levels. Thus, augmented expression of VEGF protein by breast cancer cell lines in the presence of TGFbeta appears to involve upregulation at a step beyond transcription. Moreover, the data strongly indicate that in these breast cancer cell lines, CMT 308 reduces VEGF secretion by targeting some post-transcriptional event. The capacity of CMT 308 to diminish levels of a major pro-angiogenic signal makes the nonantimicrobial tetracycline derivative an attractive candidate for anti-angiogenic therapy in management of breast cancer.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Dose-Response Relationship, Drug; Female; Humans; Neoplasm Invasiveness; Neovascularization, Pathologic; RNA, Messenger; RNA, Neoplasm; Tetracyclines; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Glycosylation; Humans; Lectins; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transforming Growth Factor beta

The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.

Topics: Aging; Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Dogs; Epithelial Cells; Gene Expression Regulation, Neoplastic; Goosecoid Protein; Humans; Mice; Neoplasm Metastasis; Organizers, Embryonic; Signal Transduction; Transforming Growth Factor beta

Prolonged propagation of primary diploid fibroblasts in culture activates an ageing process known as replicative senescence, which is considered to provide a barrier against oncogenic transformation. Remarkably, both cell autonomous tumor-suppressive and cell nonautonomous tumor-promoting effects of senescent cells have been reported. Recently, we described that the p53 target gene plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of replicative senescence. PAI-1 antagonizes the protease urokinase-type plasminogen activator (uPA). Both are secreted factors and involved in heterotypic signaling processes such as wound healing, angiogenesis and metastasis. Both uPA and PAI-1 are expressed in senescent cells and their relative abundance controls proliferation downstream of p53. Here, we present data that the effects of PAI-1 and uPA in the senescence response are not strictly cell autonomous. We discuss these findings in the context of the emerging roles of PAI-1 and uPA in heterotypic cellular signaling in senescence, wound healing and metastasis.

Topics: Animals; Breast Neoplasms; Cellular Senescence; Fibroblasts; Homeostasis; Mice; Models, Biological; Paracrine Communication; Plasminogen Activator Inhibitor 1; Stromal Cells; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Wound Healing

We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Shape; Coculture Techniques; Epidermal Growth Factor; Estradiol; Female; Fibroblasts; Genetic Markers; Hormone Antagonists; Humans; Keratins; Mammary Glands, Animal; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Mifepristone; Mutation; Neoplasm Transplantation; Receptors, Estrogen; Receptors, Progesterone; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Down-Regulation; Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Oncogene Protein p55(v-myc); p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Retinoblastoma Protein; rho-Associated Kinases; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha Chains; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Pituitary Neoplasms; RNA, Neoplasm; Tenascin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Although both the antiapoptotic function of survivin and vitamin D3 (VD3)-mediated cell growth inhibition and apoptosis have been extensively studied, it is not known whether survivin plays a role in VD3 compound-mediated cell growth inhibition and apoptosis induction. Using an isogenic model of MCF-7 breast adenocarcinoma cells (MCF-7E and MCF-7L sublines that are sensitive and resistant to VD3 compounds), we found that VD3 compounds effectively downregulated survivin in VD3-sensitive MCF-7E cells, which was associated with VD3-induced apoptosis. In contrast, VD3 compounds failed to downregulate survivin in VD3-resistant MCF-7L cells, which showed resistant to VD3-induced apoptosis. However, inhibition of survivin expression by small interfering RNA (siRNA) induced cell death per se and further sensitized VD3-induced apoptosis in MCF-7L cells, indicating that the inability of these cells to respond to VD3 is due to the failure to downregulate survivin. Forced expression of survivin not only blocked VD3-mediated G1 cell accumulation but also increased S and G2/M cell populations. VD3 treatment rapidly triggered the activation of p38 MAPK signaling in MCF-7E cells but not in MCF-7L cells. Moreover, inhibition of p38 activation diminished VD3-mediated survivin inhibition and partially rescued VD3-induced cell death. We further showed that VD3 increased the expression of TGF(beta)1 and TGF(beta) receptor 2, and that blocking the function of TGF(beta) receptor 2 diminished VD3 compound-mediated survivin downregulation. Thus, we propose that the VD3 compound-induced growth inhibition and apoptosis induction are at least partially dependent on survivin downregulation via VD3-induced TGFbeta signaling and the activation of p38 MAPK pathway. Targeting survivin through these pathways may lead to novel applications for cancer therapeutics.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cholecalciferol; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Small Interfering; Survivin; Transforming Growth Factor beta

Hepatocyte growth factor (HGF) is a multifunctional cytokine that is involved in recovery process after organ injuries.. We studied HGF and the membrane bound receptor, c-met locally in patients who suffered from chronic leg ulcers (> or =1 year) caused by venous insufficiency.. Skin biopsies from the edge of the ulcers were taken from patients (n=13) and studied by immunohistochemical staining for detection of HGF and c-met. Skin biopsies from healthy volunteers (n=10) were used as the control material. Ulcer secretion from chronic ulcers (n=11) was examined for the presence of HGF by ELISA and the concentration of HGF was compared with acute ulcers in healthy controls (n=10) and in patients operated for a non-invasive breast cancer (n=12).. We observed that c-met expression in the ulcer area increased significantly in chronic ulcers compared to controls (p=0.005). Concentration of ulcer-HGF in the patients with chronic ulcer was significantly higher than acute ulcers (p<0.01). The biological activity of HGF in ulcer secret was assessed in-vitro in transferred, mouse skin epithelial cell monolayer. Enhanced migration and morphologic changes were seen after adding ulcer secret from acute ulcers (> 1 ng/mL) that was inhibited by anti-HGF antibodies. No biological activity was observed by adding ulcer secret from chronic ulcers irrespective HGF concentration.. We conclude that in chronic skin ulcers decreased biological activity of endogenous HGF and overexpression of c-met is seen which might explain fibrosis and delayed recovery. Administration of exogenous active HGF might contribute to accelerated healing in these patients.

Topics: Adult; Animals; Biopsy; Blotting, Western; Breast Neoplasms; Case-Control Studies; Cell Line; Cell Movement; Cytokines; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Leg Ulcer; Male; Mice; Proto-Oncogene Proteins c-met; Skin; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ulcer; Venous Insufficiency; Wound Healing

Metastases in breast cancer are a vital concern in treatment, with epidermal growth factor receptor and ErbB2 strongly implicated in mediating tumor invasion and spreading. In this study, we investigated the role of decorin in suppressing both primary breast carcinomas and pulmonary metastases. We show that decorin causes marked growth suppression both in vitro and in vivo using a metastatic breast cancer cell line and an orthotopic mammary carcinoma model. Treatment with decorin protein core reduced primary tumor growth by 70% and eliminated observed metastases. An adenoviral vector containing the decorin transgene caused primary tumor retardation of 70%, in addition to greatly reducing observed metastases. Moreover, we demonstrate that ErbB2 phosphorylation and total receptor protein levels are reduced in this model system upon de novo expression of decorin under the control of a doxycycline-inducible promoter. Primary tumor growth in vivo was reduced by up to 67% upon decorin induction, and pulmonary metastases were markedly hampered as well. These effects are likely occurring through decorin's long-term downregulation of the ErbB2 tyrosine kinase cascade. These results demonstrate a novel role for decorin in reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapeutics for metastatic breast cancer.

Topics: Adenoviridae; Animals; Breast Neoplasms; Decorin; Disease Models, Animal; Extracellular Matrix Proteins; Female; Genetic Vectors; Humans; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Phosphorylation; Proteoglycans; Rats; Receptor, ErbB-2; Transforming Growth Factor beta; Tumor Cells, Cultured

The proliferation of malignant breast epithelial cells is regulated by various stimuli including cytokines and growth factors, thus the variants of those genes may modify the breast cancer risk. To evaluate the potential influences of TGF-beta1 T29C and TNF-beta A252G gene polymorphisms on breast cancer risk, a case-control study was conducted in Korea.. Histologically confirmed breast cancer cases (n=560) and controls (n=509) with no previous history of cancer were recruited from three teaching hospitals in Seoul, Korea. Genotypes were determined by PCR-CTPP (polymerase chain reaction with confronting two-pair primers) method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression model adjusting for age, body mass index, education, parity, age at first full-term pregnancy, and family history of breast cancer.. The TGF-beta1 29C-allele containing genotypes posed an increased risk of breast cancer (OR=1.3, 95% CI=1.02-1.79), especially in postmenopausal women (OR=1.6, 95% CI=1.01-2.44). Similarly, the TNF-beta 252G-allele containing genotypes posed an increased risk of postmenopausal breast cancer (OR=1.7, 95% CI=1.09-2.55). The risk of postmenopausal breast cancer increased in parallel with the number of the risk genotypes (p for trend <0.01). When data were stratified by the presumed non-genetic risk factors, TGF-beta1 C-allele containing genotypes were found to increase breast cancer risk almost two-fold in postmenopausal women with greater than median body mass index (>22.8 kg/m2) (OR=1.9, 95% CI=1.04-3.37).. The results of this study therefore suggest that polymorphisms of TGF-beta1 and TNF-beta genes may modify individual susceptibility to breast cancer in Korean women.

Topics: Breast Neoplasms; Case-Control Studies; Female; Genetic Predisposition to Disease; Humans; Korea; Likelihood Functions; Linear Models; Lymphotoxin-alpha; Middle Aged; Polymorphism, Genetic; Postmenopause; Premenopause; Risk; Transforming Growth Factor beta

There is growing evidence that common variants of the transforming growth factor-beta (TGF-beta) signaling pathway may modify breast cancer risk. In vitro studies have shown that some variants increase TGF-beta signaling, whereas others have an opposite effect. We tested the hypothesis that a combined genetic assessment of two well-characterized variants may predict breast cancer risk. Consecutive patients (n = 660) with breast cancer from the Memorial Sloan-Kettering Cancer Center (New York, NY) and healthy females (n = 880) from New York City were genotyped for the hypomorphic TGFBR1*6A allele and for the TGFB1 T29C variant that results in increased TGF-beta circulating levels. Cases and controls were of similar ethnicity and geographic location. Thirty percent of cases were identified as high or low TGF-beta signalers based on TGFB1 and TGFBR1 genotypes. There was a significantly higher proportion of high signalers (TGFBR1/TGFBR1 and TGFB1*CC) among controls (21.6%) than cases (15.7%; P = 0.003). The odds ratio [OR; 95% confidence interval (95% CI)] for individuals with the lowest expected TGF-beta signaling level (TGFB1*TT or TGFB1*TC and TGFBR1*6A) was 1.69 (1.08-2.66) when compared with individuals with the highest expected TGF-signaling levels. Breast cancer risk incurred by low signalers was most pronounced among women after age 50 years (OR, 2.05; 95% CI, 1.01-4.16). TGFBR1*6A was associated with a significantly increased risk for breast cancer (OR, 1.46; 95% CI, 1.04-2.06), but the TGFB1*CC genotype was not associated with any appreciable risk (OR, 0.89; 95% CI, 0.63-1.21). TGFBR1*6A effect was most pronounced among women diagnosed after age 50 years (OR, 2.20; 95% CI, 1.25-3.87). This is the first study assessing the TGF-beta signaling pathway through two common and functionally relevant TGFBR1 and TGFB1 variants. This approach may predict breast cancer risk in a large subset of the population.

Topics: Activin Receptors, Type I; Adult; Aged; Alleles; Breast Neoplasms; Case-Control Studies; Female; Genetic Predisposition to Disease; Genotype; Humans; Middle Aged; Neoplasm Staging; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Estrogen; Receptors, Progesterone; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

We used 2D-cocultures employing fibroblasts of different genetic backgrounds and MCF10A-derived human breast epithelial cells of increasingly malignant potential to investigate tumor-stroma interactions in breast cancer and to identify possible signaling pathways involved. Tumor cells induced expression of matrix-metalloproteinase 9 (MMP-9) in fibroblasts in a pattern dependent on the degree of their malignancy. In-situ zymography localized the main gelatinolytic activity around stromal cells in cocultures and xenografted tumors. Use of Smad3 knockout fibroblasts, small molecule inhibitors, and neutralizing antibodies showed that MMP-9 expression was induced by tumor cell-derived TNF-alpha and TGF-beta, dependent on Smad-, Ras-, and PI3-kinase-signaling, and likewise modulated by subsequent HGF- and EGF-signaling. Together, our results indicate that MMP-9 levels in tumor fibroblasts are regulated by a complex tumor-stroma cross-talk, involving multiple ligands and cellular signaling pathways.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Coculture Techniques; Epidermal Growth Factor; Epithelial Cells; Fibroblasts; Humans; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Protein Kinases; Signal Transduction; Smad3 Protein; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

BMP-2 is involved in the fetal and postnatal development of the mammary gland but has also been detected in breast cancer cells. To clarify the biological role of BMP-2 in breast cancer, we used the human breast cancer cell line MCF-7. Incubation with BMP-2 under serum-free conditions induced activation of the mitogen activated protein kinases (MAPKs) ERK1/2 and the basic helix-loop-helix transcription factors Id-1, proteins that can protect from apoptosis. Stably transfected MCF-7 cells overexpressing BMP-2 revealed significantly increased resistance to hypoxia-induced apoptosis compared to empty vector controls. Cytoplasmic BMP-2/4 protein expression was detected in carcinoma cells of 81 samples of invasive breast cancer in contrast to adjacent normal mammary epithelial cells. BMP-2/4 expression did not correlate with common prognostic parameters and was not associated with relapse-free or overall survival. We conclude that BMP-2/4 expression is reactivated in invasive breast cancer and part of an autocrine/paracrine mechanism rescuing malignant cells from hypoxic cell death via activation of the MAPK and Id-1 pathway.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Death; Cell Hypoxia; Cell Line, Tumor; Female; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Prognosis; Transforming Growth Factor beta

Recent studies suggest that normal tissue radiosensitivity is influenced by single nucleotide polymorphisms (SNPs) in certain genes. In order to seek a confirmation of these findings, this study investigated SNPs in genes TGFB1 (position -509, codon 10 and codon 25), SOD2 (codon 16), XRCC1 (codon 399), XRCC3 (codon 241), APEX (codon 148) and ATM (codon 1853) in 26 breast cancer patients with marked changes in breast appearance after radiotherapy and 26 matched controls. Statistically significant associations were found between the TGFB1 codon 10 Pro allele (P=0.005) as well as the TGFB1 position -509 T allele (P=0.018) and increased risk of altered breast appearance. No significant associations were found for the remaining SNPs.

Topics: Breast Neoplasms; Codon; Female; Humans; Neoplasm Staging; Polymorphism, Single Nucleotide; Predictive Value of Tests; Prognosis; Radiation Injuries; Radiation Tolerance; Risk Factors; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics-based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-beta1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Chickens; Cyclin-Dependent Kinase Inhibitor p21; Drug Screening Assays, Antitumor; Endothelium, Vascular; Enzyme Activation; Female; Gene Expression Profiling; Humans; Immunoglobulins; Mammary Glands, Human; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Oxadiazoles; Receptor, IGF Type 2; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Loss of actin stress fibers has been associated with cell transformation and metastasis. TGF-beta induction of stress fibers in epithelial cells requires high molecular weight tropomyosins encoded by TPM1 and TPM2 genes. Here, we investigated the mechanism underlying the failure of TGF-beta to induce stress fibers and inhibit cell migration in metastatic cells. RT-PCR analysis in carcinoma cell lines revealed a significant reduction in TPM1 transcripts in metastatic MDA-MB-231, MDA-MB-435 and SW620 cell lines. Treatment of these cells with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) increased mRNA levels of TPM1 with no effect on TPM2. Importantly, 5-aza-dC treatment of MDA-MB-231 cells restored TGF-beta induction of TPM1 and formation of stress fibers. Forced expression of TPM1 by using Tet-Off system increased stress fibers in MDA-MB-231 cells and reduced cell migration. A potential CpG island spanning the TPM1 proximal promoter, exon 1, and the beginning of intron 1 was identified. Bisulfite sequencing showed significant cytosine methylation in metastatic cell lines that correlated with a reduced expression of TPM1. Together these results suggest that epigenetic suppression of TPM1 may alter TGF-beta tumor suppressor function and contribute to metastatic properties of tumor cells.

Topics: Azacitidine; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA Methylation; Exons; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Introns; Molecular Sequence Data; Neoplasm Metastasis; Promoter Regions, Genetic; RNA, Messenger; Transforming Growth Factor beta; Tropomyosin

Transforming growth factor beta (TGF-beta) inhibits proliferation and promotes cell migration. In TGF-beta-treated MCF10A mammary epithelial cells overexpressing HER2 and by chromatin immunoprecipitation, we identified novel Smad targets including protein tyrosine phosphatase receptor type kappa (PTPRK). TGF-beta up-regulated PTPRK mRNA and RPTPkappa (receptor type protein tyrosine phosphatase kappa, the protein product encoded by the PTPRK gene) protein in tumor and nontumor mammary cells; HER2 overexpression down-regulated its expression. RNA interference (RNAi) of PTPRK accelerated cell cycle progression, enhanced response to epidermal growth factor (EGF), and abrogated TGF-beta-mediated antimitogenesis. Endogenous RPTPkappa associated with EGF receptor and HER2, resulting in suppression of basal and ErbB ligand-induced proliferation and receptor phosphorylation. In MCF10A/HER2 cells, TGF-beta enhanced cell motility, FAK phosphorylation, F-actin assembly, and focal adhesion formation and inhibited RhoA activity. These responses were abolished when RPTPkappa was eliminated by RNA interference (RNAi). In cells expressing RPTPkappa RNAi, phosphorylation of Src at Tyr527 was increased and (activating) phosphorylation of Src at Tyr416 was reduced. These data suggest that (i) RPTPkappa positively regulates Src; (ii) HER2 signaling and TGF-beta-induced RPTPkappa converge at Src, providing an adequate input for activation of FAK and increased cell motility and adhesion; and (iii) RPTPkappa is required for both the antiproliferative and the promigratory effects of TGF-beta.

Topics: Actins; Breast Neoplasms; Cell Adhesion; Cell Movement; Cells, Cultured; DNA-Binding Proteins; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Mammary Glands, Human; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Receptor, ErbB-2; RNA Interference; Signal Transduction; Smad Proteins; src-Family Kinases; Trans-Activators; Transforming Growth Factor beta; Tumor Suppressor Proteins; Up-Regulation

Breast cancer is a very heterogeneous disease considering a number of biomarkers which are under investigation. However, most important biomarkers in a clinical setting are ER (estrogen receptors) and HER-2 (human epidermal growth factor receptor 2), but still only as predictive markers for tamoxifen and trastuzumab therapy. Their prognostic role is still subject of investigation. On the other hand, although, TGF-beta1 is known as marker of invasiveness and metastatic capacity of breast cancer cells, this marker has never been considered to be introduced in routine clinical setting. TGF-beta1 is ER regulated biomarker which act synergistically with HER-2. Aim of this study is to show that clinical significance of determination ER and HER-2 status could be improved, when they are related with TGF-beta1 as additional biomarker. It seems that TGF-beta1 could make a difference regarding prognosis and prediction in breast cancer patients. This hypothesis could be easily verified in corresponding clinical research.

Topics: Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Predictive Value of Tests; Receptor, ErbB-2; Receptors, Estrogen; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta (TGF-beta) inhibits the proliferation of carcinomas in early stages of breast cancer, whereas it promotes tumor growth and metastasis in later stages of cancer. We evaluated a possible association between TGF-beta1 gene polymorphisms and breast cancer risk in a population-based case-control study of Chinese women living in Shanghai, which included 1,127 breast cancer cases and 1,228 population controls. Two polymorphisms, C-509T and T+29C, were in strong linkage disequilibrium. There were no overall differences in the genotype distribution of T+29C polymorphisms of the TGF-beta1 gene among cases and controls. However, the distribution of the high-activity C allele of T+29C polymorphisms differed by cancer stages (P(trend) = 0.02). This allele was associated with decreased risk of early-stage breast cancer [stages 0 and I; odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.54-0.99], and the OR was further reduced to 0.66 (95% CI, 0.45-0.96) for those homozygous for this allele (P(trend) = 0.03). On the other hand, the same allele was associated with nonsignificantly increased risk of breast cancer with advanced stages III and IV (OR, 1.33; 95% CI, 0.81-2.18), which differed significantly from that observed for early-stage cancer (P = 0.04). This result suggests a possible dual effect of TGF-beta1 shown by in vitro experiments and provides an explanation for some of the inconsistent findings from previous epidemiologic studies that did not evaluate this association by cancer stage.

Topics: Adult; Breast Neoplasms; Case-Control Studies; China; Female; Humans; Linkage Disequilibrium; Middle Aged; Neoplasm Staging; Odds Ratio; Polymorphism, Genetic; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Markers of angiogenesis, cell proliferation, and cytokine regulation are associated with the development and course of autoimmune and malignant diseases. We investigated associations between cytokine production genotypes in breast cancer patients compared with controls and explored associations with known prognostic indices and relapse status. Eighty-eight females with breast carcinoma (BC) were studied in this case-control study comparing the cytokine genotypes of TNF-alpha TGF-beta1, IL-10, IL-6, and IFN-gamma with controls. Cytokine polymorphisms were identified by sequence-specific primers for codons, introns, or promoters regulating cytokine production. Patient characteristics, such as estrogen and progesterone receptor status, DNA ploidy, Her-2 neu expression, lymph node involvement, tumor size, and relapse status were evaluated. Cytokine genotypes were not associated with breast cancer compared with controls. Correlations between TGF-beta1 high-production genotypes and greater than four positive lymph nodes (OR=2.3; p=ns) and TNF-alpha high-production genotype and the mean level of estrogen receptor expression (66 +/- 24 vs. 34 +/- 36, p=0.016) were identified. The median patient follow-up interval from diagnosis to evaluation was 50.1 months (range 13-387 months). Relapse status was known for 84 of the patients. The odds of relapse in TGF-beta1 codon 10 CC genotypes was 2.81 times that in TGF-beta1 high-production genotypes (OR=2.81; 95% CI for OR: 1.0, 7.8; p=0.04). Mean progesterone receptor expression was decreased in relapsed patients (40.9 +/- 29.9% vs. 23.1 +/- 24.5, p=0.05). The other cytokine genotypes studied (IL-10, IL-6, IFN-gamma, and TNF-alpha production were not associated with breast cancer overall or relapse status. In this study, TGF-beta1 low-production genotypes (TGF-beta1 10 CC) were associated with an increased odds of disease relapse. This finding should be confirmed in a longitudinal study to further investigate the regulatory function of cytokine production as a prognostic indicator of relapse.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal; Carcinoma, Lobular; Case-Control Studies; Cytokines; Female; Genotype; Humans; Interferon-gamma; Interleukin-10; Interleukin-6; Lymph Nodes; Middle Aged; Neoplasm Recurrence, Local; Ploidies; Polymorphism, Genetic; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. Here we report that CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, we identify CUTL1 as a transcriptional target of transforming growth factor beta and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease-Free Survival; DNA-Binding Proteins; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasms; NIH 3T3 Cells; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Repressor Proteins; RNA, Double-Stranded; Signal Transduction; Smad4 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Up-Regulation

The effect of X-irradiation on the expression of mRNAs coding for transforming growth factor beta (TGF-beta) and its receptors was compared in human breast carcinoma MCF-7 cells differing in their sensitivity to doxorubicin.. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the estimation of mRNAs expression for TGF-beta1, TGF-beta2 and TGF-beta type I and II receptors in the unirradiated and irradiated cells of two sublines of MCF-7 cells--sensitive (MCF-7(wt)) and resistant (MCF-7(DOX/R)) to doxorubicin.. X-irradiation caused an increase in TGF-beta1 coding for mRNA expression in both breast cancer cell lines tested and an elevation of TGF-beta2 mRNA expression in doxorubicin-resistant subline. The down-regulation of TGF-beta type I and II receptors in the irradiated MCF-7(DOX/R) cells was revealed at the transcriptional level.. The results of our study show that the development of refractoriness of used tumor cells to anti-cancer drug doxorubicin, at least partly, can be caused by the impairment in TGF-beta signaling pathway.

Topics: Activin Receptors, Type I; Antibiotics, Antineoplastic; Breast Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Female; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; X-Rays

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

Topics: Amino Acid Motifs; Animals; Blotting, Western; Breast Neoplasms; DNA-Binding Proteins; Female; Humans; Mice; Polymerase Chain Reaction; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptor, Notch4; Receptors, Cell Surface; Receptors, Notch; Signal Transduction; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Breast cancer cell lines migrated towards a BMP-2 source depending on BMP-2 concentration. After a short exposure to BMP-2, the cells were able to migrate through matrigel. MCF-7 cells transfected with the BMP-2 gene also showed enhanced migratory properties and high expression of the metastasis-related gene BCSG1. In a xenograft model without estrogen supplementation MCF-7/BMP-2 cells formed tumors. These tumors were characterised by an enhanced vasculature and the formation of chondroid and osseous structures. In conclusion elevated levels of BMP-2 enhance the tumorigenic properties of breast carcinoma cells and drive the cells towards a more aggressive phenotype with estrogen independent growth.

Topics: Animals; Antineoplastic Agents, Hormonal; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chemotactic Factors; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Invasiveness; NIH 3T3 Cells; RNA, Messenger; Tamoxifen; Time Factors; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family and play an important role in the regulation of embryonic vasculogenesis but their role in postnatal angiogenesis remains to be clarified. In this study we investigated a possible role of BMP-2 in the promotion of tumor angiogenesis.. We studied the effect of BMP-2 on human dermal microvascular endothelial cells (HDMECs) and examined a possible angiogenic activity of BMP-2 with the mouse sponge assay. The effect of BMP-2 overexpression on tumor vascularization was also analyzed in xenografts of human BMP-2 transfected MCF-7 breast cancer cells (MCF-7/BMP2) in mice.. BMP receptor activation selectively induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in contrast to the ERK1/2 MAP kinases. In keeping with this finding, BMP-2 had no significant effect on endothelial cell proliferation but promoted HDMEC tube formation in the matrigel assay. The transcription factor inhibitor of differentiation 1 (Id1), which is known to play an important role in neovascularization of tumors, was confirmed as a BMP target in HDMECs. Immunohistochemical analysis of sponge sections revealed that BMP-2 induced vascularization and showed an additive enhancement of angiogenesis with VEGF. In the murine breast cancer xenograft model, human MCF-7 cells with stable overexpression of BMP-2 developed vascularized tumors while empty vector control MCF-7 cells failed to form tumors.. We conclude that activation of the BMP pathway by BMP-2 can promote vascularization and might be involved in tumor angiogenesis possibly by stimulating the Id1 and p38 MAPK pathway.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Mice; Neovascularization, Pathologic; p38 Mitogen-Activated Protein Kinases; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation

Transforming growth factor-beta (TGF-beta) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-beta-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-beta. Recent findings suggest that therapeutics designed to antagonize TGF-beta signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-beta type II receptor antagonist that inhibits TGF-beta binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-beta, particularly its stimulation of mammary epithelial-mesenchymal transition (EMT), can be prevented by CystC.. Retroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Delta14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-beta stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-beta stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-beta.. CystC and Delta14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-beta by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-beta-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Delta14CystC also inhibited TGF-beta signaling in two tumorigenic human breast cancer cell lines.. Our findings show that TGF-beta stimulation of initiating metastatic events, including decreased cell polarization, reduced cell-cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-beta.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Cystatin C; Cystatins; Epithelial Cells; Female; Humans; Kidney; Mesoderm; Protease Inhibitors; Rats; Transforming Growth Factor beta

TGF-beta can signal by means of Smad transcription factors, which are quintessential tumor suppressors that inhibit cell proliferation, and by means of Smad-independent mechanisms, which have been implicated in tumor progression. Although Smad mutations disable this tumor-suppressive pathway in certain cancers, breast cancer cells frequently evade the cytostatic action of TGF-beta while retaining Smad function. Through immunohistochemical analysis of human breast cancer bone metastases and functional imaging of the Smad pathway in a mouse xenograft model, we provide evidence for active Smad signaling in human and mouse bone-metastatic lesions. Genetic depletion experiments further demonstrate that Smad4 contributes to the formation of osteolytic bone metastases and is essential for the induction of IL-11, a gene implicated in bone metastasis in this mouse model system. Activator protein-1 is a key participant in Smad-dependent transcriptional activation of IL-11 and its overexpression in bone-metastatic cells. Our findings provide functional evidence for a switch of the Smad pathway, from tumor-suppressor to prometastatic, in the development of breast cancer bone metastasis.

Topics: Animals; Base Sequence; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-11; Mice; Molecular Sequence Data; Neoplasm Transplantation; Promoter Regions, Genetic; Signal Transduction; Transcriptional Activation; Transforming Growth Factor beta; Tumor Suppressor Proteins

To assess if feature, extent and duration of surgery could influence levels of systemic proangiogenic cytokines vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-beta).. We collected blood samples from 82 consecutive breast cancer patients who underwent various types of surgery, classified according to the magnitude of tissue injury in: minimal (quadrantectomy), moderate (mastectomy without reconstruction), and heavy [mastectomy followed by reconstruction with transversus recto-abdominal muscle cutaneous flap (TRAM)]. Samples were collected one day before surgery (D(-1)), at the end of surgical tumor removal (D0), and on 1st (D(+1)), 2nd (D(+2)) and 5th (D(+5)) day after surgery. Serum VEGF, bFGF and TGF-beta levels were measured by the enzyme immunoassay method.. On average a continuous decrease was observed for all growth factors from the day before operation to the 5th day after operation. On day (D(+5)) an increase was observed for patients who underwent extended respect to moderate surgery. These differences were found statistically significant for bFGF and VEGF (p = 0.05 and p = 0.025 respectively). A statistically different trend for type of operation was observed also for TGF-beta at 24-48 h: a minor reduction, compared to time of operation, was observed for minimal surgery, an intermediate reduction for moderate surgery and a higher decrease for extended surgery.. Angiogenic cytokines perioperative levels could be increased on 5th day (D(+5)) by extent of surgery and should induce perioperative stimulation of residual cancer cells. A better understanding of the time interval during which the sequelae of events in wound healing occur may be the basis for defining new therapeutic strategies that can interfere with tumor outgrowth sparing wound healing processes.

Topics: Aged; Breast Neoplasms; Cytokines; Female; Fibroblast Growth Factor 2; Humans; Mastectomy; Middle Aged; Neovascularization, Pathologic; Prospective Studies; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To evaluate the changes of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer.. Sixty four patients with breast cancer, 15 patients with benign breast tumors and 9 healthy volunteers were included in this study. The proportion of CD(4)(+)CD(25)(+) T cells population in total T cells was evaluated by flow cytometric analysis. The cytokine production (TGF-beta1) was measured by ELISA.. The population of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer accounted for (5.1 +/- 2.9)% of the total amount of T lymphocytes, and was significantly higher in comparison with that in patients with benign tumors and in healthy volunteers (P < 0.05). The CD(4)(+)CD(25)(+) T cells population in breast cancer patients was positively correlated with the cancer size and with TGF-beta1 level (r = 0.511 and r = 0.253, respectively), and negatively correlated with CD(8)(+)CD(28)(+) T cells and NK cells (r = -0.243 and r = -0.301, respectively).. The CD(4)(+)CD(25)(+) regulatory T cells in peripheral blood of patients with breast cancer is significantly increased in comparison with that in patients with benign breast tumor and in healthy subjects. It may be responsible for immune suppression in breast cancer patients.

Topics: Adult; Aged; Breast Neoplasms; CD4-Positive T-Lymphocytes; Cell Count; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-2 Receptor alpha Subunit; Middle Aged; T-Lymphocyte Subsets; Transforming Growth Factor beta

TGFBR1*6A is a common polymorphism of the type I transforming growth factor beta receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown.. To determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3-amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells.. Tumor and germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004. In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A.. TGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-beta-dependent cell proliferation.. TGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-beta growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells.. TGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-beta. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-beta signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

Topics: Activin Receptors, Type I; Alleles; Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genotype; Head and Neck Neoplasms; Humans; Neoplasm Metastasis; Phenotype; Polymorphism, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Sequence Deletion; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. We used adenovirus-mediated gene transfer of these natural inhibitors in a mouse model of breast cancer to examine the roles of TGF-beta superfamily signaling in tumor growth and metastasis.. We systemically administered, by intravenous injection, adenoviruses (AdCMV) containing the mouse cDNAs for Smad7, Smad6, c-Ski, the c-Ski mutant c-Ski (ARPG), or LacZ (control) to nude mice (>19 mice/group) bearing tumors derived from mouse mammary carcinoma JygMC(A) cells, which spontaneously metastasize to lung and liver, and examined their effects on survival and metastasis. High-throughput western blotting analysis was used to examine the expression levels for 47 signal transduction proteins in JygMC(A) cells and primary tumors. We also investigated the proliferation, migration, and invasion of JygMC(A) cells that stably overexpressed Smad6 or Smad7. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon's rank sum tests. Parametric comparisons were done by one-way analysis of variance or two-sided unpaired Student's t tests. All statistical tests were two-sided.. Control mice bearing tumors derived from JygMC(A) cells showed many metastases to the lung and liver; all animals died by 50 days after cell inoculation. By contrast, mice treated with AdCMV-Smad7 or AdCMV-c-Ski demonstrated a dramatic decrease in metastasis and statistically significantly longer survival than control mice (Smad7 versus LacZ: medium survival = 55 days versus 41 days, difference = 14 days [95% confidence interval {CI} = 6 days to 22 days], P < .001), whereas mice treated with AdCMV-Smad6 or AdCMV-c-Ski (ARPG) did not. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and tight junctions, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells.. Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers.

Topics: Adenoviridae; Alanine; Animals; Arginine; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA-Binding Proteins; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Glycine; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Mutagenesis, Insertional; Neoplasm Invasiveness; Polymerase Chain Reaction; Proline; Proto-Oncogene Proteins; Repressor Proteins; Signal Transduction; Smad6 Protein; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to investigate regulation of mRNA, coding for TGFbeta ligands and specific receptors, by novel antitumor antibiotic landomycin E (LE) in human breast adenocarcinoma cells of MCF-7 line, sensitive and resistant to anti-cancer agent doxorubicin.. Semi-quantitative analysis of mRNA expression was estimated using RT-PCR and DNA gel-electrophoresis methods. For comparison, another anti-cancer drug doxorubicin (adriamycin) was utilized. THE RESULTS obtained indicate that LE induces up-regulation of mRNA coding for TGFbeta receptors type I and type II in MCF-7 cells, as well as in their doxorubicin-resistant sub-line.. Anti-tumor action of LE may be mediated by TGFbeta, and a signaling pathway of this cytokine is not affected by the development of tumor cell resistance to doxorubicin.

Topics: Adenocarcinoma; Aminoglycosides; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Electrophoresis, Gel, Two-Dimensional; Female; Gene Expression; Humans; Ligands; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-beta) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-beta1 on breast cancer cells in the presence or absence of overexpressed HER-2.. Cell proliferation assays were used to determine the effect of TGF-beta on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-beta1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-beta signaling pathway was assessed using TGF-beta1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays.. We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-beta1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-beta in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-beta induced pro-invasive and pro-metastatic gene signature.. HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-beta. In contrast, HER-2 and TGF-beta signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Proliferation; Disease Progression; Epithelial Cells; Female; Gene Expression Profiling; Genetic Engineering; Humans; Mammary Glands, Human; Mesoderm; Neoplasm Invasiveness; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

We investigated the mechanism by which cancers evade the growth inhibitory effects of TGF-beta. Using two p21-/- somatically deleted human epithelial cell lines, we find that TGF-beta serves as a growth stimulator rather than a growth suppressor to cells lacking p21. In addition, TGF-beta stimulated p21-/- cells exhibited a mesenchymal phenotype, demonstrated by an upregulation of vimentin and decreased expression of E-cadherin. Analysis of primary human breast cancers by immunohistochemical labeling confirmed a correlation between p21 loss and positive vimentin expression. These data provide a molecular mechanism explaining how nongastrointestinal cancers can escape the anti-proliferative effects of this cytokine and simultaneously use this pathway for growth advantage.

Topics: Breast Neoplasms; Cadherins; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin

MCF10A mammary epithelial cells form growth-arrested structures when cultured in three-dimensional basement membrane gels. Activation of the receptor tyrosine kinase ErbB2 induces formation of proliferative structures that share properties with noninvasive early stage lesions. We conducted a genetic screen to identify cDNAs that can cooperate with ErbB2 to induce migration in these cells, with the hypothesis that they would represent candidate "second hits" in the development of invasive breast carcinomas. We found that expression of transforming growth factor (TGF)beta1 and TGFbeta3 in cells expressing activated ErbB2 induces migration in transwell chambers and invasive behavior in both basement membrane cultures and invasion chambers. The ability of ErbB2 to cooperate with TGFbeta correlated with sustained, elevated activation of extracellular signal-regulated kinase (Erk)-mitogen-activated protein kinase. Pharmacological reduction of Erk activity inhibited the cooperative effect of TGFbeta and ErbB2 on migration and expression of activated Erk kinase was sufficient to cooperate with TGFbeta to induce migration and invasion, suggesting that sustained Erk activation is critical for ErbB2/TGFbeta cooperation. In addition, we show that costimulation of ErbB2 and TGFbeta induces autocrine secretion of factors that are sufficient to induce migration, but not invasion, by means of both epidermal growth factor receptor-dependent and -independent processes. These results support the role of TGFbeta as a pro-invasion factor in the progression of breast cancers with activated ErbB2 and suggest that activation of the Erk and epidermal growth factor receptor pathways are key in mediating these events.

Topics: Breast Neoplasms; Cell Movement; Epithelial Cells; Female; Humans; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Receptor, ErbB-2; Transforming Growth Factor beta; Tumor Cells, Cultured

Interleukin-3 (IL-3) expression by tumor-infiltrating lymphocytes (TILs) and its effects on vessel assembly were evaluated. TILs from 'in situ' human breast cancers expressed CD4/CD25 antigens and IL-3. An injection of Matrigel containing SMC and IL-3 or basic-fibroblast growth factor (bFGF) into SCID mice confirmed the neoangiogenetic effect of both factors. However, in response to IL-3, but not to bFGF, only few SMC became incorporated into the nascent vessels. To evaluate the possibility that signals emanated by the nascent vasculature in the presence of IL-3 may negatively regulate SMC recruitment, conditioned media (CM) from IL-3-treated endothelial cells (EC) or SMC were tested for their biological effects on SMC and EC. CM from IL-3-treated SMC stimulated the migration of EC. In contrast, the migration of SMC was not affected by CM from IL-3-stimulated EC; however, it was greatly enhanced by blocking transforming growth factor beta (TGF beta) activity. TGF beta immunoenzymatic assay demonstrated the following: (i) the absence of TGF beta activity in CM from IL-3-stimulated EC; (ii) a barely detectable TGF beta activity in CM from IL-3-stimulated SMC; and (iii) the presence of TGF beta activity in the supernatants of SMC stimulated with CM from IL-3-, but not from bFGF-stimulated EC. Increased TGF beta mRNA expression was only detected in SMC stimulated with CM from IL-3-treated EC. Finally, the inhibitory signals induced by IL-3 in vivo were abrogated by the addition of the neutralizing TGF beta antibody. Thus, the positive immunostaining for IL-3 by TILs in 'in situ' breast cancers sustains the possibility that early in tumor development, IL-3 can contribute to the chronic immaturity of these vessels.

Topics: Animals; Breast Neoplasms; Cell Movement; Cells, Cultured; Collagen; Culture Media, Conditioned; Drug Combinations; Endothelial Cells; Enzyme Activation; Female; Fibroblast Growth Factor 2; Humans; Interleukin-3; Laminin; Lymphocytes; Matrix Metalloproteinase 2; Mice; Mice, SCID; Mitogens; Models, Biological; Muscle, Smooth, Vascular; Neovascularization, Pathologic; Proteoglycans; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The effect of genetic polymorphisms in the TGF-beta1 gene at codon 10 (T+29C), codon 25 (G+74C), and the promoter region [C --> T at -509 from the transcription site, (C-509T)] on breast cancer survival was evaluated among a cohort of 1111 patients. The median follow-up time for the cohort was 5.17 years after cancer diagnosis. No DNA sequence variation at codon 25 of the TGF-beta1 gene was found, whereas polymorphisms in C-509T and T+29C were in strong linkage disequilibrium. Patients who carried the C allele of T+29C polymorphism had a reduced 5-year disease-free survival rate (75.6% for T/C, and 78.2% for C/C) compared with the T/T genotype (85.1%; P, 0.04); the age-adjusted hazard ratio was 1.5 (95% confidence interval, 1.1-2.2). Adjustment for clinical prognostic factors slightly attenuated the association (hazard ratio, 1.4, 95% confidence interval, 1.0-1.9). Our study suggests that genetic polymorphisms in the TGF-beta1 gene may play a role in breast cancer progression.

Topics: Adult; Breast Neoplasms; Disease-Free Survival; Female; Humans; Middle Aged; Polymorphism, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Breast Neoplasms; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Humans; Transforming Growth Factor beta

Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by stimulating osteoblastic production of IL-11. The subsequent release of PGE(2) followed by inhibition of GM-CSF production by cells within the bone microenvironment plays an important part in mediating the effects of breast cancer cells on OCL formation and their resorptive activity.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Coculture Techniques; Colorectal Neoplasms; Dinoprostone; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-11; Interleukin-6; Lung Neoplasms; Mice; Osteoclasts; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Laboratory studies suggest a dual role for the transforming growth factor-beta (TGF-beta) signaling pathway in breast cancer. The normal antiproliferative activity of TGF-beta in early breast tumor development is replaced by a promoting effect in later stages. A T29C transition polymorphism in the TGFB1 gene has been associated with higher circulating TGF-beta1 levels, and inconsistently with breast cancer risk in three recent studies. We tested the association of this variant with invasive breast cancer in a case-control study of 1123 cases and 2314 controls nested in the Multiethnic Cohort (MEC) Study. This study is a large prospective study being conducted in Hawaii and Los Angeles that includes Japanese, white, African American, Latino, and Native Hawaiian women who were predominantly postmenopausal at baseline. After adjustment for breast cancer risk factors, the odds ratio (OR) and 95% confidence interval (95% CI) for the TGFB1 29 CC genotype was 0.95 (95% confidence interval: 0.76-1.18), compared to the TT genotype. Analyses stratified by race/ethnicity, stage, or age category did not reveal any association of this variant with breast cancer. Given the strong biological rationale and the scarce and divergent epidemiologic data to date, additional investigations of the relationship between breast cancer and genetic variants in the TGF-beta signaling pathway appear warranted.

Topics: Aged; Breast Neoplasms; Case-Control Studies; Ethnicity; Female; Genotype; Hawaii; Humans; Los Angeles; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Prospective Studies; Racial Groups; Risk Factors; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Smad proteins are intracellular signalling mediators of transforming growth factor-beta (TGF-beta) superfamily. In the nucleus, activated Smad complexes regulate transcriptional responses of the target genes in cooperation with transcriptional coactivators and corepressors. To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a coactivator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a coactivator for receptor-regulated Smads (R-Smads) for TGF-beta signalling pathways. GCN5 functions like PCAF, in that it binds to TGF-beta-specific R-Smads, and enhances transcriptional activity induced by TGF-beta. In addition, GCN5, but not PCAF, interacts with R-Smads for bone morphogenetic protein (BMP) signalling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo. Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-beta. In conclusion we identified GCN5 as a Smad-binding transcriptional coactivator which positively regulates both TGF-beta and BMP signalling pathways.

Topics: Acetyltransferases; Animals; Binding Sites; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Extracts; Cell Line; Cloning, Molecular; DNA-Binding Proteins; Histone Acetyltransferases; Humans; p300-CBP Transcription Factors; Plasminogen Activator Inhibitor 1; Precipitin Tests; Promoter Regions, Genetic; Receptors, Growth Factor; Regulatory Sequences, Nucleic Acid; Signal Transduction; Smad Proteins; Smad3 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor beta (TGF-beta) is an example for a large and still-growing family of growth factors. TGF-beta1 is known to act both as a tumour suppressor and as a stimulator of tumour progression. This study examines the relationship amongst putative enhancer, promoter, 5'-untranslated-region (UTR) and exon-1 polymorphisms of the TGF-beta1 gene (region I from -1881 to -1613; region II from -1410 to -1123, and region III from -55 to +176, as per human genome organisation (HUGO) nomenclature) in 26 breast cancer patients and 97 healthy control subjects. The germline and somatic status of the four known polymorphisms was ascertained, and a significant difference was observed for the germline C/T and T/T genotype distribution between patients and controls in comparison to C/C genotypes at position -1349 (chi2 = 6.193; P = 0.009). In addition to the somatic variations observed for some of the regions studied, in 10/26 (38%) sporadic breast cancer cases, a novel somatic mutation in codon 47 of exon 1 (GenBank accession number AY059373) was also detected in tumour samples. The risk of cancer was found to be significant (OR = 4.525) for the -1349 C/T and T/T genotype background, suggesting that this genetic background may act as a risk factor for sporadic breast cancer.

Topics: 5' Untranslated Regions; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Disease Progression; DNA Mutational Analysis; DNA Primers; Enhancer Elements, Genetic; Exons; Genotype; Humans; India; Molecular Sequence Data; Mutation; Odds Ratio; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Promoter Regions, Genetic; Risk; Risk Factors; Sequence Analysis, DNA; Software; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hormones, estrogen and prolactin, transforming growth factor-beta (TGF-beta), and matrixmetalloproteinases (MMPs) may modulate breast cancer progression. The goal of this research was to examine the regulation of expression of TGF-beta and MMPs (MMP-1, 2, 9) by estrogen and prolactin, independently and in combination, at physiological doses, and at doses stimulating cancer cell (T47D) proliferation in vitro. Prolactin, and estrogen synergistically, and similarly, inhibited the expression of TGF-beta and MMPs at physiological concentrations without altering cell proliferation, indicating a beneficial role of the hormones. The growth stimulating concentration of prolactin, but not estrogen, also inhibited the TGF-beta and MMP expression.

Topics: Breast Neoplasms; Cell Survival; Drug Synergism; Drug Therapy, Combination; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Prolactin; Promoter Regions, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

We have examined overexpression of the human epidermal growth factor receptor 2 (HER2) to determine if it modifies the anti-proliferative effect of transforming growth factor (TGF)-beta against MCF-10A human mammary epithelial cells. Exogenous TGF-beta inhibited cell proliferation and induced Smad-dependent transcriptional reporter activity in both MCF-10A/HER2 and MCF-10A/vector control cells. Ligand-induced reporter activity was 7-fold higher in HER2-overexpressing cells. In wound closure and transwell assays, TGF-beta induced motility of HER2-transduced, but not control cells. The HER2-blocking antibody trastuzumab (Herceptin) prevented TGF-beta-induced cell motility. Expression of a constitutively active TGF-beta type I receptor (ALK5(T204D)) induced motility of MCF-10A/HER2 but not MCF-10A/vector cells. TGF-beta-induced motility was blocked by coincubation with either the phosphatidylinositol 3-kinase inhibitor LY294002, the mitogen-activated protein kinase (MAPK) inhibitor U0126, the p38 MAPK inhibitor SB202190, and an integrin beta(1) blocking antibody. Rac1 activity was higher in HER2-overexpressing cells, where both Rac1 and Pak1 proteins were constitutively associated with HER2. Both exogenous TGF-beta and transduction with constitutively active ALK5 enhanced this association. TGF-beta induced actin stress fibers as well as lamellipodia within the leading edge of wounds. Herceptin blocked basal and TGF-beta-stimulated Rac1 activity but did not repress TGF-beta-stimulated transcriptional reporter activity. These data suggest that 1) overexpression of HER2 in nontumorigenic mammary epithelial is permissive for the ability of TGF-beta to induce cell motility and Rac1 activity, and 2) HER2 and TGF-beta signaling cooperate in the induction of cellular events associated with tumor progression.

Topics: Actins; Activin Receptors, Type I; Adenoviridae; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Blotting, Northern; Breast Neoplasms; Bromodeoxyuridine; Butadienes; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Cell Movement; Chromones; Disease Progression; DNA, Complementary; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Imidazoles; Immunoblotting; Integrin beta1; Ligands; Luminescent Proteins; Microscopy, Fluorescence; Models, Genetic; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Precipitin Tests; Protein Serine-Threonine Kinases; Pseudopodia; Pyridines; rac1 GTP-Binding Protein; Receptor, ErbB-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Retroviridae; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Trastuzumab; Wound Healing

Transforming growth factor (TGF) beta is a pre-eminent negative growth regulator that has antiproliferative effects on a range of epithelial cells. This ability has evoked interest in this growth factor as a tumour suppressor with potential clinical significance. In the early stages of breast carcinogenesis, a growth-inhibitory response to TGFbeta is maintained, which depends on an intact TGFbeta signalling pathway. Tumour development and progression of cells along a neoplastic continuum is accompanied by loss of this growth-inhibitory response to TGFbeta, which might instead promote tumour growth indirectly through a combination of permissive effects on stromal tissue, angiogenesis, and the immune system. This review discusses the complexity of functional pleiotropy and the continually changing roles of TGFbeta as a tumour evolves, along with competing therapeutic strategies. The boosting of local endogenous amounts of TGFbeta in conjunction with enhancement of cellular responsiveness might be appropriate in early malignant disease, and anti-TGFbeta approaches could yield a therapeutic gain in metastatic states.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Disease Progression; Female; Humans; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Antiestrogens are successfully used in the treatment of breast cancer. The purpose of this study was to investigate the role of different signal transduction pathways in antiestrogen-induced growth inhibition to gain insights into mechanisms of antiestrogen resistance. We used specific MAPK inhibitors and MCF-7 carcinoma cells as a model to demonstrate that p38 MAPK is an important mediator of antiestrogen growth inhibition in breast cancer. A kinase assay showed that antiestrogens (4-hydroxytamoxifen and ICI 182.780) rapidly induce p38 activity. Overexpression of kinase-deficient mutants of p38 reduced the antiestrogen suppression of Cyclin A transcription. TGFbeta, a negative regulator of breast cancer cell growth, is induced by antiestrogens; therefore, activation of p38 could have been mediated by TGFbeta. We used a TGFbeta and antiestrogen-sensitive reporter gene assay to show that p38 activation precedes TGFbeta activation. These results were further confirmed by quantitative RT-PCR analysis of the antiestrogen-induced transcription of TGFbeta2 and TGFbeta receptor II. Inhibition of p38 reduced the induction of both genes. Finally, Western blot analysis shows that antiestrogens induce phosphorylation of Smad (mothers against decapentaplegic homolog) 2 via p38. Promoter assays with the Smad-dependent reporter p6SBE confirm participation of Smad3 and Smad4 in antiestrogen action. Taken together, our data delineate an antiestrogen signal transduction pathway involving sequential activation of p38 and TGFbeta pathways to mediate growth inhibition.

Topics: Breast Neoplasms; Cell Proliferation; Cyclin A; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Humans; Mutation; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad2 Protein; Smad3 Protein; Smad4 Protein; Tamoxifen; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Tumor progression due to loss of autocrine negative transforming growth factor-beta (TGF-beta) activity was reported in various cancers of epithelial origin. Estrogen receptor expressing (ER(+)) breast cancer cells are refractory to TGF-beta effects and exhibit malignant behavior due to loss or inadequate expression of TGF-beta receptor type II (RII). The exogenous TGF-beta effects on the modulation of cell cycle machinery were analyzed previously. However, very little is known regarding the endogenous control of cell cycle progression by autocrine TGF-beta. In this study, we have used a tetracycline regulatable RII cDNA expression vector to demonstrate that RII replacement reconstitutes autocrine negative TGF-beta activity in ER(+) breast cancer cells as evidenced by the delayed entry into S phase by the RII transfectants. Reversal of the delayed entry into S phase by the RII transfectants in the presence of tetracycline in addition to the decreased steady state transcription from a promoter containing the TGF-beta responsive element (p3TP-Lux) by TGF-beta neutralizing antibody treatment of the RII transfected cells confirmed that autocrine-negative TGF-beta activity was induced in the transfectants. Histone H1 kinase assays indicated that the delayed entry of RII transfectants into phase was associated with markedly reduced cyclin-dependent kinase (CDK)2 kinase activity. This reduction in kinase activity was due to the induction of CDK inhibitors p21/waf1/cip1 and p27/kip, and their association with CDK2. Tetracycline treatment of RII transfectants led to the suppression of p21/waf1/cip1and p27/kip expression, thus, directly demonstrating induction of CDK inhibitors by autocrine TGF-beta leading to growth control of ER(+) breast cancer cells.

Topics: Breast Neoplasms; CDC2-CDC28 Kinases; Cell Adhesion; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Disease Progression; Flow Cytometry; Humans; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Tetracycline; Transfection; Transforming Growth Factor beta; Tumor Suppressor Proteins

Although overexpression of TGF-beta1 protein has been demonstrated in advanced breast cancer (BC) patients, as well as in other solid tumours, the molecular mechanism of this process remains obscure. This paper proposes that a genetic/epigenetic alteration might occur in the TGF-beta1 gene, within the region coding for the recognition site with TGFbeta receptor type II, leading to a disruption of the ligand-receptor interaction and triggering the TGF-beta1 cascade-related BC progression. To establish the operational framework for this hypothesis, in the present study, this recognition site was identified by the Informational Spectrum Method (ISM) to comprise two TGF-beta1 peptides (positions 47-66 aa and 83-112 aa) and one receptor peptide at positions 112-151 aa of the extracellular domain of the receptor (TbetaRIIM). The TbetaRIIM locus was further evaluated by ISM-derived deletion analysis of the TbetaRII sequences. To provide experimental support for the proposed model, a pilot study of plasma TGF-beta1 analysis was performed in advanced BC patients (n = 8). Two commercial ELISA assays, one with specific alphaTGF-beta1 MAb (MAb) and other with TbetaRIIM as the immobilized phase, revealed pronounced differences in the pattern of plasma TGF-beta1 elevation. In MAb-profile, the TGF-beta1 increase was detected in 7 of 8 patients, whereas analogous TbetaRIIM-profile revealed the elevation in 3 of 8 patients, taking a 50% of maximal elevation as the cut-off value. These findings are consistent with the proposed aberration of TGF-beta1 ligand within the TbetaRII recognition site. Summarizing, this model system is a good starting point for further genetic studies, particularly on genetic/epigenetic alterations of sequences involved in TGF-beta1 and TbetaRIIM interaction, with putative prognostic value for breast cancer.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Breast Neoplasms; Female; Gene Expression Profiling; Genetic Predisposition to Disease; Humans; Molecular Sequence Data; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Analysis, Protein; Transforming Growth Factor beta; Yugoslavia

We have previously shown that transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; Collagenases; Core Binding Factor Alpha 1 Subunit; COS Cells; DNA-Binding Proteins; Gene Expression; Humans; Matrix Metalloproteinase 13; Neoplasm Proteins; Promoter Regions, Genetic; Protein Structure, Tertiary; Proto-Oncogene Proteins c-jun; Rats; Smad3 Protein; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1

Amplification and overexpression of ErbB2 (HER2/Neu) is one of the most common alterations associated with breast cancer. Activation of ErbB2 via homodimerization in a non-transformed human mammary epithelial cell line, MCF-10A, in basement membrane cultures leads to formation of proliferative structures that share properties with non-invasive early stage lesions. Recently, we have shown that activation of ErbB2 homodimers combined with expression of transforming growth factor (TGF)-beta induces invasive and migratory activity in MCF-10A cells. In this system, migration requires inputs from numerous cellular pathways. We discuss this data and a model for migration induced by ErbB2 and TGF-beta. Concurrent studies by other groups have also shown that ErbB2 and TGF-beta can cooperate to increase metastatic and invasive behavior in murine mammary tumors. Here we discuss these studies and the potential implications of this research on breast cancer therapeutics.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Female; Humans; Mice; Models, Biological; Neoplasm Metastasis; Receptor, ErbB-2; Transforming Growth Factor beta

Primary breast carcinoma are frequently infiltrated by dendritic cells (DC). The mechanisms involved in the localization and status of activation of DC within primary breast carcinoma were investigated. CCL20/MIP3alpha, a chemokine involved in immature DC and their precursors attraction, was detected by immunohistochemistry on cryopreserved tissue sections of primary breast tumors and by ELISA and biological assay in metastatic effusion fluids from breast cancer patients but not from other tumors. In vitro, irradiated breast carcinoma cell lines (BCC) as well as their conditioned media promoted CD34+ cell differentiation into CD1a+ Langerhans cells (LC) precursors as early as day 6, while at day 12, 2 different CCR6+ subpopulations of DC with a Langerhans cell (CD1a(+)Langerin(+)CD86+) and an immature DC (CD1a(high)Langerin-CD86(-)HLA-DR(low)CD40(low)) phenotype were observed. This phenomenon was partly driven by a TGFbeta-dependent mechanism since a pan TGFbeta polyclonal antibody completely blocks BCC-induced LC differentiation and partly reduces immature DC development. These DC failed to maturate in response to sCD40L or LPS stimuli and CD1a(high)Langerin(-)CD86- cells have a reduced T-cell stimulatory capacity in MLR experiments. The absolute number of T cells was reduced by 50% in both the CD4+ or CD8+ compartments, these T cells expressing lower levels of the CD25 Ag and producing less IFNgamma. These results show that breast carcinoma cells produce soluble factors, which may attract DC and their precursors in vivo, and promote the differentiation of the latter into LC and immature DC with altered functional capacities. The infiltration of BCC by these altered DC may contribute to the impaired immune response against the tumor.

Topics: Antigens, CD; Antigens, CD1; Antigens, CD34; Antigens, Surface; B7-2 Antigen; Breast Neoplasms; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Cell Movement; Chemokine CCL20; Chemokines, CC; Chemotaxis; Culture Media, Conditioned; Cytokines; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Lectins, C-Type; Macrophage Inflammatory Proteins; Mannose-Binding Lectins; Membrane Glycoproteins; Neoplasm Metastasis; Phenotype; T-Lymphocytes; Transforming Growth Factor beta

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Genital Neoplasms, Female; Humans; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Transfection; Transforming Growth Factor beta; Up-Regulation; Urokinase-Type Plasminogen Activator; Vitronectin

The loss of transforming growth factor-beta (TGF-beta) response due to the dysregulation of TGF-beta receptors type I (RI) and type II (RII) is well known for its contribution to oncogenesis. Estrogen receptor-expressing breast cancer cells are refractory to TGF-beta-mediated growth control because of the reduced expression of TGF-beta receptors. Although RII is required for the binding of TGF-beta to RI, RI is responsible for directly transducing TGF-beta signals through the Smad protein family. Treatment of estrogen receptor-expressing MCF-7L and ZR75 breast cancer cells with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) led to a dramatic induction of RI. Accumulation of acetylated histones H3 and H4 was observed in the SAHA-treated cells. Chromatin immunoprecipitation analysis followed by PCR with RI promoter-specific primers indicated an accumulation of acetylated histones in chromatin associated with the RI gene, suggesting that histone deacetylation was involved in the transcriptional inactivation of RI. SAHA treatment stimulated RI promoter activity through the inhibition of Sp1/Sp3-associated HDAC activity. Histone acetyltransferase p300 stimulated RI promoter activity, thus further confirming the involvement of HDAC activity in the transcriptional repression of RI. Significantly, SAHA-mediated RI regeneration restored the TGF-beta response in breast cancer cells.

Topics: Acetylation; Acetyltransferases; Activin Receptors, Type I; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Chromatin; DNA Methylation; DNA-Binding Proteins; Genes, Reporter; Histone Acetyltransferases; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Luciferases; Precipitin Tests; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sp1 Transcription Factor; Sp3 Transcription Factor; Time Factors; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Vorinostat

The process of cancer invasion involves a complex interplay between cell-cell and cell-medium adhesion, proteolytic enzyme secretion, cell birth and death processes, random and directed motility, and immune response, as well as many other factors. The growth factor TGF beta is known to have a complex effect on this process. It inhibits mitosis and promotes apoptosis in a concentration-dependent manner in vitro, and it is for this reason that its secretion is thought to be helpful in inhibiting tumour growth. However, recent in vitro and in vivo results have shown a significant effect of this growth factor in promoting the sensitivity of malignantly transformed cells to gradients of extracellular matrix proteins--an effect which tends to increase invasiveness. The drug tamoxifen has been demonstrated to be therapeutically effective in the treatment of patients with breast cancer; however, it is known also that many patients become resistant to the effect of this drug after a few years, and the reasons for this remain controversial. In this work we take our established model of cancer invasion (J. Theor. Biol. 216(1) (2002) 85), and extend it to include the effect of TGF beta. In so doing we demonstrate that a tamoxifen-stimulated upregulation of the secretion of TGF beta may give rise to a tumour which has a smaller number of cells but which has a greater invasiveness, greater metastatic potential, and a tumour histology which is known to correlate with a poorer prognosis. These data suggest that tamoxifen-stimulated secretion of TGF beta might explain treatment failure in some patients.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Extracellular Matrix; Female; Humans; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Tamoxifen; Transforming Growth Factor beta

TGF beta/Smad signaling pathway members are potent tumor suppressors for many types of cancers. We hypothesize that breast tumors differentially express these genes and that this expression pattern plays a role in the proliferation of breast cancer. We examined the mRNA levels of TIEG, Smad7, Smad2, and Bard1 using real-time RT/PCR in 14 normal breast, five non-invasive, 57 invasive (including 29 with outcome data), and five metastatic breast tumor tissues. TIEG and Smad7 mRNA levels were lower in non-invasive tumors compared to normal breast tissues. TIEG, Bard1, and Smad2 mRNA levels were lower in invasive cancers compared to normal breast tissues. In addition, TIEG, Smad2, and Bard1, provided discriminatory ability to potentially distinguish between normal and tumor samples, N- and N+ tumors, and N-/good (no recurrence for at least 5 years) and N-/bad (recurrence within 3 years) outcome patients. TIEG mRNA levels accurately discriminated between normal breast tissue and primary tumors with a sensitivity and specificity of 96 and 93%, respectively. TIEG, in combination with Smad2, distinguished between N+ and N- primary tumors with a sensitivity and specificity of 75 and 85%, respectively. TIEG in combination with Bard1 discriminated between N-/bad outcome from N-/good tumors with a sensitivity and specificity of 83 and 82%, respectively. Our results support the hypothesis that the differential gene expression of TIEG, Smad2, and Bard1, which are tumor suppressor genes, plays a significant role in the proliferation of breast cancer. Further investigation is necessary to validate the ability of these genes to discriminate between different populations of breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Division; DNA-Binding Proteins; Early Growth Response Transcription Factors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad7 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Zinc Fingers

The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Humans; Mice; Mice, Nude; Mutation; Neoplasm Metastasis; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad2 Protein; Smad3 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta

The aim of the study was to investigate the survival and growth of human breast carcinoma MCF-7 cells with different sensitivity to doxorubicin and production of transforming growth factor beta-(TGF-beta) in dependence on the dose- and duration of X-ray in order to check if the cross-resistance to doxorubicin and radiation effects exists.. Determination of cell number and valiability using trypan blue (0.1% (w/v)) exclusion method, Western blot analysis of p53 protein expression, biological testing of TGF-beta activity, lectinocytochemical analysis for apoptosis quantitative estimation in unirradiated and irradiated cells of both sublines of MCF-7 cells--sensitive (MCF-7(wt)) and resistant (MCF-7(DOX/R)) to doxorubicin.. It was found that doxorubicin-resistant breast cancer cells were also more refractory to X-radiation-dependent growth inhibition. There were revealed different effects of distinct doses of X-ray on p53 protein expression by cells of both sublines. The level of production of TGF-beta was compared in non-irradiated MCF-7 cells and in these cells exposed to X-radiation. It was shown that X-radiation increased TGF-beta activity in the conditioned medium of the irradiated cells of both doxorubicin-sensitive and -resistant lines.. The results of our study suggest that the biological effects of X-radiation on human breast cancer MCF-7 cells can be at least partly mediated by TGF-beta. Taking into account that TGF-beta is a potent natural immunosupressor, one may consider that an increased activity of this cytokine can intensify negative effects of X-radiation.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; Cell Survival; Doxorubicin; Drug Resistance, Neoplasm; Female; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53; X-Rays

Alterations in TGF-beta signaling appear to be associated with an altered risk of developing cancer, including breast cancer. We carried out a case-control study on 8 polymorphisms, including 5 in the TGF-beta1 gene (G-800A, C-509T, Leu10-->Pro, Arg25-->Pro and Thr263-->Ile), a polyalanine polymorphism (9A-->6A) in the TGF-betaRI gene and 2 (G-875A and A-364G) in the TGF-betaRII gene, using samples from 2 different populations, Polish familial and Finnish unselected breast cancer cases, together with ethnically and geographically matched controls. Additionally, familial breast cancer cases with respective controls from Sweden and Germany were studied in the Leu10-->Pro polymorphism, making the total number of familial cases 659. Allele, genotype and haplotype analysis on the TGF-beta1 gene as well as an analysis of the combinations of genotypes of the TGF-beta1 and its receptor genes in each individual were performed. Population differences in the allele and genotype distributions were found from 5 of the polymorphisms and 3 common haplotypes from the TGF-beta1 gene between the Finnish and other populations. However, no statistically significant difference between the breast cancer and healthy control groups was found for any of the 8 polymorphisms nor did the haplotype or genotype combination analysis reach statistical significance. Thus, none of the studied polymorphisms from the TGF-beta1 and its receptor genes was found to influence significantly susceptibility to breast cancer. The possible contribution of 6A/6A homozygosity in the TGF-betaRI gene to breast cancer needs to be confirmed in an independent study.

Topics: Activin Receptors, Type I; Adolescent; Adult; Aged; Aged, 80 and over; Alleles; Breast Neoplasms; Case-Control Studies; Female; Genotype; Haplotypes; Humans; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

The transcription factor NF-kappaB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-kappaB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-beta. We show here that the IKK-2/IkappaBalpha/NF-kappaB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-kappaB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-beta. Furthermore, inhibition of NF-kappaB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-kappaB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-kappaB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-kappaB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-beta-dependent signaling pathways in late-stage tumorigenesis depends critically on NF-kappaB activity.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Neoplastic; Cell Transformation, Viral; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Mammary Glands, Animal; Mesoderm; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B; Oncogene Protein p21(ras); Retroviridae; Time Factors; Transforming Growth Factor beta

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-beta (TGF-beta) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-beta bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-beta in each other. In cocultures, PPT-I and TGF-beta expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-beta and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5' flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-beta, and transient tranfections all showed that c-myc is required for TGF-beta-mediated induction of PPT-I in BCCs. TGF-beta was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-beta is important to the regulation of PPT-I in the early events of bone invasion by BCCs.

Topics: Bone Marrow Cells; Breast Neoplasms; Cell Line, Tumor; Coculture Techniques; Consensus Sequence; Cytokines; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Protein Precursors; Stromal Cells; Tachykinins; Transfection; Transforming Growth Factor beta

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogen Receptor Modulators; Female; Humans; Isotretinoin; Ki-67 Antigen; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Thymidine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Suppressor Protein p53

Transforming growth factor (TGF)-beta has been reported to exert growth inhibitory activity in normal epithelial cells whereas it induces cell proliferation and invasive phenotypes in advanced carcinomas. Our previous study showed that MCF10A, a spontaneously immortalized "normal" breast epithelial cell line, is resistant to TGF-beta-induced growth inhibition, suggesting that conversion of TGF-beta growth inhibitory signaling into an oncogenic pathway may occur at the early stage of tumor development/progression. To address this issue, we investigated the TGF-beta signaling pathway and its role in phenotypic transformation of MCF10A cells. TGF-beta treatment induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, accompanied with down-regulation of epithelial cell marker E-cadherin. TGF-beta treatment was sufficient to induce migrative and invasive phenotypes in these cells, an important phenotypic conversion during tumor progression. We also showed that TGF-beta treatment rapidly activated ERK-1/2 and p38 MAPK leading to upregulation of matrix metalloproteinase (MMP)-2 and MMP-9. Using chemical inhibitors and dominant negative mutants of MAPKs, we provide evidence that while both p38 MAPK and ERKs are required for TGF-beta-induced MCF10A cell migration and invasion, TGF-beta-induced MMP-2 and MMP-9 expression depends on p38 MAPK signaling, but is independent of ERK activity. This study demonstrates the roles of TGF-beta signaling pathways for induction of oncogenic signaling in preneoplastic human breast epithelial cells and will deepen our understanding of TGF-beta signaling in the progress of breast cancer.

Topics: Breast Neoplasms; Cell Movement; Cell Transformation, Neoplastic; Disease Progression; Epithelial Cells; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; p38 Mitogen-Activated Protein Kinases; Phenotype; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The anti-estrogen 4-hydroxytamoxifen (TAM) and vitamin A-related compounds, the retinoids, in combination act synergistically to inhibit growth of breast cancer cells in vitro and in vivo. To clarify the mechanism of this synergism, the effect of TAM and all trans-retinoic acid (AT) on proliferation of MCF-7 breast cancer cells was studied in vitro. TAM and AT acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. In a temporally related manner, TAM+AT acted synergistically to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis. TAM and AT each blocked cell cycle progression throughout 7 days of treatment but without any synergistic or additive effect on this process, indicating a selective synergism for apoptosis. The negative growth factor-transforming growth factor beta (TGFbeta) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM+AT on apoptosis. TAM+AT acted synergistically to induce a fivefold increase in TGFbeta1 secretion over 72 h. TGFbeta1 alone had no apoptotic effects on these cells; however, TGFbeta1 in combination with AT acted synergistically to inhibit growth, to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner comparable with that noted for TAM+AT. The synergism of both TAM+AT and TGFbeta1+AT for apoptosis was suppressed by estradiol. Co-incubation of TAM+AT with anti-TGFbeta antibody did not block down-regulation of Bcl-2 protein expression or stimulation of apoptosis. The synergistic effects of TAM+AT on apoptosis therefore occur independently of TGFbeta, although TGFbeta may interact with AT in a novel manner to provide another important anti-proliferative mechanism for breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Estrogen Receptor Modulators; Female; Humans; Stimulation, Chemical; Tamoxifen; Time Factors; Transforming Growth Factor beta; Tretinoin

Breastfeeding plays a substantial role in mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1). Mammary epithelial cells, as well as macrophages and lymphocytes, are thought to serve as sources of the virus in breast milk. Soluble factors in breast milk exert various biological functions, including immune tolerance or immune modulation, and may influence milk-borne infection with HIV-1. In this study we show that transforming growth factor beta (TGF-beta), a major cytokine in breast milk, inhibited HIV-1 infection of mammary epithelial MCF-7 cells but enhanced that of macrophages. TGF-beta downregulated the HIV-1 long terminal repeat (LTR) promoter in MCF-7 cells but upregulated it in macrophages. Stimulation with TGF-beta suppressed NF-kappaB binding to the HIV-1 LTR in MCF-7 cells, at least in part by downregulating induced IkappaB kinase expression. Cell type-dependent effects of TGF-beta on HIV-1 expression may play a role in milk-borne infection with HIV-1.

Topics: Binding Sites; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Forkhead Transcription Factors; HIV Long Terminal Repeat; HIV-1; Humans; I-kappa B Kinase; Macrophages; NF-kappa B; Protein Serine-Threonine Kinases; Smad2 Protein; Smad3 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta), tenascin-C (TN-C) and matrix metalloproteinases (MMPs) have been demonstrated independently to be associated with disease progression and poor prognosis in patients with breast cancer. The present study explored effects of TGF-beta and TN-C on MMP-9 expression and cancer invasion. An experimental study was designed to analyse MDA-MB-231 breast cancer cells, known for their high invasiveness, after stimulation with TGF-beta1 and/or TN-C. TGF-beta1 stimulated TN-C expression in the cells. Co-stimulation of MDA-MB-231 cells with TN-C and TGF-beta increased MMP-9 expression at both the gene (28-fold) and the protein levels. The in vitro invasion also increased (4-fold). GM6001 inhibited the invasion induced by the co-stimulation. The combined effect of TN-C and TGF-beta resulted in enhanced MMP-9 expression and cancer invasion in vitro.

Topics: Breast Neoplasms; Carcinogens; Cell Line, Tumor; Female; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tenascin; Transforming Growth Factor beta; Up-Regulation

The purpose of the present study was to evaluate breast carcinoma samples before and two days after treatment with tamoxifen in order to analyse early histopathological alterations--particularlynuclear alterations-- as well as immunohistochemical expression of Ki-67, Erb-B2, VEGF, TGF-beta1 and ILK proteins. Twenty one cases of invasive ductal and lobular breast carcinoma were studied. Patients were submitted to biopsy of the lesion and, after confirmation of the diagnosis, they received 20 mg of tamoxifen a day, beginning two days before surgery. The samples obtained during biopsy and after surgery were stained with HE for histopathological diagnosis. Estrogen receptor was positive in 18 cases and negative in 3. The immunohistochemical method was applied for the detection of Ki-67, Erb-B2, protein, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta1) and integrin linked kinase (ILK). Two days after tamoxifen treatment, the following results were observed: 1) decrease in the cell volume, chomatine condensation, nucleoli less evident and clearly defined nuclear limits; 2) significant reduction in the expression of Erb-B2 protein and significant increase in the expression of TGF-beta1 protein; 3) expression of others proteins (Ki-67, VEGF and ILK) was not altered during the indicated time frame. Our results suggest that analyzing nuclear alterations and expression of Erb-B2 and TGF-beta1 proteins would be useful to assess the initial response to tamoxifen.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cell Nucleus; Humans; Immunohistochemistry; Ki-67 Antigen; Protein Serine-Threonine Kinases; Receptor, ErbB-2; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factors

Bone is a primary target for colonization of metastatic breast cancer cells. Once present, the breast cancer cells activate osteoclasts, thereby stimulating bone loss. Bone degradation is accompanied by pain and increased susceptibility to fractures. However, targeted inhibition of osteoclasts does not completely prevent lesion progression, nor does it heal the lesions. This suggests that breast cancer cells may also affect osteoblasts, cells that build bone. The focus of this study was to determine the ability of breast cancer cells to alter osteoblast function. MC3T3-E1 osteoblasts were cultured with conditioned medium from MDA-MB-231 breast cancer cells and subsequently assayed for changes in differentiation. Osteoblast differentiation was monitored by expression of osteocalcin, bone sialoprotein and alkaline phosphatase, and by mineralization. Osteoblasts cultured with MDA-MB-231 conditioned medium did not express these mature bone proteins, nor did they mineralize a matrix. Inhibition of osteoblast differentiation was found to be due to transforming growth factor beta present in MDA-MB-231 conditioned medium. Interestingly, breast cancer conditioned medium also altered cell adhesion. When osteoblasts were assayed for adhesion properties using interference reflection microscopy and scanning acoustic microscopy, there was a reduction in focal adhesion plaques and sites of detachment were clearly visible. F-actin was disassembled and punctate in osteoblasts cultured with MDA-MB-231 conditioned medium rather than organized in long stress fibers. Taken together, these observations suggest that metastatic breast cancer cells alter osteoblast adhesion and prevent differentiation. These affects could account for the continued loss of bone after osteoclast inhibition in patients with bone-metastatic breast cancer.

Topics: Alkaline Phosphatase; Bone Neoplasms; Breast Neoplasms; Calcification, Physiologic; Cell Adhesion; Cell Differentiation; Culture Media, Conditioned; Female; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta

p38 belongs to a family of mitogen-activated protein kinases, which transfer extracellular signals into intracellular responses. p38 is also frequently detected in clinical breast cancer specimens, but its role as a prognostic factor is not known. Of the various p38 isoforms, p38alpha has been shown to mediate the in vitro invasiveness of breast cancer cells through up-regulation of urokinase plasminogen activator (uPA). We studied the role of p38alpha in breast cancer bone metastases, using dominant negative blockade approach. Human MDA-MB-231 breast cancer clones stably expressing dominant negative p38alpha (p38/AF) exhibited decreased basal MMP-9 activity. TGF-beta1-induced MMP-9 activity was also blunted in these clones, as compared with controls in which TGF-betal up-regulated MMP-9 activity. Consistent with these findings, SB202190, a specific p38 inhibitor, also inhibited TGF-beta1-induced MMP-9 activity in parental cells. The p38/AF clones exhibited also reduced uPA production after growth on vitronectin and decreased cell motility, as compared with controls. VEGF production levels in all the studied clones were similar. The p38/AF clone, which had similar in vitro growth rate as the control pcDNA3 clone, formed significantly less bone metastases in a mouse model, as compared with the control clone. In conclusion, inhibition of the p38alpha pathway results in decreased MMP-9 activity, impaired uPA expression and decreased motility, all of which may contribute to the decreased formation of bone metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Movement; Down-Regulation; Enzyme Inhibitors; Female; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Vitronectin