transforming-growth-factor-beta and Neoplasm-Metastasis

Although many studies have investigated the role of cytokines in bone metastases, our knowledge of their function in spine metastasis is limited. Therefore, we performed a systematic review to map the available evidence on the involvement of cytokines in spine metastasis in solid tumors. A PubMed search identified 211 articles demonstrating a functional link between cytokines/cytokine receptors and bone metastases, including six articles confirming the role of cytokines/cytokine receptors in spine metastases. A total of 68 cytokines/cytokine receptors were identified to mediate bone metastases; 9 (mostly chemokines) played a role in spine metastases: CXC motif chemokine ligand (CXCL) 5, CXCL12, CXC motif chemokine receptor (CXCR) 4, CXCR6, interleukin (IL) 10 in prostate cancer, CX3C motif chemokine ligand (CX3CL) 1 and CX3C motif chemokine receptor (CX3CR) 1 in liver cancer, CC motif chemokine ligand (CCL) 2 in breast cancer, and transforming growth factor (TGF) β in skin cancer. Except for CXCR6, all cytokines/cytokine receptors were shown to operate in the spine, with CX3CL1, CX3CR1, IL10, CCL2, CXCL12, and CXCR4 mediating bone marrow colonization, CXCL5 and TGFβ promoting tumor cell proliferation, and TGFβ additionally driving bone remodeling. The number of cytokines/cytokine receptors confirmed to mediate spinal metastasis is low compared with the vast spectrum of cytokines/cytokine receptors participating in other parts of the skeleton. Therefore, further research is needed, including validation of the role of cytokines mediating metastases to other bones, to precisely address the unmet clinical need associated with spine metastases.

Topics: Bone Neoplasms; Chemokine CXCL12; Cytokines; Humans; Ligands; Male; Neoplasm Metastasis; Receptors, CXCR4; Receptors, Cytokine; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) mechanism is responsible for metastasis of tumor cells and their spread to various organs and tissues of body, providing undesirable prognosis. In addition to migration, EMT increases stemness and mediates therapy resistance. Hence, pathways involved in EMT regulation should be highlighted. STAT3 is an oncogenic pathway that can elevate growth rate and migratory ability of cancer cells and induce drug resistance. The inhibition of STAT3 signaling impairs cancer progression and promotes chemotherapy-mediated cell death. Present review focuses on STAT3 and EMT interaction in modulating cancer migration. First of all, STAT3 is an upstream mediator of EMT and is able to induce EMT-mediated metastasis in brain tumors, thoracic cancers and gastrointestinal cancers. Therefore, STAT3 inhibition significantly suppresses cancer metastasis and improves prognosis of patients. EMT regulators such as ZEB1/2 proteins, TGF-β, Twist, Snail and Slug are affected by STAT3 signaling to stimulate cancer migration and invasion. Different molecular pathways such as miRNAs, lncRNAs and circRNAs modulate STAT3/EMT axis. Furthermore, we discuss how STAT3 and EMT interaction affects therapy response of cancer cells. Finally, we demonstrate targeting STAT3/EMT axis by anti-tumor agents and clinical application of this axis for improving patient prognosis.

Topics: Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasms; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

TGF-β plays a prominent role as an inducer of epithelial-mesenchymal transitions (EMTs) during development and wound healing and in disease conditions such as fibrosis and cancer. During these processes EMT occurs together with changes in cell proliferation, differentiation, communication, and extracellular matrix remodeling that are orchestrated by multiple signaling inputs besides TGF-β. Chief among these inputs is RAS-MAPK signaling, which is frequently required for EMT induction by TGF-β. Recent work elucidated the molecular basis for the cooperation between the TGF-β-SMAD and RAS-MAPK pathways in the induction of EMT in embryonic, adult and carcinoma epithelial cells. These studies also provided direct mechanistic links between EMT and progenitor cell differentiation during gastrulation or intra-tumoral fibrosis during cancer metastasis. These insights illuminate the nature of TGF-β driven EMTs as part of broader processes during development, fibrogenesis and metastasis.

Topics: Epithelial-Mesenchymal Transition; Fibrosis; Humans; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta

Malignant pleural mesothelioma (MPM) is an aggressive tumor mainly associated with asbestos exposure and is characterized by a very difficult pharmacological approach. One of the molecular mechanisms associated with cancer onset and invasiveness is the epithelial-to-mesenchymal transition (EMT), an event induced by different types of inducers, such as transforming growth factor β (TGFβ), the main inducer of EMT, and oxidative stress. MPM development and metastasis have been correlated to EMT; On one hand, EMT mediates the effects exerted by asbestos fibers in the mesothelium, particularly via increased oxidative stress and TGFβ levels evoked by asbestos exposure, thus promoting a malignant phenotype, and on the other hand, MPM acquires invasiveness via the EMT event, as shown by an upregulation of mesenchymal markers or, although indirectly, some miRNAs or non-coding RNAs, all demonstrated to be involved in cancer onset and metastasis. This review aims to better describe how EMT is involved in driving the development and invasiveness of MPM, in an attempt to open new scenarios that are useful in the identification of predictive markers and to improve the pharmacological approach against this aggressive cancer.

Topics: Biomarkers, Tumor; Epithelial-Mesenchymal Transition; Humans; Mesothelioma, Malignant; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Pleural Neoplasms; RNA, Neoplasm; Transforming Growth Factor beta

A plethora of cytokines are produced in the tumor microenvironment (TME) those play a vital role in cancer prognosis. Though it is completely contextual, cytokines produced from an inflammatory micro-environment can either modulate cancer progression at early stages of tumor development or in later stages cytokine derived cues can in turn control tumor cell invasion and metastasis. Therefore, understanding the crosstalk between the key cytokines regulating cancer prognosis is critical for the development of an effective therapy. In this regard, the role of transforming growth factor-beta (TGF-β) in cancer is controversially discussed in general inhibition of TGF-β promotes de novo tumorigenesis whereas paradoxically, TGF-β can promote malignancy in already established tumors. Another important cytokine, TNF-α have intense crosstalk with TGF-β from the fact that in a non-cancer context, TGF-β promotes fibrosis whereas TNF-α has anti-fibrotic activity. We have recently reported that TGF-β-induced differentiation of epithelial cells to mesenchymal type is suppressed by TNF-α through regulation of cellular homeostatic machinery- autophagy. Moreover, there are also rare reports of synergy between these two cytokines as well. The crosstalk between TGF-β and TNF-α is not only limited to regulating cancer cell differentiation and proliferation but also includes involvement in cell death. In this review, we hence summarize the molecular mechanisms by which these two important cytokines, TGF-β and TNF-α control cancer prognosis.

Topics: Animals; Apoptosis; Autophagy; Cell Death; Cell Differentiation; Cell Proliferation; Cellular Senescence; Cytokines; Epithelial-Mesenchymal Transition; Homeostasis; Humans; Inflammation; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Prognosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Deregulation of the mRNA translational process has been observed during tumorigenesis. However, recent findings have shown that deregulation of translation also contributes specifically to cancer cell spread. During metastasis, cancer cells undergo changes in cellular state, permitting the acquisition of features necessary for cell survival, dissemination, and outgrowth. In addition, metastatic cells respond to external cues, allowing for their persistence under significant cellular and microenvironmental stresses. Recent work has revealed the importance of mRNA translation to these dynamic changes, including regulation of cell states through epithelial-to-mesenchymal transition and tumor dormancy and as a response to external stresses such as hypoxia and immune surveillance. In this review, we focus on examples of altered translation underlying these phenotypic changes and responses to external cues and explore how they contribute to metastatic progression. We also highlight the therapeutic opportunities presented by aberrant mRNA translation, suggesting novel ways to target metastatic tumor cells.

Topics: Carcinogenesis; Cell Movement; Cell Survival; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Phenotype; Protein Biosynthesis; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Escape; Tumor Hypoxia; Tumor Microenvironment

Extracellular signal-regulated kinase (ERK5) is an essential regulator of cancer progression, tumor relapse, and poor patient survival. Epithelial to mesenchymal transition (EMT) is a complex oncogenic process, which drives cell invasion, stemness, and metastases. Activators of ERK5, including mitogen-activated protein kinase 5 (MEK5), tumor necrosis factor α (TNF-α), and transforming growth factor-β (TGF-β), are known to induce EMT and metastases in breast, lung, colorectal, and other cancers. Several downstream targets of the ERK5 pathway, such as myocyte-specific enhancer factor 2c (MEF2C), activator protein-1 (AP-1), focal adhesion kinase (FAK), and c-Myc, play a critical role in the regulation of EMT transcription factors SNAIL, SLUG, and β-catenin. Moreover, ERK5 activation increases the release of extracellular matrix metalloproteinases (MMPs), facilitating breakdown of the extracellular matrix (ECM) and local tumor invasion. Targeting the ERK5 signaling pathway using small molecule inhibitors, microRNAs, and knockdown approaches decreases EMT, cell invasion, and metastases via several mechanisms. The focus of the current review is to highlight the mechanisms which are known to mediate cancer EMT via ERK5 signaling. Several therapeutic approaches that can be undertaken to target the ERK5 pathway and inhibit or reverse EMT and metastases are discussed.

Topics: Animals; Cell Adhesion; Cytoskeleton; Disease Progression; Epithelial-Mesenchymal Transition; Extracellular Matrix; Humans; Mitogen-Activated Protein Kinase 7; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. Late-stage diagnosis with significant tumor burden, accompanied by recurrence and chemotherapy resistance, contributes to this poor prognosis. These morbidities are known to be tied to events associated with epithelial-mesenchymal transition (EMT) in cancer. During EMT, localized tumor cells alter their polarity, cell-cell junctions, cell-matrix interactions, acquire motility and invasiveness and an exaggerated potential for metastatic spread. Key triggers for EMT include the Transforming Growth Factor-β (TGFβ) family of growth factors which are actively produced by a wide array of cell types within a specific tumor and metastatic environment. Although TGFβ can act as either a tumor suppressor or promoter in cancer, TGFβ exhibits its pro-tumorigenic functions at least in part via EMT. TGFβ regulates EMT both at the transcriptional and post-transcriptional levels as outlined here. Despite recent advances in TGFβ based therapeutics, limited progress has been seen for ovarian cancers that are in much need of new therapeutic strategies. Here, we summarize and discuss several recent insights into the underlying signaling mechanisms of the TGFβ isoforms in EMT in the unique metastatic environment of EOCs and the current therapeutic interventions that may be relevant.

Topics: Carcinoma, Ovarian Epithelial; Disease Progression; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Metastasis; Ovarian Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

When treating diseases related primarily to tissue remodeling and fibrosis, it is desirable to regulate TGFβ concentration and modulate its biological effects. The highest cellular concentrations of TGFβ are found in platelets, with about 40% of all TGFβ found in peripheral blood plasma being secreted by them. Therefore, an understanding of the mechanisms of TGFβ secretion from platelets may be of key importance for medicine. Unfortunately, despite the finding that platelets are an important regulator of TGFβ levels, little research has been carried out into the development of platelet-directed therapies that might modulate the TGFβ-dependent processes. Nevertheless, there are some very encouraging reports suggesting that platelet TGFβ may be specifically involved in cardiovascular diseases, liver fibrosis, tumour metastasis, cerebral malaria and in the regulation of inflammatory cell functions. The purpose of this review is to briefly summarize these few, extremely encouraging reports to indicate the state of current knowledge in this topic. It also attempts to better characterize the influence of TGFβ on platelet activation and reactivity, and its shaping of the roles of blood platelets in haemostasis and thrombosis.

Topics: Blood Platelets; Cardiovascular Diseases; Hemostasis; Humans; Inflammation; Liver Cirrhosis; Malaria, Cerebral; Neoplasm Metastasis; Platelet Activation; Thrombosis; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) signalling pathway switches between anti-tumorigenic function at early stages of cancer formation and pro-tumorigenic effects at later stages promoting cancer metastasis. A similar contrasting role has been uncovered for reactive oxygen species (ROS) in pancreatic tumorigenesis. Down-regulation of ROS favours premalignant tumour development, while increasing ROS level in pancreatic ductal adenocarcinoma (PDAC) enhances metastasis. Given the functional resemblance, we propose that ROS-mediated processes converge with the spatial and temporal activation of TGFβ signalling and thereby differentially impact early tumour growth versus metastatic dissemination. TGFβ signalling and ROS could extensively orchestrate cellular processes and this concerted function can be utilized by cancer cells to facilitate their malignancy. In this article, we revisit the interplay of canonical and non-canonical TGFβ signalling with ROS throughout pancreatic tumorigenesis and metastasis. We also discuss recent insight that helps to understand their conflicting effects on different stages of tumour development. These considerations open new strategies in cancer therapeutics.

Topics: Humans; Neoplasm Metastasis; Pancreatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

In the process of cancer EMT, some subgroups of cancer cells simultaneously exhibit both mesenchymal and epithelial characteristics, a phenomenon termed partial EMT (pEMT). pEMT is a plastic state in which cells coexpress epithelial and mesenchymal markers. In squamous cell carcinoma (SCC), pEMT is regulated, and the phenotype is maintained via the HIPPO pathway, NOTCH pathway and TGF-β pathways and by microRNAs, lncRNAs and the cancer microenvironment (CME); thus, SCC exhibits aggressive tumorigenic properties and high stemness, which leads collective migration and therapy resistance. Few studies have reported therapeutic interventions to address cells that have undergone pEMT, and this approach may be an effective way to inhibit the plasticity, drug resistance and metastatic potential of SCC.

Topics: Carcinoma, Squamous Cell; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; MicroRNAs; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Bladder cancer (BC) is the 11th most common diagnosed cancer, and a number of factors including environmental and genetic ones participate in BC development. Metastasis of BC cells into neighboring and distant tissues significantly reduces overall survival of patients with this life-threatening disorder. Recently, studies have focused on revealing molecular pathways involved in metastasis of BC cells, and in this review, we focus on microRNAs (miRNAs) and their regulatory effect on epithelial-to-mesenchymal transition (EMT) mechanisms that can regulate metastasis. EMT is a vital process for migration of BC cells, and inhibition of this mechanism restricts invasion of BC cells. MiRNAs are endogenous non-coding RNAs with 19-24 nucleotides capable of regulating different cellular events, and EMT is one of them. In BC cells, miRNAs are able to both induce and/or inhibit EMT. For regulation of EMT, miRNAs affect different molecular pathways such as transforming growth factor-beta (TGF-β), Snail, Slug, ZEB1/2, CD44, NSBP1, which are, discussed in detail this review. Besides, miRNA/EMT axis can also be regulated by upstream mediators such as lncRNAs, circRNAs and targeted by diverse anti-tumor agents. These topics are also discussed here to reveal diverse molecular pathways involved in migration of BC cells and strategies to target them to develop effective therapeutics.

Topics: Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; MicroRNAs; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Transcription Factors; Transforming Growth Factor beta; Urinary Bladder Neoplasms; Wnt Signaling Pathway

Thyroid cancer has been rapidly increasing in prevalence among humans in last 2 decades and is the most prevalent endocrine malignancy. Overall, thyroid-cancer patients have good rates of long-term survival, but a small percentage present poor outcome. Thyroid cancer aggressiveness is essentially related with thyroid follicular cell loss of differentiation and metastasis. The discovery of oncogenes that drive thyroid cancer (such as RET, RAS, and BRAF), and are aligned in the MAPK/ERK pathway has led to a new perspective of thyroid oncogenesis. The uncovering of additional oncogene-modulated signaling pathways revealed an intricate and active signaling cross-talk. Among these, microRNAs, which are a class of small, noncoding RNAs, expanded this cross-talk by modulating several components of the oncogenic network - thus establishing a new layer of regulation. In this context, TGFβ signaling plays an important role in cancer as a dual factor: it can exert an antimitogenic effect in normal thyroid follicular cells, and promote epithelial-to-mesenchymal transition, cell migration, and invasion in cancer cells. In this review, we explore how microRNAs influence the loss of thyroid differentiation and the increase in aggressiveness of thyroid cancers by regulating the dual function of TGFβ. This review provides directions for future research to encourage the development of new strategies and molecular approaches that can improve the treatment of aggressive thyroid cancer.

Topics: Cell Transformation, Neoplastic; Disease Progression; Humans; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Thyroid Gland; Thyroid Neoplasms; Transforming Growth Factor beta

The epithelial mesenchymal transition (EMT) is a highly complex and dynamic morphogenetic process in which epithelial cells change their cellular characteristics and transform to mesenchymal cells. It plays a key role in tissue remodeling, not only during embryonic development and stem cell biology but also during wound healing, fibrosis, and cancer progression. In EMT, under different extracellular cell signals and stimuli, epithelial cells undergo an orchestrated series of morphological, cellular, biochemical, and molecular changes. The mechanism of cancer cell metastasis is similar to embryonic development, like epithelial cells migrating and differentiating into all tissues of the embryo. In this period, epithelial cells differentiate into mesenchymal cells, migrate, and return to the epithelial cells when necessary. The important cell signaling pathways in embryonic development, such as growth factor receptor tyrosine kinases (FGF, HGF, TGF-α), TGF-β, Wnt, NOTCH, MAPK, JAK/STAT and inflammatory cytokines such as NF-κB, TNF-α, IL6, cathepsin and HIF-1α. All play crucial roles in EMT during cancer cell invasion and metastasis. In cancer progression, similar cell signaling pathways and molecular activation occur in embryonic development; therefore, it is important to understand the molecular mechanisms of cell signaling pathways related to cancer metastasis and improve new diagnostic and therapeutic approaches for resistance to drugs.

Topics: Animals; Biomarkers; Cell Movement; Epithelial-Mesenchymal Transition; Extracellular Space; Gene Expression Regulation; Humans; Neoplasm Metastasis; Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factors; Transforming Growth Factor beta; Wnt Signaling Pathway

Epithelial to mesenchymal transition (EMT) is a process during which cancer cells lose epithelial features, cytoskeletal architecture is re-organized, cell shape changes and cells activate genes that help to define mesenchymal phenotype, what leads to an increased cell motility and dissemination of tumor to distant metastatic sites. This review describes different signaling networks between microRNAs and proteins that regulate EMT in tumor growth. Activation of EMT is mediated via series of paracrine signaling molecules. WNT, TGF-b, NOTCH and Shh signaling pathways play crucial roles in activation of EMT-related transcription factors, such as SNAIL, SLUG, ZEB1/2 or TWIST. Recent data provide evidence that crosstalk between microRNAs, long non-coding RNAs and EMT-transcription factors is crucial event in EMT regulation. MicroRNAs affect also level of proteins responsible for cellular contact, adhesion and cytoskeletal proteins, what induces changes of epithelial to mesenchymal phenotype. Understanding of those signaling networks may help to identify novel biomarkers or develop new treatment strategies based on microRNA therapeutics in future.

Topics: Cell Movement; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasms; Receptors, Notch; Snail Family Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1; Wnt Signaling Pathway; Zinc Finger E-box-Binding Homeobox 1

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

This review aims to provide an overview of recent developments regarding the roles of MMPs in tumour invasion and metastasis. Much of the mortality burden belonging to cancer relates to its ability to invade adjacent tissue and form metastases at distant sites. This would not be possible without remodelling of the ECM, a process which is enabled by the functions of MMPs. Recent studies provide a better understanding of the importance of the biophysical nature of the ECM, how this influences cancer cell motility, and how MMPs act to modify matrix stiffness. The regulation of MMPs and the role of immune cell generated MMPs has also become better understood. All of this provides a framework for the therapeutic targeting of MMPs and recent advances in the development of selective MMPs inhibitors are also reviewed. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Antigens, CD; beta-D-Galactoside alpha 2-6-Sialyltransferase; Extracellular Matrix; Humans; Immune System; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Receptors, G-Protein-Coupled; Sialyltransferases; Terminology as Topic; Thrombospondins; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) is a secreted cytokine that regulates cell proliferation, migration, and the differentiation of a plethora of different cell types. Consistent with these findings, TGF-β plays a key role in controlling embryogenic development, inflammation, and tissue repair, as well as in maintaining adult tissue homeostasis. TGF-β elicits a broad range of context-dependent cellular responses, and consequently, alterations in TGF-β signaling have been implicated in many diseases, including cancer. During the early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inducing cytostasis and the apoptosis of normal and premalignant cells. However, at later stages, when cancer cells have acquired oncogenic mutations and/or have lost tumor suppressor gene function, cells are resistant to TGF-β-induced growth arrest, and TGF-β functions as a tumor promotor by stimulating tumor cells to undergo the so-called epithelial-mesenchymal transition (EMT). The latter leads to metastasis and chemotherapy resistance. TGF-β further supports cancer growth and progression by activating tumor angiogenesis and cancer-associated fibroblasts and enabling the tumor to evade inhibitory immune responses. In this review, we will consider the role of TGF-β signaling in cell cycle arrest, apoptosis, EMT and cancer cell metastasis. In particular, we will highlight recent insights into the multistep and dynamically controlled process of TGF-β-induced EMT and the functions of miRNAs and long noncoding RNAs in this process. Finally, we will discuss how these new mechanistic insights might be exploited to develop novel therapeutic interventions.

Topics: Animals; Carcinogenesis; Epithelial-Mesenchymal Transition; Humans; Immune Evasion; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) is well recognized as playing a double role in tumor progression. Its antitumor role takes place in the early stages of cancer development, when TGF-β acts as a repressor of epithelial tumor growth. In advanced stages of cancer development, TGF-β has a tumor stimulating role, acting concomitantly with the increase of cancer cell migration and metastasis. One of the critical features of cancer cells is their ability to migrate and invade the surrounding tissues leading to metastases in different organs. Cancer cells that leave the tumor to infiltrate neighboring tissues and ultimately overtake a distant organ, need a complex and fine-regulated mechanism to move through the barrier imposed by the extracellular matrix (ECM). Therefore, cancer cells express a set of proteinases which are involved in the degradation and turnover of ECM. In particular, the urokinase type plasminogen activator (uPA) and the uPA cell surface receptor play key cellular roles in the enhancement of cell malignance during tumor progression. In normal cells uPA system is finely regulated, while in tumor cells its expression and activity are dysregulated in a way to enhance cells' invasion capacity during tumor progression. TGF-β strongly regulates uPA in cancer from transcriptional expression to enzyme activity. In turn, uPA participates in the activation of secreted latent TGF-β, thus producing a malicious loop which contributes to tumor progression and metastasis. In this review we will analyze the main molecular mechanisms implicated in uPA regulation by TGF-β. Moreover, the specific roles and interaction between TGF-β and uPA system in cancer cells and their impact on tumorigenesis will be portrayed.

Topics: Animals; Carcinogenesis; Cell Membrane; Cell Movement; Extracellular Matrix; Humans; Neoplasm Metastasis; Neoplasms; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

The transforming growth factor (TGF)-β signaling events are well known to control diverse processes and numerous responses, such as cell proliferation, differentiation, apoptosis, and migration. TGF-β signaling plays context-dependent roles in cancer: in pre-malignant cells TGF-β primarily functions as a tumor suppressor, while in the later stages of cancer TGF-β signaling promotes invasion and metastasis. Recent studies have also suggested that the cross-talk between TGF-β signaling and other signaling pathways, such as Hippo, Wnt, EGFR/RAS, and PI3K/AKT pathways, may substantially contribute to our current understanding of TGF-β signaling and cancer. As a result of the wide-ranging effects of TGF-β, blockade of TGF-β and its downstream signaling components provides multiple therapeutic opportunities. Therefore, the outlook for anti-TGF-β signaling therapy for numerous diseases appears bright and will provide valuable information and thinking on the drug molecular design. In this review, we focus on recent insights into the regulation of TGF-β signaling in cancer metastasis which may contribute to the development of novel cancer-targeting therapies.

Topics: Antineoplastic Agents; Disease Progression; Humans; Models, Biological; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

Current immunotherapy has limited efficacy on metastatic castrate-resistant prostate cancer (mCRPC). We therefore sought to improve the antitumor ability of mCRPC patient-derived CD8. We investigated the role of a novel chimeric antigen receptor T-immunotherapy based on autologous metastatic castrate-resistant prostate cancer patient-derived prostate-specific membrane antigen (PSMA)-specific, transforming growth factor-ß insensitive CD8

Topics: Animals; Antigens, Surface; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Glutamate Carboxypeptidase II; Humans; Immunotherapy; Male; Mice; Mice, Nude; Molecular Targeted Therapy; Neoplasm Metastasis; Prostate-Specific Antigen; Prostatic Neoplasms, Castration-Resistant; Transforming Growth Factor beta; Treatment Outcome

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFβ) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFβ signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFβ signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis.

Topics: Animals; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasms; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Circulating tumor cells (CTCs) are a subset of cancer cells that are shed from the primary or metastatic tumors into the bloodstream. CTCs are responsible for the establishment of blood-borne distant metastases but their rarity, estimated at one CTC per billion blood cells, presents the biggest technical barrier to their functional studies. Recent advances in CTC isolation technology have allowed for the reliable capture of CTCs from the whole blood of cancer patients. The ability to derive clinically relevant information from CTCs isolated through a blood draw allows for the monitoring of active disease, avoiding the invasiveness inherent to traditional biopsy techniques. This review will summarize recent developments in CTC isolation technology; the development of CTC-derived models; the unique molecular characteristics of CTCs at the transcriptomic, genomic, and proteomic levels; and how these characteristics have been correlated to prognosis and therapeutic efficacy. Finally, we will summarize the recent findings on several signaling pathways in CTCs and metastasis. The study of CTCs is central to understanding cancer biology and promises a "liquid biopsy" that can monitor disease status and guide therapeutic management in real time.

Topics: Cell Count; Cell Separation; Epithelial-Mesenchymal Transition; Humans; Liquid Biopsy; Neoplasm Metastasis; Neoplastic Cells, Circulating; Receptor, ErbB-2; Transforming Growth Factor beta; Wnt Signaling Pathway

Metastasis of tumor cells from primary sites of malignancy to neighboring stromal tissue or distant localities entails in several instances, but not in every case, the epithelial-mesenchymal transition (EMT). EMT weakens the strong adhesion forces between differentiated epithelial cells so that carcinoma cells can achieve solitary or collective motility, which makes the EMT an intuitive mechanism for the initiation of tumor metastasis. EMT initiates after primary oncogenic events lead to secondary secretion of cytokines. The interaction between tumor-secreted cytokines and oncogenic stimuli facilitates EMT progression. A classic case of this mechanism is the cooperation between oncogenic Ras and the transforming growth factor β (TGFβ). The power of TGFβ to mediate EMT during metastasis depends on versatile signaling crosstalk and on the regulation of successive waves of expression of many other cytokines and the progressive remodeling of the extracellular matrix that facilitates motility through basement membranes. Since metastasis involves many organs in the body, whereas EMT affects carcinoma cell differentiation locally, it has frequently been debated whether EMT truly contributes to metastasis. Despite controversies, studies of circulating tumor cells, studies of acquired chemoresistance by metastatic cells, and several (but not all) metastatic animal models, support a link between EMT and metastasis, with TGFβ, often being a common denominator in this link. This article aims at discussing mechanistic cases where TGFβ signaling and EMT facilitate tumor cell dissemination.

Topics: Animals; Epithelial-Mesenchymal Transition; Humans; Models, Biological; Neoplasm Metastasis; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Colorectal cancer is one of the most common malignancies and is the second-leading cause of cancer-related death world-wide, which is linked to genetic mutations, epigenetic alterations, and oncogenic signaling activation. MicroRNAs, one of the categories of epigenetics, have been demonstrated significant roles in carcinogenesis and progression through regulating of oncogenic signaling pathways, stem cells, epithelial-mesenchymal transition, and metastasis. This review summarizes the roles of microRNAs in the regulating of Wnt, Ras, TGF-β, and inflammatory signaling pathways, stemness, and epithelial-mesenchymal transition, for carcinogenesis and metastasis in colorectal cancer. Improving our understanding of the mechanisms of regulatory interactions of microRNAs with signaling pathways in colorectal cancer formation and progression will aid in determining the genes responsible for colorectal cancer initiation, progression, metastasis, and recurrence and, finally, in developing personalized approaches for cancer prevention and therapy.

Topics: Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Humans; MicroRNAs; Neoplasm Metastasis; Neoplastic Stem Cells; ras Proteins; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Hairy and Enhancer-of-split related with YRPW motif (Hey) transcription factors are important regulators of stem cell embryogenesis. Clinical relevance shows that they are also highly expressed in malignant carcinoma. Recent studies have highlighted functions for the Hey factors in tumor metastasis, the maintenance of cancer cell self-renewal, as well as proliferation and the promotion of tumor angiogenesis. Pathways that regulate

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Carcinoma; Cell Cycle Proteins; Cell Proliferation; Histone Deacetylases; Humans; Neoplasm Metastasis; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

Wilms tumor is the most common kidney malignancy in children, especially in children aged less than 6 years. Although therapeutic approach has reached successful rates, there is still room for improvement. Considering the tumor microenvironment, cytokines represent important elements of interaction and communication between tumor cells, stroma, and immune cells. In this regard, the transforming growth factor beta (TGF-β) family members play significant functions in physiological and pathological conditions, particularly in cancer. By regulating cell growth, death, and immortalization, TGF-β signaling pathways exert tumor suppressor effects in normal and early tumor cells. Thus, it is not surprising that a high number of human tumors arise due to alterations in genes coding for various TGF-β signaling components. Understanding the ambiguous role of TGF-β in human cancer is of paramount importance for the development of new therapeutic strategies to specifically block the metastatic signaling pathway of TGF-β without affecting its tumor suppressive effect. In this context, this review attempt to summarize the involvement of TGF-β in Wilms tumor.

Topics: Animals; Child, Preschool; Humans; Kidney Neoplasms; Neoplasm Metastasis; Predictive Value of Tests; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment; Wilms Tumor

Colorectal cancer (CRC) is one of the most frequent tumor types in Western countries. Approximately 20 % of patients show metastasis at the time of diagnosis, with the liver being one of the most affected organs. Transforming growth factor-beta (TGF-β) plays a regulatory role not only in the physiology of the normal colon but also in the development of CRC and its metastatic process. In this review, we analyze the molecular mechanisms leading to TGF-β dysregulation in tumor and stroma cells and the modification of the microenvironment that fosters CRC metastasis. Recent genomic studies have identified a CRC subtype with a mesenchymal and aggressive phenotype having TGF-β as a hub gene of this signature. Consistent with these findings, the inhibition of TGF-β signaling has been shown to impair experimental CRC metastasis to the liver. Based on these and other results conducted in various tumor types, the pharmaceutical industry has developed a variety of strategies to target TGF-β. We provide up-to-date information of these therapies, which are currently in preclinical or clinical trials.

Topics: Animals; Colon; Colorectal Neoplasms; Drug Discovery; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplastic Stem Cells; Rectum; Signal Transduction; Transforming Growth Factor beta

Metastasis of malignant cells to vital organs remains the major cause of mortality in many types of cancers. The tumour invasion-metastasis cascade is a stepwise and multistage process whereby tumour cells disseminate from primary sites and spread to colonize distant sites through the systemic haematogenous or lymphatic circulations. The general steps of metastasis may be similar in almost all tumour types, but metastasis to different tissues seems to require distinct sets of regulators and/or an 'educated' microenvironment which may facilitate the infiltration and colonization of tumour cells to specific tissues. Moreover, interactions of tumour cells with stromal cells, endothelial cells, and immune cells that they encounter will also aid them to gain survival advantages, evade immune surveillance, and adapt to the new host microenvironment. Due to the high correlation between tumour metastasis and survival rate of patients, a deeper understanding of the molecular participants and processes involved in metastasis could pave the way towards novel, more effective and targeted approaches to prevent and treat tumour metastasis. In this review, we provide an update on the regulation networks orchestrated by the dominant regulators of different stages throughout the metastatic process including, but not limited to, epithelial-mesenchymal transition in local invasion, resistance to anoikis during migration, and colonization of different distant sites. We also put forward some suggestions and problems concerning the treatment of tumour metastasis that should be solved and/or improved for better therapies in the near future. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Anoikis; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Models, Biological; Neoplasm Metastasis; Neoplasms; Receptors, Notch; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Wnt Signaling Pathway

Transforming growth factor-β (TGF-β) induces a pleiotropic pathway that is modulated by the cellular context and its integration with other signaling pathways. In cancer, the pleiotropic reaction to TGF-β leads to a diverse and varied set of gene responses that range from cytostatic and apoptotic tumor-suppressive ones in early stage tumors, to proliferative, invasive, angiogenic, and oncogenic ones in advanced cancer. Here, we review the knowledge accumulated about the molecular mechanisms involved in the dual response to TGF-β in cancer, and how tumor cells evolve to evade the tumor-suppressive responses of this signaling pathway and then hijack the signal, converting it into an oncogenic factor. Only through the detailed study of this complexity can the suitability of the TGF-β pathway as a therapeutic target against cancer be evaluated.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Movement; Disease Progression; Epigenesis, Genetic; Humans; Killer Cells, Natural; Macrophages; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phosphorylation; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment

Metastasis remains the major driver of mortality in patients with cancer. The multistep metastatic process starts with the dissemination of tumor cells from a primary site and leading to secondary tumor development in an anatomically distant location. Although significant progress has been made in understanding the molecular characteristics of metastasis, many questions remain regarding the intracellular mechanisms governing transition through the various metastatic stages. The runt-related transcription factor 3 (RUNX3) is a downstream effector of the transforming growth factor-β (TGF-β) signaling pathway, and has critical roles in the regulation of cell death by apoptosis, and in angiogenesis, epithelial-to-mesenchymal transition (EMT), cell migration and invasion. RUNX3 functions as a bona fide initiator of carcinogenesis by linking the Wnt oncogenic and TGF-β tumor suppressive pathways. RUNX3 is frequently inactivated in human cancer cell lines and cancer samples by hemizygous deletion of the Runx3 gene, hypermethylation of the Runx3 promoter, or cytoplasmic sequestration of RUNX3 protein. Inactivation of RUNX3 makes it a putative tumor suppressor in human neoplasia. In the present review, we summarize the proposed roles of RUNX3 in metastasis and, when applicable, highlight the mechanism by which they function.

Topics: Apoptosis; Carcinogenesis; Cell Movement; Core Binding Factor Alpha 3 Subunit; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

In recent years, the homeobox gene superfamily has been introduced as a master regulator in downstream target genes related to cell development and proliferation. An indispensable role of this family involved in organogenesis development has been widely demonstrated since expression of Six family led to a distinct increase in development of various organs. These functions of Six family genes are primarily based on structure as well as regulatory role in response to external or internal stimuli. In addition to these roles, mutation or aberrant expression of Six family plays a fundamental role in initiation of carcinogenesis, a multistep process including transformation, proliferation, angiogenesis, migration, and metastasis. This suggests that the Six superfamily members can be considered as novel target molecules to inhibit tumor growth and progression. This review focuses on the structure, function, and mechanisms of the Six family in cancer processes and possible strategies to apply these family members for diagnostic, prognostic, and therapeutic purposes.

Topics: Animals; Carcinogenesis; Cell Cycle; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Homeodomain Proteins; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Nerve Tissue Proteins; Prognosis; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Up-Regulation

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

The epithelial-mesenchymal transition (EMT) is a complex cellular program involved in the progression of epithelial cancers to a metastatic stage. Along this process, epithelial traits are repressed in favor of a motile mesenchymal phenotype. A detailed characterization and monitoring of EMT-related processes is required for the design of screening strategies needed to develop novel antimetastatic therapies. Overexpression of the canonical EMT biomarker vimentin correlates with increased tumor growth and invasiveness, as well as with reduced patient survival across various epithelial cancers. Moreover, recent findings have demonstrated an active role of vimentin in regulating and reorganizing the cellular architecture toward a migratory and invasive phenotype. However, current studies suffer from a lack of appropriate methods to trace the induction and dynamics of vimentin in cell-based assays. Recently, we have reported a novel intrabody (chromobody)-based approach to study the spatiotemporal organization of endogenous vimentin upon induction of EMT by high-content imaging. In this review, we discuss the relevance of the chromobody technology with regard to the visualization of EMT-related processes in living systems. Cancer Res; 76(19); 5592-6. ©2016 AACR.

Topics: Animals; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Transforming Growth Factor beta; Vimentin

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

FKBP51 (FKBP5 Official Symbol) is large molecular weight member of the FK506 binding protein family, a subfamily of the immunophilin proteins. FKBP51 exerts multiple biological functions in the cell, including modulation of steroid hormone response, immune regulation, cell proliferation, regulation of pAkt levels and control of NF-κB activation. Several lines of evidence support a role for this protein in cancer biology, especially in resistance to chemo- and radio-therapy. Recent research studies highlighted functions of FKBP51 in promoting the epithelial to mesenchymal transition (EMT) transdifferentiation program in melanoma. This process, which is classically regulated by Transforming Growth Factor (TGF)-β, enables cancer cells to disseminate from primary tumors and spread to distant locations, acquiring resistance to therapy and self-renewal capability. This last, in turn, is crucial to their subsequent expansion at sites of dissemination. The aim of the present article is to review recent literature data that involve FKBP51 in the mechanisms that switch the TGF-β from a tumor suppressor to a pro-metastatic invader.

Topics: Animals; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Humans; Melanoma; Neoplasm Metastasis; Tacrolimus Binding Proteins; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their morphology and function and gradually transformed into mesenchymal-like cells. It is considered that EMT is the main cause for tumor recurrence and metastasis. Many factors are involved in the regulation of EMT, such as E-cadherin, transforming growth factor-β, Wnt signaling pathway, microRNA and EMT-related transcription factors. This article reviews the research progress on EMT and the involved mechanisms, and thus to provide a new perspective on cancer therapy in the future.

Topics: Cadherins; Epithelial-Mesenchymal Transition; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasms; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Wnt Signaling Pathway

The β6 subunit of the αvβ6 integrin heterodimer has long been an enigma in cancer biology though recent research has provided many new insights into its biology. Collectively, these findings include discovery of the transcriptional, translational and cell biological mechanisms by which β6 acts, the identification of the cellular influences β6 exerts upon the cell proteome, the characterisation of multiple β6-centric pro-metastatic signalling systems and the search for pharmacological therapies (industry and academia) targeted against β6. Once expressional restriction is overcome in early colorectal cancer (CRC), epithelial cell surface restricted αvβ6 can physically interact with, and activate, known oncoproteins, and has the potential to enable the cross-talk through non-canonical signal transduction pathways, resulting in the adoption of an invasive/metastatic phenotype. This recent research has identified numerous interconnections and potential feedback loops, highlighting the fact that the expression of the β6 subunit may initiate a cascade of downstream effects on the CRC cell rather than acting through a single mechanism. We here review these recent studies and postulate that the existence of a cell surface uPAR/αvβ6/TGFβ "metastasome" interactome in/on a proportion of colorectal cancer cells, where β6 expression sequesters and activates multiple systems at the invasive front of tumour lesions, promoting cancer metastasis and hence explaining why β6 has been correlated with reduced patient survival in CRC.

Topics: Antigens, Neoplasm; Cell Movement; Chemokine CXCL12; Colorectal Neoplasms; Eukaryotic Initiation Factor-4E; Humans; Integrins; Neoplasm Metastasis; Promoter Regions, Genetic; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

The epithelial-mesenchymal transition (EMT) is a cellular process though which an epithelial phenotype can be converted into a phenotype of mesenchymal cells. Under physiological conditions EMT is important for embryogenesis, organ development, wound repair and tissue remodeling. However, EMT may also be activated under pathologic conditions, especially in carcinogenesis and metastatic progression. Major signaling pathways involved in EMT include transforming growth factor β(TGF-β), Wnt, Notch, Hedgehog and other signaling pathways. These pathways are related to several transcription factors, including Twist, Smads and zinc finger proteins snail and slug. These interact with each other to provide crosstalk between the relevant signaling pathways. This review lays emphasis on studying the relationship between EMT and signaling pathways in carcinogenesis and metastatic progression.

Topics: Animals; Carcinogenesis; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Neoplasm Metastasis; Neoplasms; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

Epithelial-mesenchymal transition (EMT) is a physiological process that plays an important role in embryonic development and wound healing and is appropriated during pathological conditions including fibrosis and cancer metastasis. EMT can be initiated by a variety of factors, including transforming growth factor (TGF)-β, and is characterized by loss of epithelial features including cell-cell contacts and apicobasal polarity and acquisition of a motile, mesenchymal phenotype. A key feature of EMT is reorganization of the cytoskeleton and recent studies have elucidated regulation mechanisms governing this process. This review describes changes in gene expression patterns of cytoskeletal associated proteins during TGFβ-induced EMT. It further reports TGFβ-induced intracellular signaling cascades that regulate cytoskeletal reorganization during EMT. Finally, it highlights how changes in cytoskeletal architecture during EMT can regulate gene expression, thus further promoting EMT progression.

Topics: Cytoskeleton; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide with a multifactorial, multistep, complex process and poor prognosis. Its early diagnosis and metastasis monitoring are of the utmost importance. Hepatoma tissues synthesize various tumor-related proteins, genes, enzymes, microRNA, etc. and then secrete into the blood. Detections of circulating biomarkers are useful to find tumor at an early stage or monitor metastasis after postoperative treatment. This paper summarizes recent studies of specific biomarkers at early diagnosis or in monitoring metastasis or postoperative recurrence of HCC.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; gamma-Glutamyltransferase; Glypicans; Heat-Shock Proteins; Humans; Insulin-Like Growth Factor II; Isoenzymes; Liver Neoplasms; MicroRNAs; Neoplasm Metastasis; Telomerase; Transforming Growth Factor beta

Soy isoflavones are dietary components for which an association has been demonstrated with reduced risk of prostate cancer (PCa) in Asian populations. However, the exact mechanism by which these isoflavones may prevent the development or progression of PCa is not completely understood. There are a growing number of animal and in vitro studies that have attempted to elucidate these mechanisms. The predominant and most biologically active isoflavones in soy products, genistein, daidzein, equol, and glycetin, inhibit prostate carcinogenesis in some animal models. Cell-based studies show that soy isoflavones regulate genes that control cell cycle and apoptosis. In this review, we discuss the literature relevant to the molecular events that may account for the benefit of soy isoflavones in PCa prevention or treatment. These reports show that although soy isoflavone-induced growth arrest and apoptosis of PCa cells are plausible mechanisms, other chemo protective mechanisms are also worthy of consideration. These possible mechanisms include antioxidant defense, DNA repair, inhibition of angiogenesis and metastasis, potentiation of radio- and chemotherapeutic agents, and antagonism of estrogen- and androgen-mediated signaling pathways. Moreover, other cells in the cancer milieu, such as the fibroblastic stromal cells, endothelial cells, and immune cells, may be targeted by soy isoflavones, which may contribute to soy-mediated prostate cancer prevention. In this review, these mechanisms are discussed along with considerations about the doses and the preclinical models that have been used.

Topics: Angiogenesis Inhibitors; Animals; Antioxidants; Apoptosis; Autophagy; Cell Cycle; Cell Differentiation; DNA Repair; Epigenesis, Genetic; Glycine max; Humans; Insulin-Like Growth Factor I; Isoflavones; Male; MicroRNAs; Neoplasm Metastasis; Neoplastic Stem Cells; Prostaglandins; Prostatic Neoplasms; Protein-Tyrosine Kinases; Radiation-Sensitizing Agents; Receptors, Androgen; Receptors, Estrogen; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

TGF-beta signaling is one of the major pathways controlling cell and tissue behavior not only in homeostasis but also in disease. During tumorigenesis TGF-beta orchestrated processes are key due to its dual role as tumor suppressor and tumor promoter. Important functions of this pathway have been described in a context-dependent manner both in epithelial cancer cells and in the tumor microenvironment during tumor progression. Carcinoma-associated fibroblasts (CAFs) are one of the most abundant stromal cell types in virtually all solid tumors. CAFs favor malignant progression by providing cancer cells with proliferative, migratory, survival and invasive capacities. A complex network of signaling pathways underlying their tumor-promoting properties is beginning to take shape. In this review, we examine current evidence on the emerging mechanisms involving TGF-beta in CAF-mediated cancer progression, and discuss their potential as therapeutic targets.

Topics: Animals; Fibroblasts; Humans; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasms; Paracrine Communication; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Tumor Microenvironment

Targeting prostate cancer metastasis has very high therapeutic potential. Prostate cancer is the second most common cause of cancer death among men in the USA, and death results from the development of metastatic disease. In order to metastasize, cancer cells must complete a series of steps that together constitute the metastatic cascade. Each step therefore offers the opportunity for therapeutic targeting. However, practical limitations have served as limiting roadblocks to successfully targeting the metastatic cascade. They include our still-emerging understanding of the underlying biology, as well as the fact that many of the dysregulated processes have critical functionality in otherwise normal cells. We provide a discussion of the underlying biology, as it relates to therapeutic targeting. Therapeutic inroads are rapidly being made, and we present a series of case studies to highlight key points. Finally, future perspectives related to drug discovery for antimetastatic agents are discussed.

Topics: Antineoplastic Agents; Epithelial-Mesenchymal Transition; Humans; Integrins; Male; Matrix Metalloproteinases; Neoplasm Metastasis; Prostatic Neoplasms; Signal Transduction; 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

Gastric cancer represents an important problem for the public health, being one of the main causes of mortality. At present, it represents the second cause of mortality due to cancer, after the bronchopulmonary cancer in men and the fourth cause of mortality in women. Important progresses have been made in the last couple of years in determining the neoplastic etiopathogenesis, but it cannot be affirmed that the genetic mutations chain, which leads to the appearance of the malignant cell, has been fully understood.

Topics: Apoptosis; Biomarkers, Tumor; Cadherins; Carcinogenesis; Catenins; Cell Proliferation; Cyclins; Female; Helicobacter pylori; Humans; Intercellular Signaling Peptides and Proteins; Male; Microsatellite Repeats; Neoplasm Metastasis; Stomach Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Protein p53

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

Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade downstream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.

Topics: Disease Progression; Endocytosis; Endosomes; Epidermal Growth Factor; Fibroblast Growth Factors; Hepatocyte Growth Factor; Humans; Neoplasm Metastasis; Neoplasms; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

Metastatic dissemination represents the true cause of the malignant character of cancers. Its targeting is much more difficult than that of cell proliferation, because metastasis, like angiogenesis, involves a number of complex interactions between tumour and stroma; the contribution of adhesion and motility pathways is added to that of proliferation and survival pathways. Long distance extension, discontinuous in respect to the primitive tumour, is a major feature of cancer and the main cause of patients' death. Cancer cells use two main dissemination pathways: the lymphatic pathway, leading to the invasion of the lymph nodes draining the organs where the tumour evolves; and the blood pathway, leading to the invasion of distant organs such as liver, brain, bone or lung. Metastasis is inscribed within the properties of the primitive tumour, as shown by the comparative molecular analysis of the primitive tumour and its own metastases: their similarity is always more important than what could be expected from the general activation of "metastasis genes" or the inhibition of "metastasis suppressor genes". Among the signalling pathways involved in metastasis, one can mention the integrin pathway, the transforming growth factor beta (TGFβ) pathway, the chemokine pathway, the dependence receptor pathway and many others. These pathways allow the possibility of therapeutic targeting, thanks to therapeutic antibodies or small molecules inhibiting the kinases involved in these signalling pathways, but not a single properly anti-metastatic drug has yet been proposed: the complexity and the diversity of the processes allowing metastasis emergence, as well as the fact that the activation mechanisms are more often epigenetic than genetic and are generally physiological processes misled by the malignant cell, render especially difficult the therapeutic approach of metastasis.

Topics: Apoptosis Regulatory Proteins; Chemokines; Genes, Tumor Suppressor; Humans; Integrins; Lymphatic Metastasis; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Signal Transduction; Transforming Growth Factor beta

Dissemination of cancer cells to distant organ sites is the leading cause of death due to treatment failure in different types of cancer. Mehlen and Puisieux have reviewed the importance of the development of inappropriate cell survival signaling for various steps in the metastatic process and have noted the particular importance of aberrant cell survival to successful colonization at the metastatic site. Therefore, the understanding of mechanisms that govern cell survival fate of these metastatic cells could lead to the understanding of a new paradigm for the control of metastatic potential and could provide the basis for developing novel strategies for the treatment of metastases. Numerous studies have documented the widespread role of Akt in cell survival and metastasis in colorectal cancer, as well as many other types of cancer. Akt acts as a key signaling node that bridges the link between oncogenic receptors to many essential pro-survival cellular functions, and is perhaps the most commonly activated signaling pathway in human cancer. In recent years, Akt2 and Akt3 have emerged as significant contributors to malignancy alongside the well-characterized Akt1 isoform, with distinct non-overlapping functions. This review is aimed at gaining a better understanding of the Akt-driven cell survival mechanisms that contribute to cancer progression and metastasis and the pharmacological inhibitors in clinical trials designed to counter the Akt-driven cell survival responses in cancer.

Topics: Antineoplastic Agents; Cell Survival; Colorectal Neoplasms; Humans; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Protein Isoforms; Proto-Oncogene Proteins c-akt; 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

Topics: Animals; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Insulin-Like Growth Factor I; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

Smad7 was initially identified as an inhibitor of Transforming growth factor (TGF)-β due mainly to its ability to bind TGF-β receptor type I and prevent TGF-β-associated Smad signaling. More recently, it has been demonstrated that Smad7 can interact with other intracellular proteins and regulate also TGF-β-independent signaling pathways thus making a valid contribution to the neoplastic processes in various organs. In particular, data emerging from experimental studies indicate that Smad7 may differently modulate the course of various tumors depending on the context analyzed. These observations, together with the demonstration that Smad7 expression is deregulated in many cancers, suggest that therapeutic interventions around Smad7 can help interfere with the development/progression of human cancers. In this article we review and discuss the available data supporting the role of Smad7 in the modulation of cancer growth and progression.

Topics: Humans; Neoplasm Metastasis; Neoplasms; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Extracellular matrix (ECM) has both structural and regulatory roles. This update reviews the representative recent developments in diverse aspects of ECM biology relevant to inflammation, tissue destruction, fibrosis, and regeneration.. Biological regulation by ECM is emerging as a major research area, driven by several new directions. Sensing of mechanical cues provided by ECM was found to be crucial in regulating cell differentiation. Transforming growth factor-β (TGF-β) is a pivotal agent in fibrosis and inflammation. A combination of structural biology and cell biology provided novel insights on the mechanisms of its activation by cellular traction and ECM. Improved understanding of how fibrillin microfibrils and associated proteins regulated TGF-β sequestration and activation was achieved by analysis of inherited connective tissue disorders having TGF-β dysregulation as an underlying pathologic mechanism. Insights on microRNA-mediated ECM regulation suggest a key role for miR-29, for which potential therapeutic roles are emerging. Advances in understanding the ECM turnover by proteinases provided novel insights on cell regulation and identified useful disease biomarkers.. As a crucial modulator of cell behavior, ECM has exceptionally strong relevance and translational implications for human disease, opening novel opportunities for mechanistic understanding of disease pathogenesis as well as treatment.

Topics: Animals; Biomarkers; Extracellular Matrix; Humans; Inflammation; Mechanotransduction, Cellular; MicroRNAs; Neoplasm Metastasis; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a potent pleiotropic cytokine that regulates mammalian development, differentiation, and homeostasis in essentially all cell types and tissues. TGF-β normally exerts anticancer activities by prohibiting cell proliferation and by creating cell microenvironments that inhibit cell motility, invasion, and metastasis. However, accumulating evidence indicates that the process of tumorigenesis, particularly that associated with metastatic progression, confers TGF-β with oncogenic activities, a functional switch known as the "TGF-β paradox." The molecular determinants governing the TGF-β paradox are complex and represent an intense area of investigation by researchers in academic and industrial settings. Recent findings link genetic and epigenetic events in mediating the acquisition of oncogenic activity by TGF-β, as do aberrant alterations within tumor microenvironments. These events coalesce to enable TGF-β to direct metastatic progression via the stimulation of epithelial-mesenchymal transition (EMT), which permits carcinoma cells to abandon polarized epithelial phenotypes in favor of apolar mesenchymal-like phenotypes. Attempts to deconstruct the EMT process induced by TGF-β have identified numerous signaling molecules, transcription factors, and microRNAs operant in mediating the initiation and resolution of this complex transdifferentiation event. In addition to its ability to enhance carcinoma cell invasion and metastasis, EMT also endows transitioned cells with stem-like properties, including the acquisition of self-renewal and tumor-initiating capabilities coupled to chemoresistance. Here, we review recent findings that delineate the pathophysiological mechanisms whereby EMT stimulated by TGF-β promotes metastatic progression and disease recurrence in human carcinomas.

Topics: Animals; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; MicroRNAs; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

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

Characterized by immunosuppression regulatory T cells (Tregs) play a key role in maintaining immune tolerance. A growing number of tumours have been found with Tregs accumulating in microenvironment and patients with high density of Tregs in tumour stroma get a worse prognosis, which suggests that Tregs may inhibit anti-tumour immunity in stroma, resulting in a poor prognosis. In this paper, we demonstrate the accumulation of Tregs in tumour stroma and the possible suppressive mechanisms. We also state the immunotherapy that has being used in animal and clinical trials.

Topics: CD8-Positive T-Lymphocytes; Dendritic Cells; Humans; Immune Tolerance; Immunotherapy; Interleukin-10; Killer Cells, Natural; Neoplasm Metastasis; Neoplasms; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment

Transforming growth factor-β (TGF-β) elicits both tumor-suppressive and tumor-promoting functions during cancer progression. Here, we describe the tumor-promoting functions of TGF-β and how these functions play a role in cancer progression. Normal epithelial cells undergo epithelial-mesenchymal transition (EMT) through the action of TGF-β, while treatment with TGF-β and fibroblast growth factor (FGF)-2 results in transdifferentiation into activated fibroblastic cells that are highly migratory, thereby facilitating cancer invasion and metastasis. TGF-β also induces EMT in tumor cells, which can be regulated by oncogenic and anti-oncogenic signals. In addition to EMT promotion, invasion and metastasis of cancer are facilitated by TGF-β through other mechanisms, such as regulation of cell survival, angiogenesis, and vascular integrity, and interaction with the tumor microenvironment. TGF-β also plays a critical role in regulating the cancer-initiating properties of certain types of cells, including glioma-initiating cells. These findings thus may be useful for establishing treatment strategies for advanced cancer by inhibiting TGF-β signaling.

Topics: Animals; Cell Differentiation; Disease Progression; Epithelial-Mesenchymal Transition; Fibroblast Growth Factor 2; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; 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

Transforming growth factor β (TGFβ) is implicated in human malignancy. Tumors may escape the tumor suppressor activity of TGFβ by mutating some of its signaling components. Carcinoma and stromal cells produce high amounts of TGFβ which promotes epithelial-mesenchymal transition (EMT), tumor cell invasiveness and tumor angiogenesis, while suppressing immune responses against the tumor. Thus, TGFβ has tumor suppressive as well as tumor promoting effects supporting metastasis. TGFβ elicits the EMT response by activating complementary signaling cascades that mobilize embryonic transcription factors that reprogram the epithelial cell so that it acquires both progenitor-like, pro-motility and mesenchymal features. Such nuclear reprogramming of carcinoma cells involves epigenetic and transcriptional regulation, the activity of miRNAs, and modulation of RNA splicing and mRNA translation, leading to the expression of key intracellular and membrane proteins together with a large pool of secreted factors that mediate and account for the phenotypic changes that accompany EMT.

Topics: Animals; Epithelial-Mesenchymal Transition; Extracellular Matrix; Gene Regulatory Networks; Humans; Membrane Proteins; MicroRNAs; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Protein Biosynthesis; RNA Splicing; Signal Transduction; Transforming Growth Factor beta

The capacity of the immune system to distinguish foreign from self-antigen, and to subsequently eliminate the threat of disease without injuring the host is crucial for survival. It also serves to defend against tumor formation and progression via a process termed cancer immunosurveillance. Innate and adaptive immune cell types and effector molecules collectively function as extrinsic tumorsuppressor mechanisms. However, tumors may escape immunesurveillance through a variety of mechanisms that create a local microenvironment that is unfavorable for effective tumor immunity. Transforming growth factor β (TGF-β) has pleiotropic effects on the immune system, and is recognized as one of the most potent immunosuppressive agents in facilitating oncogenesis. The TGF-β pathway promotes cancer progression by concomitantly enhancing tumor metastases while inhibiting the protective host immunity. In this review, we discuss mechanisms through which TGF-β interferes with the development of an anti-tumor immunity and potential means through which to circumvent its activity in order to define more effective cancer immunotherapies.

Topics: Adaptive Immunity; Animals; Disease Progression; Humans; Immunity, Innate; Immunologic Surveillance; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) represent the majority of hepatic malignancies and are among the most frequent causes of cancer deaths worldwide with a rising incidence in western countries. Upon progression of liver cancer, the epithelial to mesenchymal transition (EMT) is considered a key process that drives intrahepatic metastasis. EMT is the transformation of epithelial cells to a mesenchymal phenotype exacerbating motility and invasiveness of various epithelial cell types. In this review we focus on EMT in hepatic fibrosis, HCC and CCC that is governed by the transforming growth factor (TGF)-β signaling. This cytokine has been shown to play diverse and conflicting roles in malignant development, acting as a tumor-suppressor in early cancerogenesis but enhancing tumor dissemination in later stages of tumor progression. Importantly, TGF-β can induce EMT in a variety of cancers including HCC and CCC, even though the complex molecular mechanisms underlying this process are not yet fully understood. We aim at collecting recent findings on the impact of TGF-β-induced EMT in liver carcinoma progression and at discussing new insights on promising drugable targets for future therapeutic approaches against CCC and HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cholangiocarcinoma; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Liver Cirrhosis; Liver Neoplasms; Neoplasm Metastasis; Signal Transduction; 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

Transforming growth factor-β (TGFβ) suppresses tumor formation since it inhibits cell growth and promotes apoptosis. However, in advanced cancers TGFβ elicits tumor promoting effects through its ability to induce epithelial-mesenchymal transition (EMT) which enhances invasiveness and metastasis; in addition, TGFβ exerts tumor promoting effects on non-malignant cells of the tumor, including suppression of immune surveillance and stimulation of angiogenesis. TGFβ promotes EMT by transcriptional and posttranscriptional regulation of a group of transcription factors that suppresses epithelial features, such as expression of components of cell junctions and polarity complexes, and enhances mesenchymal features, such as production of matrix molecules and several cytokines and growth factors that stimulate cell migration. The EMT program has certain similarities with the stem cell program. Inducers and effectors of EMT are interesting targets for the development of improved diagnosis, prognosis and therapy of cancer.

Topics: Animals; Cell Adhesion; Cell Nucleus; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Inflammation; MAP Kinase Signaling System; Mice; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Platelet-Derived Growth Factor; RNA Processing, Post-Transcriptional; RNA Splicing; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) system signals via protein kinase receptors and SMAD mediators to regulate a large number of biological processes. Alterations of the TGF-β signalling pathway are implicated in human cancer. Prior to tumour initiation and early during progression, TGF-β acts as a tumour suppressor; however, at later stages, it is often a tumour promoter. Knowledge about the mechanisms involved in TGF-β signal transduction has allowed a better understanding of cancer progression, invasion, metastasis and epithelial-to-mesenchymal transition. Furthermore, several molecular targets with great potential in therapeutic interventions have been identified. This review discusses the TGF-β signalling pathway, its involvement in cancer and current therapeutic approaches.

Topics: Animals; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Mutation; Neoplasm Metastasis; Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment

The molecular mechanisms underlying cancer progression and metastasis are still poorly understood. In recent years, the epithelial-to-mesenchymal transition (EMT), a traditional phenomenon revealed in embryonic development, has been gradually accepted as a potential mechanism underlying cancer progression and metastasis. Many cell signaling pathways involved in development have been shown to contribute to EMT. An increasing number of genetic and epigenetic elements have been discovered, and their cross-talk relationship in EMT remains to be explored. In addition, accumulating experimental evidence suggests that EMT plays a critical role in different aspects of cancer progression, such as metastasis, stem cell traits, and chemoresistance. However, there are some disagreements and debate about these studies, which raise critical questions worthy of further investigation. Solving these questions will lead to a more complete understanding of cancer metastasis. Due to the close relationship of EMT to cancer metastasis and chemoresistance, targeting EMT or reversing EMT is likely to lead to novel therapeutic approaches for the treatment of human cancers.

Topics: Animals; DNA Methylation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Histones; Humans; MicroRNAs; Neoplasm Metastasis; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a biological process that drives polarized, immotile epithelial cells to undergo multiple biochemical changes to acquire a mesenchymal cell phenotype. The characteristic features of EMT are cell apolarity, loss of cellular adhesion, reduced expression of E-cadherin and increased migratory capacity, as well as invasiveness. EMT is a physiological process that is essential for normal embryonic development. Additionally, abnormal activation of EMT contributes to some human pathologies such as tissue fibrosis, cancer cell invasion and metastasis. In both situations, the basic molecular mechanisms are similar, but lead to different effects depending on cell type and biological conditions of the environment. TGF-β is a multifunctional cytokine that controls proliferation, differentiation and other functions in many cell types. It has been found that neoplastic development converts TGF-β into an oncogenic cytokine. It activates various molecular processes, which are engaged in EMT initiation. All that makes TGF-β a key regulator of EMT.  

Topics: Animals; Cadherins; Cell Adhesion; Cell Differentiation; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Mesenchymal Stem Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Precancerous Conditions; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) signalling pathway plays a critical and dual role in the progression of human cancer. During the early phase of tumour progression, TGF-β acts as a tumour suppressor, exemplified by deletions or mutations in the core components of the TGF-β signalling pathway. On the contrary, TGF-β also promotes processes that support tumour progression such as tumour cell invasion, dissemination, and immune evasion. Consequently, the functional outcome of the TGF-β response is strongly context-dependent including cell, tissue, and cancer type. In this review, we describe the molecular signalling pathways employed by TGF-β in cancer and how these, when perturbed, may lead to the development of cancer. Concomitantly with our increased appreciation of the molecular mechanisms that govern TGF-β signalling, the potential to therapeutically target specific oncogenic sub-arms of the TGF-β pathway increases. Indeed, clinical trials with systemic TGF-β signalling inhibitors for treatment of cancer patients have been initiated. However, considering the important role of TGF-β in cardiovascular and many other tissues, careful screening of patients is warranted to minimize unwanted on-target side effects.

Topics: Disease Progression; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Metastatic, rather than primary tumours are responsible for ninety percent cancer deaths. Despite significant advances in the understanding of molecular and cellular mechanisms in tumour metastases, there are limitations in preventive treatment of metastatic tumours. Much evidence arising from laboratory and clinical studies suggests that growth factors and their receptors are implicated in cancer metastases development. We review the origin and production of growth factors and their receptors in all stages of cancer metastases including epithelial-mesenchymal transition, cancer cell invasion and migration, survival within the circulation, seeding at distant organs and metastatic tumour angiogenesis. The functions of growth factors and their receptors are also discussed. This review presents the efforts made in understanding this challenge to aid in the development of new treatment strategies for cancer metastases.

Topics: Angiopoietins; Animals; Apoptosis; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Glucose-6-Phosphate Isomerase; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Interleukin-8; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptor, IGF Type 1; Receptors, Growth Factor; Ribonuclease, Pancreatic; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The most ominous stage of cancer progression is metastasis, or the dissemination of carcinoma cells from the primary site into distant organs. Metastases are often resistant to current extirpative therapies and even the newest biological agents cure only a small subset of patients. Therefore a greater understanding of tumor biology that integrates properties intrinsic to carcinomas with tissue environmental modulators of behavior is needed. In no aspect of tumor progression is this more evident than the acquisition of cell motility that is critical for both escape from the primary tumor and colonization. In this overview, we discuss how this behavior is modified by carcinoma cell phenotypic plasticity that is evidenced by reversible switching between epithelial and mesenchymal phenotypes. The presence or absence of intercellular adhesions mediate these switches and dictate the receptivity towards signals from the extracellular milieu. These signals, which include soluble growth factors, cytokines, and extracellular matrix embedded with matrikines and matricryptines will be discussed in depth. Finally, we will describe a new mode of discerning the balance between epithelioid and mesenchymal movement.

Topics: Cadherins; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Cytokines; Desmosomes; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Gap Junctions; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Integrins; Neoplasm Metastasis; Neoplasms; Phenotype; Signal Transduction; Tight Junctions; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Acquisition of invasive phenotypes of cancer cells is one of the key steps to promote metastasis. It has been reported that fibroblastic cells are involved in enhancement of proliferation and invasion of cancer cells. Thus, it is important to know the molecular mechanisms of EMT (epithelial-mesenchymal transition) for developing diagnosis and cancer therapy. Since TGF-βis a key mediator of EMT and frequently expressed in various tumors, it may regulate not only the EMT of cancer cells as they acquire metastatic properties, but also the EMT of normal epithelial cells that are adjacent to tumors. In this review, we will discuss the EMT induced by TGF-β.

Topics: Epithelial-Mesenchymal Transition; Humans; Molecular Targeted Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Protein Splicing; RNA-Binding Proteins; Signal Transduction; Transforming Growth Factor beta

Cancer stem cells (CSCs) , which are subset of tumor cells resistant to radiation and chemotherapy, are associated with malignant characteristics of tumor and possess both self-renewal ability and pluripotency for tumor formation. In the process of generating non-CSCs from CSCs, gene mutations and epigenetic changes are induced in those cells, resulting in composition of tumor tissue with heterogeneous cell population. CSCs have been recognized as the source of metastatic foci. Epithelial-mesenchymal transition (EMT) is a change in cellular phenotype characterized by the loss of cell-to-cell adhesions and the gain of migratory behaviors,which has been shown to be a critical factor for initiating cancer invasion and metastasis. However, some recent studies suggest that EMT is not essential requirement for tumor invasion and metastasis. Herein, we discuss the biological significance of CSCs and EMT in tumor invasion and metastasis.

Topics: Cell Adhesion; Cell Movement; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Humans; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Transforming Growth Factor beta

During the past few years, Epithelial-Mesenchymal Transition (EMT) has emerged as one of the most hot spots in clinical research. Its existence in human tumors can form the basis for explaining characteristics of cancer progression and metastasis, as well as certain cases of drug resistance and relapses after treatment. These cellular responses are tightly regulated by intracellular signaling pathways evoked by humoral factors that include growth factors, chemokines and cytokines. Indeed, several gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. A deeper understanding of the cellular and molecular basis of these different programs should aid in both the development of better diagnosis methods, as well as of specific treatments for invasive cancer. In this review we set out to summarize recent novel insights into the molecular players underlying EMT and its relation with cancer progression and metastasis.

Topics: Animals; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Matrix Metalloproteinases; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

Pancreatic cancer has high incidence and mortality rates, and effective treatment remains a clinical challenge. As deregulation of the cytokine transforming growth factor beta (TGF-β) contributes to the progression of pancreatic carcinoma, the TGF-β pathway has been targeted using various strategies, including small molecule inhibitors of TGF-βRI, TGF-β-specific neutralizing antibodies and antisense compounds. As increased TGF-β2 levels in serum or tumor tissue of patients with pancreatic cancer correlated with poor prognosis, inhibition of TGF-β2 synthesis via the antisense oligonucleotide trabedersen (AP 12009) is a promising approach.

Topics: Animals; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor beta

Bone is one of the most common organs to be affected in patients with metastatic cancer. These bone metastases are often accompanied by bone destruction, bone fractures, pain, and hypercalcemia. Transforming growth factor-β (TGF-β) is a major bone-derived factor that is released in active form upon osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic cells to secrete factors that further drive osteolytic destruction of the bone adjacent to the tumor, categorizing TGF-β as a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between tumor cells and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. In this review, preclinical and clinical data are evaluated for the potential use of TGF-β inhibitors in clinical practice to treat bone metastases.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Humans; Neoplasm Metastasis; Transforming Growth Factor beta

Human malignant melanoma is highly resistant to chemotherapy and current immunotherapeutic approaches induce long term remission only in the minority of patients. The transforming growth factor-β (TGF-β) has attracted much attention as a therapeutic target because it plays an important and pleiotropic role in melanoma progression. TGF-β is a multifunctional cytokine involved in the regulation of many cellular processes including cell proliferation, differentiation and survival. Resistance to the growth inhibitory effects of TGF-β without alterations of TGF-β signaling molecules is characteristic of cutaneous melanoma. Melanoma produces increasing amounts of TGF-β with disease progression, inhibiting immune responses and providing an optimal microenvironment for undisturbed tumor growth. In addition, TGF-β exerts its tumor promoting functions via direct effects on tumor cell motility and invasiveness and indirectly by modulating tumor stroma and extracellular matrix, supporting angiogenesis and inhibiting immune surveillance. TGF-β acts through multiple intracellular signaling pathways and the outcome of TGF-β signaling is context-dependent. Defining the impact of the different TGF-β signaling pathways on melanoma progression will help to identify suitable therapeutic targets. Here we review the current knowledge of TGF-β in melanoma and discuss recent therapeutic approaches targeting the TGF-β pathway.

Topics: Animals; Humans; Immunosuppression Therapy; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Skin Neoplasms; 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

Transforming growth factor-β (TGF-β) is a multifunctional cytokine, with important roles in maintaining tissue homeostasis. TGF-β signals via transmembrane serine/threonine kinase receptors and intracellular Smad transcriptional regulators. Perturbed TGF-β signaling has been implicated in a large variety of pathological conditions. Increased TGF-β levels have been found in patients with cancer, fibrosis, and systemic sclerosis, and were correlated with disease severity. In cancer, TGF-β mediates tumor invasion and metastasis by affecting both tumor cells and the tumor microenvironment including fibroblast activation and immune suppression. Furthermore, TGF-β is a strong stimulator of extracellular matrix deposition. On the basis of these observations, small molecule inhibitors of the TGF-β receptor kinases, neutralizing antibodies that interfere with ligand?receptor interactions, antisense oligonucleotides reducing TGF-β expression, and soluble receptor ectodomains that sequester TGF-β have been developed to intervene with excessive TGF-β signaling activity in the aforementioned disorders. Here, we review the current state of anti-TGF-β therapy in clinical trials.

Topics: Antibodies, Neutralizing; Fibrosis; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

Topics: Animals; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Ovarian cancer is a highly metastatic disease and the leading cause of death from gynecologic malignancy. Hence, and understanding of the molecular changes associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions. The conversion of an epithelial cell to a mesenchymal cell plays a key role both in the embryonic development and cancer invasion and metastasis. Cells undergoing epithelial-mesenchymal transition (EMT) lose their epithelial morphology, reorganize their cytoskeleton and acquire a motile phenotype through the up- and down-regulation of several molecules including tight and adherent junctions proteins and mesenchymal markers. EMT is believed to be governed by signals from the neoplastic microenvironment including a variety of cytokines and growth factors. In ovarian cancer EMT is induced by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and endothelin-1 (ET-1). Alterations in these cellular pathways candidate them as useful target for ovarian cancer treatment.

Topics: Bone Morphogenetic Protein 4; Cadherins; Cell Differentiation; Endothelin-1; Epidermal Growth Factor; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Mesoderm; Neoplasm Metastasis; Ovarian Neoplasms; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that regulates cell growth and differentiation, apoptosis, cell motility, extracellular matrix production, angiogenesis, and cellular immune responses. TGF-beta demonstrates paradoxical action whereby it can function to suppress early tumorigenesis; however, it can also facilitate malignant transformation and stimulate tumor growth by manipulating a more hospitable environment for tumor invasion and the development of metastases. Given the integral role of TGF-beta in transformation and cancer progression, various components of the TGF-beta signaling pathway offer potentially attractive therapeutic targets for cancer treatment. This review focuses on the role of TGF-beta in cancer and discusses both small and large molecule drugs currently in development that target TGF-beta, its receptor and important down stream steps along its signaling pathway.

Topics: Antineoplastic Agents; Drug Design; Humans; Immunosuppressive Agents; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor beta (TGF-beta) has been studied with regard to the regulation of cell behavior for over three decades. A large body of research has been devoted to the regulation of epithelial cell and derivative carcinoma cell populations in vitro and in vivo. TGF-beta has been shown to inhibit epithelial cell cycle progression and promote apoptosis that together significantly contribute to the tumor suppressive role for TGF-beta during carcinoma initiation and progression. TGF-beta is also able to promote an epithelial to mesenchymal transition that has been associated with increased tumor cell motility, invasion and metastasis. However, it has now been shown that loss of carcinoma cell responsiveness to TGF-beta stimulation can also promote metastasis. Interestingly, enhanced metastasis in the absence of a carcinoma cell response to TGF-beta stimulation has been shown to involve increased chemokine production resulting in recruitment of pro-metastatic myeloid derived suppressor cell (MDSC) populations to the tumor microenvironment at the leading invasive edge. When present, MDSCs enhance angiogenesis, promote immune tolerance and provide matrix degrading enzymes that promote tumor progression and metastasis. Further, the recruitment of MDSC populations in this context likely enhances the classic role for TGF-beta in immune suppression since the MDSCs are an abundant source of TGF-beta production. Importantly, it is now clear that carcinoma-immune cell cross-talk initiated by TGF-beta signaling within the carcinoma cell is a significant determinant worth consideration when designing therapeutic strategies to manage tumor progression and metastasis.

Topics: Animals; Epithelial Cells; Humans; Inflammation; Macrophages; Models, Biological; Monocytes; Myeloid Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

The histologic distinction between bronchioloalveolar carcinoma and other adenocarcinomas is tissue invasion. The clinical importance of lung adenocarcinoma invasion is supported by several recent studies indicating that the risk of death in nonmucinous bronchioloalveolar carcinoma is significantly lower than that of pure invasive tumors and in tumors with greater than 0.5 cm of fibrosis or linear invasion. Using microarray gene expression profiling of human tumors, dysregulation of transforming growth factor-beta signaling was identified as an important mediator of tumor invasion. Subsequent studies showed that the CC chemokine regulated on activation, normal T cell expressed, and presumably secreted was up-regulated in invasive tumors and was required for invasion in cells with repressed levels of the transforming growth factor-beta type II receptor. Taken together, these studies illustrate how information gained from global expression profiling of tumors can be used to identify key pathways and genes mediating tumor growth, invasion, and metastasis.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Chemokine CCL5; Gene Expression Profiling; Genomics; Humans; Lung Neoplasms; Microarray Analysis; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta signalling plays a dichotomous role in tumour progression, acting as a tumour suppressor early and as a pro-metastatic pathway in late-stages. There is accumulating evidence that advanced-stage tumours produce excessive levels of TGF-beta, which acts to promote tumour growth, invasion and colonisation of secondary organs. In light of the pro-metastasis function, many strategies are currently being explored to antagonise the TGF-beta pathway as a treatment for metastatic cancers. Strategies such as using large molecule ligand traps, reducing the translational efficiency of TGF-beta ligands using antisense technology, and antagonising TGF-beta receptor I/II kinase function using small molecule inhibitors are the most prominent methods being explored today. Administration of anti-TGF-beta therapies alone, or in combination with immunosuppressive or cytotoxic therapies, has yielded promising results in the preclinical and clinical settings. Despite these successes, the temporal- and context-dependent roles of TGF-beta signalling in cancer has made it challenging to define patient subgroups that are most likely to respond, and the therapeutic regimens that will be most effective in the clinic. Novel mouse models and diagnostic tools are being developed today to circumvent these issues, which may potentially expedite anti-TGF-beta drug development and clinical application.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Antisense Elements (Genetics); Cell Transformation, Neoplastic; Combined Modality Therapy; Disease Progression; Humans; Mice; Neoplasm Metastasis; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) has seemingly contradictory roles in tumor progression: it can promote metastatic invasion but also act as a tumor suppressor. Recently, two studies have used intravital imaging to unravel the role of TGFbeta at different stages of the metastatic process. TGFbeta promotes single cell motility, which enables invasion into blood vessels. However the activation of TGFbeta signaling is a transient event and is not maintained at distant sites. The downregulation of TGFbeta signaling at secondary sites then permits growth of secondary tumors. In the absence of TGFbeta, cells are restricted to collective movement and lymphatic spread. Here, we discuss these findings and their potential implications.

Topics: Animals; Cell Movement; Humans; Molecular Imaging; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Dysregulated transforming growth factor beta (TGFbeta) signaling is observed in a variety of human cancers. TGFbeta is produced in large quantities by many tumor types and is known to be pro-oncogenic. Therapeutic strategies directed against TGFbeta signaling using neutralizing antibodies and small molecular inhibitors have been developed. However, TGFbeta is also found to function as a tumor suppressor. This switch from a tumor suppressor in premalignant stages of tumorigenesis to a tumor promoter in later stages of the disease poses great challenges in TGFbeta-targeted cancer therapy. It remains unclear what mechanisms underlie the dual role of TGFbeta and what factors mediate the switch. In the past, most work on dissecting underlying mechanisms was focused on differential regulation of signaling pathways by tumor cell autonomous TGFbeta signaling. Recent progress in elucidating TGFbeta effects on host immune/inflammatory reactions in the tumor microenvironment and distant organs brings exciting new perspectives to the field.

Topics: Genes, Tumor Suppressor; Humans; Inflammation; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Alterations in TGFbeta signaling are common in human cancers. TGFbeta has significant impact on tumor initiation and progression. Therapeutic strategies including neutralizing antibodies and small molecular inhibitors have been developed to target TGFbeta signaling. However, TGFbeta can work as both a tumor suppressor and a tumor promoter. A significant challenge to the development of successful TGFbeta antagonism treatment is understanding how and when TGFbeta switches its function from a tumor suppressor to a tumor promoter. Recent studies demonstrate that TGFbeta regulates the infiltration of inflammatory cells and cancer associated fibroblasts into the tumor microenvironment, resulting in changes in signaling cascade in tumor cells. Additionally, TGFbeta exerts systemic immune suppression and significantly inhibits host tumor immune surveillance. Neutralizing TGFbeta in preclinical mouse models enhances CD8+ T-cell and natural killer cell-mediated anti-tumor immune response. This new understanding of TGFbeta signaling in regulation of tumor microenvironment and immune response may provide useful information, particularly for patient selection and inflammation/immune biomarkers for TGFbeta antagonism therapy in clinical trials.

Topics: Animals; Carcinogens; Humans; Immune Tolerance; Inflammation; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

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

The distortion of growth factor signalling is the most important prerequisite in tumour progression. Transforming growth factor-beta (TGFbeta) signalling regulates tumour progression by a tumour cell-autonomous mechanism or through tumour-stroma interaction, and has either a tumour-suppressing or tumour-promoting function depending on cellular context. Such inherent complexity of TGFbeta signalling results in arduous, but promising, assignments for developing therapeutic strategies against malignant tumours. As numerous cellular context-dependent factors tightly maintain the balance of TGFbeta signalling and contribute to the regulation of TGFbeta-induced cell responses, in this Review we discuss how they maintain the balance of TGFbeta signalling and how their collapse leads to tumour progression.

Topics: Animals; Disease Progression; Humans; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta

The two dominant models of carcinogenesis postulate stochastic (clonal evolution) or hierarchic organization of tumor (cancer stem cell model). According to the latter, at the germinal center of tumor evolution is a cancer stem cell (CSC) which, similar to normal adult stem cells, possesses the capacity of self-renewal and a differentiation potential. Over the past few years, compelling evidence has emerged in support of the hierarchic cancer model for many solid tumors including hepatocellular cancers. The CSCs are posited to be responsible not only for tumor initiation but also for the generation of distant metastasis and relapse after therapy. These characteristics are particularly relevant for a multi-resistant tumor entity like human hepatocellular carcinoma and may herald a paradigm shift in the management of this deadly disease. Identification and detailed characterization of liver CSCs is therefore imperative for improving prevention approaches, enhancing early detection, and extending the limited treatment options. Despite the current progress in understanding the contribution of CSCs to the generation of heterogeneity of tumors, the molecular complexity and exact regulation of CSCs is poorly understood. This review focuses on the genetic and epigenetic mechanisms that regulate and define the unique CSC properties with an emphasis on key regulatory pathways of liver CSCs and their clinical significance.

Topics: AC133 Antigen; Aldehyde Dehydrogenase; Antigens, CD; Antigens, Neoplasm; beta Catenin; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Division; Cell Separation; Epigenesis, Genetic; Epithelial Cell Adhesion Molecule; Genes, myc; Glycoproteins; Hedgehog Proteins; Humans; Liver Neoplasms; MicroRNAs; Models, Biological; Neoplasm Metastasis; Neoplastic Stem Cells; Nuclear Proteins; Peptides; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Receptors, Notch; Repressor Proteins; RNA, Neoplasm; Signal Transduction; Thy-1 Antigens; Transforming Growth Factor beta; Wnt Proteins

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 setup of tumorigenesis processes is generally associated with various events leading to abnormal expression of oncogenes and/or tumor suppressor genes. Recently, the expression and/or activity of a range of molecules involved in these processes were reported to require proteolytic processing of their precursor proteins by the serine pro-protein convertases (PCs) in order to mediate their biological functions. These include adhesion molecules, proteases, growth factors, cytokines and their receptors. Since their discovery, the identification of new PCs substrates and specific PCs inhibitors became an attractive strategy in cancer therapy. In this review, we will report the implication of these enzymes and the processing of their substrates in tumor progression and metastasis. Newly reported studies on the potential use of the PCs as new therapeutic targets will be also discussed.

Topics: Animals; Antineoplastic Agents; Humans; Neoplasm Metastasis; Neoplasms; Platelet-Derived Growth Factor; Proprotein Convertases; Protease Inhibitors; Proto-Oncogene Proteins c-sis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

TGFbeta is a pleiotropic cytokine that has regulatory roles during embryonic development and adult tissue homeostasis, and is especially important for epithelial, neural and immune system differentiation and function. An increasing number of literature reports support a role for autocrine and paracrine TGFbeta-mediated processes in cancer. Progress in delineating the effects of TGFbeta in different tumors and stages of cancer has stimulated the development of TGFbeta-targeted therapies. This review describes the causes of TGFbeta malfunction in cancer, the rationale for therapeutic targeting of TGFbeta in human neoplasia, and the molecular strategies to modulate the signal.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Drug Design; Humans; Ligands; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Receptors, Transforming Growth Factor beta; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor beta

Topics: Animals; Carcinoma; Cell Differentiation; Cell Movement; Humans; Melanoma; Models, Biological; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta

Topics: Adenocarcinoma; Bone Morphogenetic Proteins; Cadherins; Claudin-1; Colorectal Neoplasms; Epigenesis, Genetic; Humans; Intercellular Junctions; Membrane Proteins; Neoplasm Metastasis; Neoplastic Processes; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.

Topics: Epigenesis, Genetic; Humans; Melanins; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Nodal Protein; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.

Topics: Animals; Antibodies, Neoplasm; Apoptosis; Carcinogens; Cell Cycle; Cell Transformation, Neoplastic; Disease Models, Animal; Epigenesis, Genetic; Epithelial Cells; Humans; Mesoderm; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Oncogenes; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

Aggressive tumour cells share many characteristics with embryonic progenitors, contributing to the conundrum of tumour cell plasticity. Recent studies using embryonic models of human stem cells, the zebrafish and the chick have shown the reversion of the metastatic phenotype of aggressive melanoma cells, and revealed the convergence of embryonic and tumorigenic signalling pathways, which may help to identify new targets for therapeutic intervention. This Review will summarize the embryonic models used to reverse the metastatic melanoma phenotype, and highlight the prominent signalling pathways that have emerged as noteworthy targets for future consideration.

Topics: Animals; Cell Communication; Cell Differentiation; Cell Line, Tumor; Cell Movement; Chick Embryo; Embryo, Mammalian; Embryo, Nonmammalian; Embryonic Stem Cells; Humans; Melanoma; Models, Animal; Neoplasm Metastasis; Neoplasm Transplantation; Neural Crest; Nodal Protein; Signal Transduction; Transforming Growth Factor beta; Zebrafish

Signaling of transforming growth factor beta (TGF-beta) is mediated through a heteromeric complex of two types of transmembrane receptors and downstream intracellular proteins known as Smads. Alterations of TGF-beta signaling underlie various forms of human cancer and developmental diseases. Human genetic studies have revealed both point mutations and deletions of Smad2 or Smad4 in several types of cancers. However, the role of Smad3 in tumorigenesis is not clear. Recent data indicate that Smad3 also functions as a tumor suppressor by inhibiting cell proliferation and promoting apoptosis. In addition, Smad3 is essential for TGF-beta-mediated immune suppression, and it plays an important role in regulating transcriptional responses that are favorable to metastasis. Therefore, through regulating different transcriptional responses, Smad3 functions as both a negative and positive regulator of carcinogenesis depending on cell type and clinical stage of the tumor.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Humans; Liver Neoplasms; Mice; Mutation; Neoplasm Metastasis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) family members are polypeptides with dual tumor suppressive and oncogenic effects. They signal through serine/threonine kinase receptor complexes, which phosphorylate cytoplasmic mediators, the Smads. Upon phosphorylation, Smads translocate to the nucleus and associate with transcriptional coactivators or corepressors, and regulate the transcriptional activation of various TGF-beta responsive genes. In addition, TGF-beta activates cellular mitogen-activated protein kinase signaling pathways, which crosstalk with Smad signaling and regulate growth, survival and motility of cells. During tumorigenesis, malignantly transformed cells often lose the response to the tumor suppressive effects of TGF-beta, which, in turn, starts to act as an autocrine tumor promoting factor by enhancing cancer invasion and metastasis. In this review, we summarize current view on the role of TGF-beta signaling in tumorigenesis, with emphasis on cancer invasion and metastasis. On the basis of these recent observations, we discuss new therapeutic strategies targeting TGF-beta signaling at distinct levels as a basis for inhibiting tumor growth, angiogenesis, invasion and metastasis.

Topics: Animals; Cell Transformation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Most cancers are characterized by excessive transforming growth factor-beta production by tumors, which can promote tumor growth and mediate epithelial-to-mesenchymal transition. Transforming growth factor-beta also has the ability to overproduce extracellular matrix components in response to injury and other stimuli. There are many strategies undergoing current evaluation for inhibiting the deleterious biological effects of transforming growth factor-beta by disrupting its signaling at various levels. The current review focuses on the recent advances made in this area, and the potential of these strategies in the clinical treatment of cancer and fibrosis.. Four main strategies used most recently for disrupting transforming growth factor-beta signaling are brought into focus in this review: inhibition or sequestration of the transforming growth factor-beta protein ligands, inhibition of transforming growth factor-beta receptor kinase activity, inhibition of SMAD signaling downstream of transforming growth factor-beta kinase activity and restoration of antitumor immunity upon transforming growth factor-beta inhibition. Various techniques currently used to employ these four strategies are discussed.. Several lines of evidence suggest that altered transforming growth factor-beta signaling contributes to tumor progression and metastasis as well as development of fibrosis. Accumulating data from preclinical and clinical studies indicate that antagonizing aberrant transforming growth factor-beta signaling is a promising novel therapeutic approach in cancer and fibrotic disorders.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Disease Progression; Extracellular Matrix; Fibrosis; Gene Expression Regulation, Neoplastic; Humans; Ligands; Models, Biological; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Several common cancers often metastasize to the skeleton in advanced disease. Bone metastases are incurable and cause protracted, severe symptoms. Growth of tumor in bone is driven by a vicious cycle: tumor-secreted factors stimulate bone cells, which in turn release growth factors and cytokines. The bone-derived factors fuel the vicious cycle by acting back on the tumor cells. The vicious cycle offers novel targets for the treatment of advanced cancers. Treatments can inhibit bone cells (osteoclasts and osteoblasts) that are stimulated by tumor-secreted factors. Drugs can also inhibit tumor responses to factors enriched in the bone microenvironment, such as transforming growth factor-beta. Animal models show that these approaches, especially combination treatments, can reduce tumor burden. The results suggest a novel paradigm in which tumor growth can be effectively inhibited by drugs that target cells in the bone microenvironment and not the tumor cells themselves.

Topics: Bone Neoplasms; Forecasting; Humans; Models, Biological; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteolysis; Transforming Growth Factor beta; Tumor Burden

Transforming growth factor-beta (TGF-beta) is a key player in malignant disease through its actions on host tissues and cells. Malignant cells often secrete large amounts of TGF-beta that act on nontransformed cells present in the tumor mass as well as distal cells in the host to suppress antitumor immune responses creating an environment of immune tolerance, augmenting angiogenesis, invasion and metastasis, and increasing tumor extracellular matrix deposition. Cells of the innate immune system contribute to the high concentrations of TGF-beta found in tumor masses. In addition, dendritic cell subpopulations secreting TGF-beta contribute to the generation of regulatory T cells that actively inhibit the activity of other T cells. Elevated levels of plasma TGF-beta are associated with advanced stage disease and may separate patients into prognostically high-risk populations. Anti-TGF-beta therapy could reverse the immunosuppressive effects of this cytokine on the host as well as decrease extracellular matrix formation, decrease angiogenesis, decrease osteolytic activity, and increase the sensitivity of the malignant cells to cytotoxic therapies and immunotherapies. Phase I clinical trials of an inhibitor of TGF-beta receptor type I kinase activity and a TGF-beta neutralizing antibody are under way.

Topics: Animals; Humans; Immune System; Immune Tolerance; Immunosuppressive Agents; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Prognosis; T-Lymphocytes; Transforming Growth Factor beta

Since the cloning in 1997 of SEL1L, the human ortholog of the sel-1 gene of C. elegans, most studies have focused on its role in cancer progression and have provided significant evidences to link its increased expression to a decrease in tumor aggressiveness. SEL1L resides on a "Genome Desert area" on chromosome 14q24.3-31 and is highly conserved in evolution. The function of the SEL1L encoded protein is still very elusive although, several evidences from lower organisms indicate that it plays a major role in protein degradation using the ubiquitin-proteosome system. SEL1L has a very complex structure made up of modules: genomically it consists of 21 exons featuring several alternative transcripts encoding for putative protein isoforms. This structural complexity ensures protein flexibility and specificity, indeed the protein was found in different sub-cellular compartments and may turn on a particular transcript in response to specific stimuli. The overall architecture of SEL1L guarantees an exquisite regulation in the expression of the gene.

Topics: Amino Acid Sequence; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Chromosome Deletion; Chromosomes, Human, Pair 14; Disease Progression; DNA Mutational Analysis; DNA, Neoplasm; Exons; Fetus; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Polymorphism, Genetic; Protein Isoforms; Proteins; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

Topics: Antineoplastic Agents; Apoptosis; Genes, Tumor Suppressor; Humans; Ligands; Models, Biological; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory polypeptide that is the prototypical member of a large family of cytokines that controls many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. The actions of TGF-beta are dependent on several factors including cell type, growth conditions, and the presence of other polypeptide growth factors. One of the biological effects of TGF-beta is the inhibition of proliferation of most normal epithelial cells using an autocrine mechanism of action, and this suggests a tumor suppressor role for TGF-beta. Loss of autocrine TGF-beta activity and/or responsiveness to exogenous TGF-beta appears to provide some epithelial cells with a growth advantage leading to malignant progression. This suggests a pro-oncogenic role for TGF-beta in addition to its tumor suppressor role. During the early phase of epithelial tumorigenesis, TGF-beta inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis. In late stages of tumor progression when tumor cells become resistant to growth inhibition by TGF-beta due to inactivation of the TGF-beta signaling pathway or aberrant regulation of the cell cycle, the role of TGF-beta becomes one of tumor promotion. Resistance to TGF-beta-mediated inhibition of proliferation is frequently observed in multiple human cancers, as are various alterations in the complex TGF-beta signaling and cell cycle pathways. TGF-beta can exert effects on tumor and stromal cells as well as alter the responsiveness of tumor cells to TGF-beta to stimulate invasion, angiogenesis, and metastasis, and to inhibit immune surveillance. Because of the dual role of TGF-beta as a tumor suppressor and pro-oncogenic factor, members of the TGF-beta signaling pathway are being considered as predictive biomarkers for progressive tumorigenesis, as well as molecular targets for prevention and treatment of cancer and metastasis.

Topics: Antineoplastic Agents; Disease Progression; Humans; Neoplasm Metastasis; Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Suppressor Proteins

Transforming growth factor beta (TGF-beta) is a ubiquitous and essential regulator of cellular and physiologic processes including proliferation, differentiation, migration, cell survival, angiogenesis, and immunosurveillance. Alterations in the TGF-beta signaling pathway, including mutation or deletion of members of the signaling pathway and resistance to TGF-beta-mediated inhibition of proliferation are frequently observed in human cancers. Although these alterations define a tumor suppressor role for the TGF-beta pathway in human cancer, TGF-beta also mediates tumor-promoting effects, either through differential effects on tumor and stromal cells or through a fundamental alteration in the TGF-beta responsiveness of the tumor cells themselves. TGF-beta and members of the TGF-beta signaling pathway are being evaluated as prognostic or predictive markers for cancer patients. Ongoing advances in understanding the TGF-beta signaling pathway will enable targeting of this pathway for the chemoprevention and treatment of human cancers.

Topics: Apoptosis; DNA-Binding Proteins; Humans; Neoplasm Metastasis; Neoplasms; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

The molecular mechanisms of epithelial-mesenchymal transformation (EMT) have long been studied to gain a greater understanding of this distinct change in cellular morphology. Early studies of the developing embryo have designated the involvement of Wnt signaling in EMT, through an activated complex of the lymphoid-enhancing factor-1 (LEF-1) transcription factor and the cell adhesion molecule beta-catenin. However, more recent studies have implicated a significant role of the transforming growth factor-beta (TGF-beta) in causing EMT in both development and pathology. The ability of TGF-beta isoforms to signal through a variety of molecules such as Smads, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) creates an incredible complexity as to their role in this transition. Here we assess the biochemical signaling pathways of TGF-beta and their potential cross-interaction with traditional Wnt signaling molecules to bring about EMT during embryogenesis and tumor metastasis.

Topics: Animals; beta Catenin; Cytoskeletal Proteins; DNA-Binding Proteins; Embryonic Development; Epithelium; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lymphoid Enhancer-Binding Factor 1; MAP Kinase Signaling System; Mesoderm; Models, Biological; Neoplasm Metastasis; Neoplasms; Neural Crest; Neurons; Phosphorylation; Signal Transduction; Smad Proteins; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

Members of the integrin family recognize a variety of spatially-restricted extracellular ligands. Classically, ligation of integrins activates cytoplasmic signals in the integrin-expressing cell and contributes to cell adhesion, migration, proliferation and survival. At least two members of this family, alphavbeta6 and alphavbeta8 perform an additional function, activation of latent complexes of transforming growth factor beta. In effect, this process allows integrins on one cell to activate signals on adjacent (in the case of alphavbeta6) or nearby cells (in the case of alphavbeta8). Integrin-mediated TGFbeta activation has been shown to play important roles in modulating tissue fibrosis, acute lung injury and pulmonary emphysema. Given the important roles that TGFbeta plays in modulating epithelial cell growth, epithelial-to-mesenchymal transformation and tumor invasion and metastasis, integrin-mediated TGFbeta activation is likely to play important roles in tumor growth and metastasis.

Topics: Animals; Antigens, Neoplasm; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cytoplasm; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Integrins; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta

TGFbeta1 was initially identified in culture media from transformed cells as part of a factor that could produce a transformed phenotype in a nontransformed cell line. Subsequently this activity was separated into TGFbeta and TGFalpha an EGF receptor ligand. With the discovery that TGFbeta1 was a potent growth inhibitor of epithelial cells, and the identification of inactivating mutations within the TGFbeta1 signaling pathway in cancers it became clear that TGFbeta1 signaling is a tumor suppressor pathway for early stages of cancer. However many human carcinomas overexpress TGFbeta1 and this is associated with poor patient prognosis and increased frequency of metastasis. Similar results have been obtained with tumor cell lines and experimental animal models. Thus stage specific duality of function is the emerging paradigm for the role of TGFbeta1 in cancer. This review will focus on the evidence for TGFbeta1 as a tumor promoting and metastasis factor and examine the biological and molecular basis for these effects. It is proposed that the switch from tumor suppressor to oncogene reflects genetic or epigenetic alterations in signaling pathways in tumor cells that alter the readout from the TGFbeta1 pathway.

Topics: Animals; Carcinoma; Disease Models, Animal; Disease Progression; Humans; Immune Tolerance; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Phenotype; Prognosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Despite major improvements in patient management, the prognosis for patients with lung cancer remains dismal. As our knowledge of the molecular biology of cancers has increased, new targets for therapeutic interventions have been identified. In this article, we discuss some of the more recent developments in this field. They include revisiting some of the established concepts, such as retinoid metabolism and the inhibition of cyclooxygenase-2 metabolism. In addition, newer targets, such as transforming growth factor-beta signaling, Janus-activated kinase/signal transducers and activators of transcription pathway, and cell invasion are discussed. These studies demonstrate that multiple, often overlapping, mechanisms of disruption are present in lung cancer cells, presenting a plethora of molecular targets.

Topics: Cyclooxygenase 2; Humans; Isoenzymes; Lung Neoplasms; Membrane Proteins; Molecular Biology; Neoplasm Invasiveness; Neoplasm Metastasis; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

Stroma cells, together with extracellular matrix components, provide the microenvironment that is pivotal for cancer cell growth, invasion and metastatic progression. Characteristic stroma alterations accompany or even precede the malignant conversion of epithelial cells. Crucial in this process are fibroblasts, also termed myofibroblasts or cancer-associated fibroblasts (CAFs) that are located in the vicinity of the neoplastic epithelial cells. They are able to modify the phenotype of the epithelial cells by direct cell-to-cell contacts, through soluble factors or by modification of extracellular matrix components. Seminal functional studies in various cancer types, including breast, colon, prostate and lung cancer, have confirmed the concept that fibroblasts can determine the fate of the epithelial cell, since they are able to promote malignant conversion as well as to revert tumour cells to a normal phenotype. This review focuses on characteristic changes of fibroblasts in cancer and provides the experimental background elucidating functional properties of CAFs in the carcinogenic process. A possible implication in lung carcinogenesis is emphasised. Finally, a laser-capture- and microarray-based approach is presented, which comprehensively characterises carcinoma-associated fibroblasts in their in vivo environment for the identification of potential targets for anti-cancer therapy.

Topics: Animals; Antineoplastic Agents; Epithelial Cells; Fibroblasts; Humans; Lung Neoplasms; Muscle Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Platelet-Derived Growth Factor; Stromal Cells; Transforming Growth Factor beta

The role of transforming growth factor-beta (TGF-beta) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-beta can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-beta are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-beta signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-beta to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

Topics: Animals; Carcinoma; Cell Transformation, Neoplastic; DNA-Binding Proteins; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Pancreatic cancer ranks fifth as a cause of cancer-related death in the world with an overall 5-year survival rate of less than 1% and a median survival of less than a year after tumour detection. Most of these patients have already metastases at the time of diagnosis. The oncologic strategies such as chemotherapy, radiotherapy, antihormonal modalities or the systemic use of specific monoclonal antibodies have not achieved a significant improvement in the survival of pancreatic cancer patients. Recent studies suggest that alterations in molecular pathways, particularly in growth factor mediated mechanisms, that regulate cell proliferation and differentiation play a pivotal role in the pathogenesis of this cancer. The molecular knowledge regarding changes in the expression of growth factors in pancreatic cancer has the potential to improve diagnostic and therapeutic treatment strategies in the near future.

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Nerve Growth Factor; Pancreatic Neoplasms; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-beta (TGF-beta ) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-Ji-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-beta to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-fl signaling pathway. SKI also facilitates cell-cycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21(Waf-1) This results in increased CDK2 activity, RB phosphorylation,and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 (INK4a) loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition,the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-beta signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

Topics: Animals; Cell Division; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Down-Regulation; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, Mutant Strains; Neoplasm Metastasis; Proto-Oncogene Proteins; Repressor Proteins; Research; Signal Transduction; Transforming Growth Factor beta

The immune system is responsible for the early detection and destruction of newly transformed malignant cells. Some transformed cells become immunologically invisible by passive avoidance of immune surveillance (i.e., when tumor cells are immunologically indistinguishable from normal cells). Other transformed cells actively secrete cytokines that effectively blind the immune system to the presence of abnormal antigens on the tumor cell surface. Transforming growth factor-beta ("TGF-beta"), which is expressed by a majority of malignant tumors, is the most potent immunosuppressor and therefore, the most likely cytokine to be responsible for the latter phenomenon. In addition to playing a key role in tumor-induced immunosuppression, TGF-beta stimulates angiogenesis. Interestingly, tumor cells eventually become refractory to TGF-beta-mediated growth arrest, either due to loss of TGF-beta receptors or due to dysregulation in TGF-beta signaling pathways. Neutralization of TGF-beta or inhibition of its production is an effective method of cancer treatment in variety of animal models. Several agents targeting TGF-beta are in the early stages of development and include anti-TGF-beta antibodies, small molecule inhibitors of TGF-beta, Smad inhibitors and antisense gene therapy. Since tumors may express more than one isoform of TGF-beta, these new drugs should target all three TGF-beta isoforms produced by human tumors. The effects of therapies targeting TGF-beta are likely to be synergistic with cytotoxic chemotherapy and immunotherapy. Reversal of TGF-beta-induced immunosuppression is a new and promising approach to cancer therapy, with potential applications in other diseases such as AIDS.

Topics: Animals; Antibodies, Monoclonal; DNA-Binding Proteins; Humans; Immune Tolerance; Immunotherapy, Active; Neoplasm Metastasis; Neoplasms; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Stromal changes at the invasion front include the appearance of myofibroblasts, cells sharing characteristics with fibroblasts and smooth muscle cells. The main precursors of myofibroblasts are fibroblasts. The transdifferentiation of fibroblasts into myofibroblasts is modulated by cancer cell-derived cytokines, such as transforming growth factor-beta (TGF-beta). TGF-beta causes cancer progression through paracrine and autocrine effects. Paracrine effects of TGF-beta implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts. Autocrine effects of TGF-beta in cancer cells with a functional TGF-beta receptor complex may be caused by a convergence between TGF-beta signalling and beta-catenin or activating Ras mutations. Experimental and clinical observations indicate that myofibroblasts produce pro-invasive signals. Such signals may also be implicated in cancer pain. N-Cadherin and its soluble form act as invasion-promoters. N-Cadherin is expressed in invasive cancer cells and in host cells such as myofibroblasts, neurons, smooth muscle cells, and endothelial cells. N-Cadherin-dependent heterotypic contacts may promote matrix invasion, perineural invasion, muscular invasion, and transendothelial migration; the extracellular, the juxtamembrane and the beta-catenin binding domain of N-cadherin are implicated in positive invasion signalling pathways. A better understanding of stromal contributions to cancer progression will likely increase our awareness of the importance of the combinatorial signals that support and promote growth, dedifferentiation, invasion, and ectopic survival and eventually result in the identification of new therapeutics targeting the stroma.

Topics: Cadherins; Epithelial Cells; Extracellular Matrix; Fibroblasts; Humans; Muscle Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Pain; Receptors, Transforming Growth Factor beta; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Topics: Animals; Female; Genes, Tumor Suppressor; Humans; Mammary Neoplasms, Experimental; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms; Oncogenes; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

In contrast to the aberrant control of proliferation, apoptosis, angiogenesis and lifespan, the cellular mechanisms that cause local invasion and metastasis of tumour cells are still poorly understood. New experimental approaches have identified different types of epithelial-plasticity changes in tumour cells towards fibroblastoid phenotypes as crucial events that occur during metastasis, and many molecules and signalling pathways cooperate to trigger these processes.

Topics: Animals; Cell Culture Techniques; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Gene Expression Profiling; Humans; Neoplasm Metastasis; Phenotype; ras Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor beta

Topics: Apoptosis; Disease Progression; Homeostasis; Humans; Immunosuppression Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Furin catalyses a simple biochemical reaction--the proteolytic maturation of proprotein substrates in the secretory pathway. But the simplicity of this reaction belies furin's broad and important roles in homeostasis, as well as in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever. This review summarizes various features of furin--its structural and enzymatic properties, intracellular localization, trafficking, substrates, and roles in vivo.

Topics: Amino Acid Sequence; Disease; Embryonic and Fetal Development; Endocytosis; Furin; Golgi Apparatus; Homeostasis; Humans; Models, Biological; Molecular Sequence Data; Molecular Structure; Neoplasm Metastasis; Signal Transduction; Subtilisins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

During the past two years, new molecular targets have been discovered which link cell cycle, cell proliferation and cellular growth. It has become more and more evident that whereas gain-of-function mutations in specific genes can lead to cancer, genomic instability plays also an important role in tumour progression. With examples taken from the recent literature, we describe in this short review crucial findings on the molecular mechanisms controlling cell cycle and proliferation. We illustrate how specific combinations of proto-oncogenes alterations can result in tissue-specific tumours. Finally, impairment of the interactions of a cancer cell with its surrounding neighbours is also shown to participate in the progression toward aggressive phenotypes.

Topics: Animals; Antineoplastic Agents, Phytogenic; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Disease Progression; Drug Resistance; Fusion Proteins, bcr-abl; Humans; Mice; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Neural Cell Adhesion Molecules; Paclitaxel; Protein Serine-Threonine Kinases; Radiation Tolerance; Rats; Thrombospondins; Transforming Growth Factor beta; Tuberous Sclerosis

Topics: Animals; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Integrin alphaVbeta3; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta

Transforming growth factor-beta is believed to play a dual role in carcinogenesis. Through its ability to inhibit cellular proliferation it suppresses tumor development in its early stages, but in the course of tumor progression malignant cells often acquire resistance to growth inhibition by transforming growth factor-beta and themselves secrete large amounts of this cytokine. Transforming growth factor-beta furthers malignant progression in two ways: for one, it acts on nontransformed cells present in the tumor mass to suppress antitumor immune responses and to augment angiogenesis. Secondly, it promotes invasion and the formation of metastases in a cell-autonomous manner that requires transforming growth factor-beta signaling activity, albeit at reduced levels, to be present in the tumor cells themselves.

Topics: Cell Transformation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) is an effective and ubiquitous mediator of cell growth. The significance of this cytokine in cancer susceptibility, cancer development and progression has become apparent over the past few years. TGF-beta plays various roles in the process of malignant progression. It is a potent inhibitor of normal stromal, hematopoietic, and epithelial cell growth. However, at some point during cancer development the majority of transformed cells become either partly or completely resistant to TGF-beta growth inhibition. There is growing evidence that in the later stages of cancer development TGF-beta is actively secreted by tumor cells and not merely acts as a bystander but rather contributes to cell growth, invasion, and metastasis and decreases host-tumor immune responses. Subtle alteration of TGF-beta signaling may also contribute to the development of cancer. These various effects are tissue and tumor dependent. Identifying and understanding TGF-beta signaling pathway abnormalities in various malignancies is a promising avenue of study that may yield new modalities to both prevent and treat cancer. The nature, prevalence, and significance of TGF-beta signaling pathway alterations in various forms of human cancer as well as potential preventive and therapeutic interventions are discussed in this review.

Topics: Animals; Cell Division; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

TGF-beta1 is an important regulator of the normal and malignant prostate. In the non-malignant prostate, TGF-beta1 stimulates cell differentiation, inhibits epithelial cell proliferation, and induces epithelial cell death. TGF-beta1 is secreted into semen where it is an important immunosuppressive factor. Prostate cancer cells express high levels of TGF-beta1, which seems to enhance prostate cancer growth and metastasis by stimulating angiogenesis and by inhibiting immune responses directed against tumour cells. Prostate cancer cells frequently lose their TGF-beta receptors and acquire resistance to the anti-proliferative and pro-apoptotic effects of TGF-beta1. Accordingly, high expression of TGF-beta1 and loss of TGF-beta receptor expression have been associated with a particularly bad prognosis in human prostate cancer patients. TGF-beta1 also seems to be a mediator of castration-induced apoptosis in androgen dependent normal and malignant prostate epithelial cells. The ability of some prostate tumours to avoid castration-induced apoptosis may not, however, be simply due to loss of TGF-beta receptor type I or type II expression in the tumour cells. It may also be related to an inability of these cells to up-regulate TGF-beta receptor levels in response to castration or possibly due to defects downstream of the receptors. Short-term therapy-induced changes in the TGF-beta system in prostate tumours can probably be used to predict the long-term response to androgen ablation treatment. Further investigations into the TGF-beta system in the prostate are needed, however, to elucidate how alterations in this system affect the behaviour of prostate tumours, and whether this system can be manipulated for therapeutical purposes.

Topics: Animals; Humans; Immune Tolerance; Male; Neoplasm Metastasis; Neovascularization, Pathologic; Prostatic Neoplasms; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF) beta1 is a potent growth inhibitor, with tumor-suppressing activity. Cancers are often refractile to this growth inhibition either because of genetic loss of TGF-beta signaling components or, more commonly, because of downstream perturbation of the signaling pathway, such as by Ras activation. Carcinomas often secrete excess TGF-beta1 and respond to it by enhanced invasion and metastasis. Therapeutic approaches should aim to inhibit the TGF-beta-induced invasive phenotype, but also to retain its growth-inhibitory and apoptosis-inducing effects.

Topics: Animals; Disease Progression; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta

Malignant cells survive and thrive by expressing growth and invasion 'programs' that many normal cell types recognize and respond to in 'programmed' patterns. An early event in the molecular evolution of many malignancies loss of response to growth control by transforming growth factor-beta (TGF-beta) frequently due to mutation in the type I or type II TGF-beta receptor or a Smad protein. The malignant cells secrete TFG-beta that acts on the host to suppress antitumor immune responses, to enhance extracellular matrix production and to augment angiogenesis. These activities resemble those induced by TGF-beta during embryonic development and account in part for the 'de-differentiated' nature of malignant disease. Clinically, TGF-beta1 is often elevated in the plasma of breast cancer patients, lung cancer patients, hepatocellular carcinoma patients, and prostate cancer patients. Preclinically, several breast cancer models and prostate cancer models in vivo have demonstrated a connection between TGF-beta expression and increased tumorigenicity, increased invasion and drug resistance. In other diseases such as colon, gastric, endometrial, ovarian, and cervical cancers and gliomas and melanoma, loss of response to TGF-beta as a growth inhibitor and increased expression of TGF-beta have been associated with malignant conversion and progression. Elevated levels of TGF-beta are measurable in nude mice bearing a wide variety of human tumor xenografts; thus, these tumor models may serve as useful mimics of the human disease with respect to the TGF-beta pathway. Cancer cure may be approached by blocking several of the major normal pathways used for tumor growth and survival in combination with cytotoxic therapies.

Topics: Animals; Cell Differentiation; Cell Division; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Arteriosclerosis; Cell Cycle; Embryonic and Fetal Development; Fibrosis; Humans; Immune Tolerance; Neoplasm Metastasis; Neoplasms; Signal Transduction; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Topics: Adenocarcinoma; Animals; Disease Progression; Fibroblast Growth Factor 2; Gene Expression; Histocytochemistry; Humans; Male; Neoplasm Metastasis; Neovascularization, Physiologic; Paracrine Communication; Prostatic Neoplasms; RNA, Messenger; Transforming Growth Factor beta

The mouse skin model of chemical carcinogenesis has been very well characterized with respect to epigenetic changes, which occur during tumour cell initiation, promotion and progression. The use of transgenic and gene knock-out mice has contributed greatly to knowledge in this area. The H-ras genetic locus has been shown to undergo multiple genetic changes, including mutagenic activation, amplification of the mutant gene, and loss of the normal allele. These different genetic events lead to thresholds of ras activity which contribute to different stages along the pathway to neoplasia. The genetic and epigenetic events which lead to tumour invasion and metastasis have been less well characterized than studies on tumour initiation and promotion, despite the fact that it is metastases which ultimately kill the animal/patient. In the mouse skin model, loss of p53 contributes to malignant conversion. Gene deletion of the INK4 locus is associated with transformation to a highly invasive spindle cell tumor phenotype. This spindle cell transformation can also be induced in vitro or in vivo by TGF beta 1, possible by synergizing with mutant H-ras. TGF beta can have both positive and negative effects on tumourigenesis, acting early as a tumour suppresser, but later as a stimulator of tumour invasion. It is this latter effect which may be clinically more significant, since many human tumours overexpress TGF beta, yet the majority still retain the intracellular signaling systems necessary for the cell to respond to this growth factor.

Topics: Animals; Cell Transformation, Neoplastic; Disease Progression; Epithelium; Genes, p53; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Skin Neoplasms; Transforming Growth Factor beta

The growth factor TGFbeta (transforming growth factor beta) was initially characterized as a repressor of cellular proliferation. However, studies over the last few years have highlighted another striking property of TGFbeta, which is its capacity to enhance development of the extracellular matrix and formation of metastases from primary tumors. Our understanding of TGFbeta signaling mechanisms has advanced substantially with the identification of the SMAD proteins that transduce TGFbeta signals from the cell membrane to the nucleus where they regulate transcription. Activation of these inducible SMADs occurs through a series of serine phosphorylations mediated by TGFbeta receptors. Other members of the SMAD family act antagonistically downstream of TGFbeta and participate in feedback regulation loops. The fact that members of the TGFbeta family are involved in biological processes as diverse as development, cell proliferation and the immune response can be explained by the intricate regulation of TGFbeta signaling, which involves tissue specificity as well as synergy with distinct signaling pathways. The dual role of TGFbeta as regulator of cellular proliferation and metastasis inducer opens novel possibilities for the use of TGFbeta signaling as a target for cancer therapy.

Topics: Cell Division; DNA-Binding Proteins; Extracellular Matrix; Ligands; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Receptors, Transforming Growth Factor beta; Signal Transduction; Trans-Activators; 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

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

The invasion-suppressor molecule E-cadherin (E-CAD) can be regulated at multiple levels: synthesis, processing and stability of mRNA; synthesis, processing and stability of protein; localization and posttranslational modification of protein; binding to catenins (E-CAD-associated proteins); and size and charge of cell surface glycosaminoglycans. Loss of E-CAD antigen and of E-CAD function in vivo has been observed with cell lines that homogeneously expressed functional E-CAD in vitro. These observations led to the idea that factors in the host may downmodulate E-CAD on the cancer cells, thereby promoting cell invasion. Nude mouse cancers that were homogeneously E-CAD-positive and noninvasive in vitro, formed by epithelioid MDCK or NMuMG cells, stained heterogeneously for E-CAD; such cancers were invasive and metastatic. The in vivo downmodulation appeared to be transient. Ex vivo cultures from primary cancers, as well as from metastases, produced homogeneously E-CAD-positive and noninvasive cells. Downmodulation did not occur when cells were micro-encapsulated and then implanted in the mouse, suggesting a role for immediate cancer cell-host cell contact. Similar in vitro/in vivo/ex vivo experiments with mouse MO4 fibrosarcoma cells, transfected with E-CAD cDNA under the control of a b-actin promotor, showed downregulation at the transcriptional or mRNA stability level. This downregulation was rapidly reversible upon ex vivo culture of the tumor cells. TGF-bl and IGF-I were found, respectively, to downregulate and upregulate the expression or the function of E-CAD. We speculate that IGF-1 restores the function of E-CAD through interaction of the IGF-I tyrosine kinase receptor with the catenin-actin cytoskeletal complex. In human cancers, immunohistochemistry has revealed changes in E-cadherin that agree with the experimental data on transient downmodulation of the invasion-suppressor function of E-cadherin by host factors.

Topics: alpha Catenin; Animals; beta Catenin; Cadherins; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Several steps during cancer progression have been distinguished on the basis of anatomo-pathological observations and experimental data. The first step, which consists of the detachment of the cancer cells from the primary tumor prior to their migration, has received much attention. Several lines of evidence have indicated that inducer molecules of tumor cell dispersion are scatter factors which are similar or identical to some growth factors. Our studies have focused on the dispersing effect of growth factors, such as acidic FGF (aFGF) on a rat bladder carcinoma cell line. These studies demonstrated that specific extracellular matrix components might contribute to the scattering effect of soluble growth factors. Additionally, our results indicated that the dispersing action of aFGF is counterbalanced by its mitogenic effect, since these two functions of aFGF cannot be observed simultaneously for the same cell. Depending on its location in the cell collective, a given cell chooses to enter mitosis or to scatter in response to aFGF. The choice between the two responses is apparently driven by molecules belonging to the transducing pathways of aFGF signaling. Finally, our data indicated that aFGF-induced tumor cell scattering leads to increased in vitro invasiveness and in vivo metastasis. Interestingly, the presence of few aFGF-producing tumor cells in a population of non-producing cells dramatically enhances the growth rate and the metastatic properties of the whole tumor, suggesting that a low proportion of highly metastatic cells in a heterogeneous cell population might modify the behavior of the tumor mass.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Differentiation; Coculture Techniques; Cyclic AMP; Desmosomes; Disease Progression; Epithelium; Fibroblast Growth Factor 1; Fibroblasts; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Recombinant Fusion Proteins; Signal Transduction; Spheroids, Cellular; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Members of the beta 1 subfamily of integrins contribute to cell adhesion, cytoskeletal organization and signal transduction processes. In some transformed cell lines and tumors, a correlation has been established between the level of expression of the alpha 5/beta 1 fibronectin receptor and neoplastic behavior. In other instances, normal and neoplastic tissue differ in beta 1 integrin expression or sub-cellular distribution. The level of expression of beta 1 integrins in tumor cells may affect tumor growth properties in several ways, including: (a) effects on anchorage dependence of growth; (b) direct signaling processes; (c) organization of the extracellular matrix and presentation of matrix bound growth factors; (d) effects on the functions of host defense cells. Thus the interplay between integrin expression and tumor behavior is complex and might be viewed as a series of interactive feedback loops rather than in terms of a straightforward cause and effect relationship.

Topics: Animals; Cell Transformation, Neoplastic; Humans; Integrin beta1; Integrins; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta

Recent studies have begun to elucidate the molecular events involved in the development of ovarian cancer. First, it has been shown that epithelial ovarian cells both produce and have receptors for many peptide growth factors. It is possible that these growth factors may participate in autocrine and paracrine growth-regulatory pathways in these cells. Increased activity of stimulatory factors, eg, transforming growth factor-alpha, or decreased activity of inhibitor factors, eg, transforming growth factor-beta, may facilitate malignant transformation. In addition, it has been shown that ovarian cancer cells often have acquired the ability to degrade extracellular matrix and invade the underlying tissues. Finally, alterations in several oncogenes and tumor-suppressor genes, including HER2/neu, c-myc, and p53, have been found in ovarian cancers. Although exciting insights into the molecular pathology of ovarian cancer have been gained, we remain far from a comprehensive understanding of the biology of this highly lethal disease.

Topics: Female; Genes, Tumor Suppressor; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Oncogenes; Ovarian Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta

Polypeptide growth factors are a diverse group of biological regulators. Because they are fundamentally involved in the cellular processes that are important for transformation and progression to malignancy, alterations in growth factor control and in their signal pathways are often observed in tumor cells. In this review, we consider the participation of growth factors and the mechanisms by which they effect tumor progression, using as examples members of the transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families. We explore the hypothesis that although abrogation of TGF-beta negative growth regulation is necessary for transformation, in the later stages of tumor progression, TGF-beta plays a direct role in the enhancement of invasion and metastasis as an autocrine stimulator of these processes. In addition, we present evidence that demonstrates both the potential and the importance of members of the FGF family in transformation and induction of metastasis. Several models of growth factor regulation of malignancy are presented in which we demonstrate (1) a link between TGF-beta 1 mitogenic stimulation of malignant cells and alterations in the expression of ribonucleotide reductase, a key rate-limiting step in the synthesis of DNA and in cell proliferation; (2) autocrine and/or intracrine FGF mitogenic stimulation of malignant cell proliferation and metastasis; and (3) autocrine TGF-beta regulation of malignant cell locomotion and invasion through elevated proteolytic activity and increased synthesis of hyaluronan and RHAMM, a novel hyaluronan cell surface receptor.

Topics: Animals; Cell Division; Cell Movement; Fibroblast Growth Factors; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Transforming Growth Factor beta

Topics: Animals; Embryo Implantation; Endopeptidases; Female; Low Density Lipoprotein Receptor-Related Protein-1; Macrophage Colony-Stimulating Factor; Neoplasm Metastasis; Receptors, Immunologic; Transforming Growth Factor beta; Trophoblasts

The process of metastasis is not random. Rather, the outcome depends on the interaction of unique metastic cells with different organ microenvironments. Although the ability of some tumor cells to reach and proliferate in the parenchyma of some organs is responsible for the development of site-specific metastasis, the molecular basis of organ-specific metastasis is unclear. This article will address the molecular mechanisms involved in the interaction between favored tumor cells and a compatible organ environment in terms of growth factors and those receptors participating in paracrine and autocrine signal transduction pathways.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factors; Growth Substances; Humans; Mice; Neoplasm Metastasis; Platelet-Derived Growth Factor; Receptor, Insulin; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Receptors, Somatomedin; Signal Transduction; Somatomedins; Transforming Growth Factor beta

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

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

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

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

Activation of coagulation appears to play a role in tumor progression. This report describes the preliminary results of a phase II study using docetaxel plus enoxaparin in 15 patients with stage IV non-small cell lung cancer (NSCLC). Time to progression was the primary endpoint. Several surrogate markers of coagulation and angiogenesis were evaluated. Enoxaparin was administered at a daily dose of 1 mg/kg (subcutaneously). The initial dose of docetaxel was 100 mg/m2, given as a 60 min infusion every 21 days with prophylactic dexamethasone. Eight patients achieved an objective response (53%) and four had stable disease, with a median duration of 3.5 months. The median time to progression was 5 months (range, 2 to >15 months). The median survival was 11 months. The most frequent toxicities were neutropenia and asthenia. No significant bleeding or thrombotic events were observed. Eleven patients had elevated D-dimer plasma levels prior to therapy, and seven of these patients with a response or stable disease had a significant decline of the D-dimer during therapy. There were no consistent changes of the plasma levels of the angiogenic factors, except for transforming growth factor-beta-1 (TGF-beta1). The median baseline level of TGF-beta1 prior to therapy was 34,867 pg/ml. Twelve out of 13 patients who achieved a response or stable disease had a significant reduction of the TGF-beta1 levels during therapy. Enoxaparin in combination with chemotherapy was safe and well tolerated in patients with advanced NSCLC. This preliminary data suggests that enoxaparin may prolong the time to progression, and therefore justify the continuation of this trial.

Topics: Aged; Anticoagulants; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Disease Progression; Docetaxel; Enoxaparin; Female; Humans; Infusions, Intravenous; Injections, Subcutaneous; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neutropenia; Taxoids; Transforming Growth Factor beta; Transforming Growth Factor beta1

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant metastasis is the main cause of death of TNBC patients. Better understanding of the mechanisms underlying TNBC metastasis may lead to new strategies of early diagnosis and more efficient treatment. In our study, we uncovered that the autophagy receptor optineurin (OPTN) plays an unexpected role in TNBC metastasis. Data mining of publicly available data bases revealed that the mRNA level of OPTN in TNBC patients positively correlates with relapse free and distance metastasis free survival. Importantly, in vitro and in vivo models demonstrated that OPTN suppresses TNBC metastasis. Mechanistically, OPTN inhibited the pro-oncogenic transforming growth factor-β (TGFβ) signaling in TNBC cells by interacting with TGFβ type I receptor (TβRI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGFβ gene response signature and expression of proto-typic TGFβ target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGFβ receptor/SMAD signaling and suppresses metastasis in TNBC.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Humans; Membrane Transport Proteins; Neoplasm Metastasis; Neoplasm Recurrence, Local; RNA, Messenger; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

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

Perturbations in transforming growth factor-β (TGF-β) signaling can lead to a plethora of diseases, including cancer. Mutations and posttranslational modifications (PTMs) of the partner of SMAD complexes contribute to the dysregulation of TGF-β signaling. Here, we reported a PTM of SMAD4, R361 methylation, that was critical for SMAD complexes formation and TGF-β signaling activation. Through mass spectrometric, co-immunoprecipitation (Co-IP) and immunofluorescent (IF) assays, we found that oncogene protein arginine methyltransferase 5 (PRMT5) interacted with SMAD4 under TGF-β1 treatment. Mechanically, PRMT5 triggered SMAD4 methylation at R361 and induced SMAD complexes formation and nuclear import. Furthermore, we emphasized that PRMT5 interacting and methylating SMAD4 was required for TGF-β1-induced epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis, and SMAD4 R361 mutation diminished PRMT5 and TGF-β1-induced metastasis. In addition, highly expressed PRMT5 or high level of SMAD4 R361 methylation indicated worse outcomes in clinical specimens analysis. Collectively, our study highlights the critical interaction of PRMT5 and SMAD4 and the roles of SMAD4 R361 methylation for controlling TGF-β signaling during metastasis. We provided a new insight for SMAD4 activation. And this study indicated that blocking PRMT5-SMAD4 signaling might be an effective targeting strategy in SMAD4 wild-type CRC.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Humans; Neoplasm Metastasis; Protein-Arginine N-Methyltransferases; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination

Topics: Adenocarcinoma of Lung; Animals; Cell Cycle; Killer Cells, Natural; Lung Neoplasms; Mice; Neoplasm Metastasis; Neoplasm Recurrence, Local; T-Lymphocytes; Transforming Growth Factor beta

Metastasis is a leading cause of mortality in patients with lung adenocarcinoma. Histone deacetylases have emerged as promising targets for anti-tumor drugs, with histone deacetylase inhibitors (HDACi) being an active area of research. However, the precise mechanisms by which HDACi inhibits lung cancer metastasis remain incompletely understood. In this study, we employed a range of techniques, including qPCR, immunoblotting, co-immunoprecipitation, chromatin-immunoprecipitation, and cell migration assays, in conjunction with online database analysis, to investigate the role of HDACi and HDAC2/YY1 in the process of lung adenocarcinoma migration. The present study has demonstrated that both trichostatin A (TSA) and sodium butyrate (NaBu) significantly inhibit the invasion and migration of lung cancer cells via Histone deacetylase 2 (HDAC2). Overexpression of HDAC2 promotes lung cancer cell migration, whereas shHDAC2 effectively inhibits it. Further investigation revealed that HDAC2 interacts with YY1 and deacetylates Lysine 27 and Lysine9 of Histone 3, thereby inhibiting Cdh1 transcriptional activity and promoting cell migration. These findings have shed light on a novel functional mechanism of HDAC2/YY1 in lung adenocarcinoma cell migration.

Topics: Adenocarcinoma of Lung; Animals; Antigens, CD; Butyric Acid; Cadherins; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Mice; Neoplasm Metastasis; Protein Binding; Transcription, Genetic; Transforming Growth Factor beta; YY1 Transcription Factor

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

Metastasis is the leading cause of cancer-related death, where TGFβ-induced epithelial-mesenchymal transition (EMT) process confers on cancer cells increased metastatic potential. However, the involvement of circRNAs in this process is still obscure. Here, we identify a TGFβ-induced circRNA called circITGB6 as an indispensable factor during the TGFβ-mediated EMT process. circITGB6 is significantly upregulated in metastatic cancer samples and its higher abundance is closely correlated to worse prognosis of colorectal cancer (CRC) patients. Through gain- and loss-of-function assays, circITGB6 is found to potently promote EMT process and tumor metastasis in various models in vitro and in vivo. Mechanistically, circITGB6 enhances the mRNA stability of PDPN, an EMT-promoting gene, by directly interacting with IGF2BP3. Notably, interfering circITGB6 with PEI-coated specific siRNA effectively represses liver metastasis. Therefore, our study reveals the function of a TGFβ-regulated circRNA in tumor metastasis and suggests that targeting circITGB6 is a promising strategy for cancer therapy.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Neoplasm Metastasis; RNA, Circular; Transforming Growth Factor beta

Prolyl 4-hydroxylase subunit alpha 3 (P4HA3) has been known to be associated with a variety of human cancers. However, the role of P4HA3 on colon cancer growth and metastasis is unclear. In this study, we investigated the effect of P4HA3 on the growth and metastasis of colon cancer and its possible molecular mechanism. First of all, we demonstrated that P4HA3 expression was greatly higher in cells and tissues of colon cancer than that in non-tumor tissues and cells, and the prognosis of patients who had higher P4HA3 was distinctively poorer than patients who had lower level of P4HA3. Second, it was shown that P4HA3 knockdown strongly inhibited the migration, proliferation and invasion ability of colon cancer cells. However, P4HA3 over-expression accelerated the abilities. Meanwhile, P4HA3 could promote subcutaneous tumorigenesis in nude mice in vivo. In addition, P4HA3 knockdown significantly decreased mesenchymal markers Vimentin, N-cadherin and Snail expression and increased epithelial marker E-cadherin expression. And conversely, over-expression of P4HA3 produced the opposite effects. In the current study, there was further evidence that down-regulating P4HA3 significantly reduced both TGF-β and its following molecules including p-Smad2 as well as p-Smad3. However, overexpression of P4HA3 showed the opposite effect. In conclusion, this study shows that P4HA3 promotes the human colon cancer growth and metastasis by affecting TGF-β/Smad signaling pathway. P4HA3 may become a new target for early diagnosis, treatment and prognosis assessment of colon cancer.

Topics: Animals; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Procollagen-Proline Dioxygenase; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcriptome; Transforming Growth Factor beta

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

Bone metastasis is closely related to tumor death in prostate cancer (PC). Long noncoding RNA small nucleolar RNA host gene 3 (SNHG3) has been implicated in the initiation and progression of multiple human cancers. Nevertheless, the biological function of SNHG3 in PC has not been elucidated. Our results indicated that SNHG3 was upregulated in bone metastasis-positive PC tissues compared to bone metastasis-negative PC tissues and adjacent normal tissues. High expression of SNHG3 indicates advanced clinicopathological features and predicts poor prognosis in patients with PC. Meanwhile, SNHG3 knockdown suppressed the proliferation, migration, and invasion abilities of PC cells and inhibited PC cell metastasis to the bone. Mechanistically, SNHG3 enhanced the expression of transforming growth factor beta receptor 1 (TGFBR1) and activated transforming growth factor-Beta (TGF-β) signaling by targeting miR-214-3p. Our study demonstrated the novel role of the SNHG3/miR-214-3p/TGF-β axis in tumor growth and bone metastasis in PC, indicating that SNHG3 may act as a biomarker and promising therapeutic target against PC.

Topics: Animals; Bone Neoplasms; Humans; Male; Mice; Neoplasm Metastasis; Neoplasm Proteins; PC-3 Cells; Prostatic Neoplasms; RNA, Long Noncoding; RNA, Neoplasm; Signal Transduction; Transforming Growth Factor beta

Postoperative abdominal infectious complication (AIC) is associated with metastasis in locally advanced gastric cancer (GC) patients after radical gastrectomy. However, the underlying mechanism remains unclear. Herein, we report that neutrophil extracellular traps (NETs), the DNA meshes released by neutrophils in response to infection, could promote GC cells proliferation, invasion, migration and epithelial-mesenchymal transition dependent on TGF-β signaling. Then we model nude mice with cecal puncture without ligation to simulate postoperative AIC and find that NETs in peripheral blood and ascites fluid facilitate GC cells extravasation and implantation into liver and peritoneum for proliferation and metastasis. Notably, TGF-β signaling inhibitor LY 2157299 could effectively impede liver and peritoneal metastasis but not concurrently aggravate sepsis in those AIC-bearing nude mice. These findings implicate that targeting downstream effectors of NETs such as TGF-β signaling might provide potential therapeutic prospect to reduce the risk of GC metastasis.

Topics: Animals; Extracellular Traps; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neutrophils; Stomach Neoplasms; Transforming Growth Factor beta

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-β to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-β reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-β. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL7; Chemokine CXCL5; Colorectal Neoplasms; Humans; Kruppel-Like Transcription Factors; Mesenchymal Stem Cells; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Microenvironment

Gamma synuclein (SNCG) is a neuronal protein that is also aberrantly overexpressed in various types of human cancer. SNCG overexpression promotes cancer invasion and metastasis. However, the mechanisms that drive cancer metastasis upon SNCG expression remain elusive. Elucidation of the mechanisms underlying the promotion of cancer metastasis by SNCG may help discover therapeutic avenues for SNCG-overexpressed cancer. Here, we show that SNCG promotes transforming growth factor-β (TGF-β)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Mechanistically, SNCG promotes p38MAPK phosphorylation by interacting with the MAPK kinase 3/6 (MKK3/6) and prevents their degradation. SNCG knockdown leads to a decrease in TGF-β-induced phosphorylation of MKK3/6; and abrogates the induction of matrix metalloproteinase (MMP)-9 expression by TGF-β and its target gene Twist1. Furthermore, p38MAPK inhibition abrogates the promotion of MMP-9 expression and cancer cell invasion by SNCG. Both p38MAPK and MMP inhibitors can suppress the promotion of cancer cell invasion by SNCG. Finally, overexpression of SNCG in liver cancer cells promotes lung metastasis, which can be suppressed by the p38MAPK inhibitor. Together, our data uncover a previously unknown role of SNCG in promoting TGF-β-MKK3/6-p38MAPK signaling. This study highlights the critical role of p38MAPK in the promotion of cancer metastasis by SNCG, and indicates that p38MAPK inhibitor may serve as a potential therapeutic for SNCG-overexpressed cancer.

Topics: gamma-Synuclein; Humans; MAP Kinase Kinase 3; MAP Kinase Kinase 6; MAP Kinase Signaling System; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Transforming Growth Factor beta

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

Bone-related events caused by breast cancer bone metastasis substantially compromise the survival and quality of life of patients. Because triple-negative breast cancer (TNBC) lacks hormone receptors and Her2-targeted therapeutic options, progress in the treatment of TNBC bone metastasis has been very slow. Intercellular adhesion molecule 1 (ICAM1) is highly expressed in various cancers and plays an important role in tumorigenesis and metastasis. However, the effect and mechanism of ICAM1 in TNBC bone metastasis are still unknown. We found that ICAM1 was highly expressed in TNBC and correlated with prognosis in TNBC patients. Cell lines with high expression of ICAM1 exhibited enhanced bone metastasis in tumor-bearing mice, and silencing ICAM1 expression significantly inhibited bone metastasis in mice. ICAM1 interacted with integrins to activate the epithelial-to-mesenchymal transition program through TGF-β/SMAD signaling, ultimately enhancing cell invasiveness. Therefore, the findings of the present study provide a strong rationale for the application of ICAM1-targeted therapy in TNBC patients with bone metastasis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Integrins; Intercellular Adhesion Molecule-1; Mice; Neoplasm Metastasis; Quality of Life; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

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

This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

Topics: Cell Line; Colorectal Neoplasms; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Humans; Neoplasm Metastasis; Neoplastic Stem Cells; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Metastasis is the primary cause of cancer-related mortality. Metastasis involves a complex multistep process during which individual tumor cells spread primarily through destruction of the endothelial barrier, entering the circulatory system to colonize distant organs. However, the role of the endothelial barrier as the rate-limiting process in tumor metastasis and how these processes affect the regulation of the host microenvironment at the molecular level are poorly understood.. Here, we analyzed differentially expressed genes in breast cancer and lung adenocarcinoma, including metastatic and recurrent specimens, using TCGA dataset. The effects of EMCN on endothelial cells in vitro and in vivo were analyzed by assessing angiogenesis and vascular permeability, respectively. We established a syngeneic mouse model of endothelial cell-specific knockout of EMCN (EMCN. We present a new translational strategy of EMCN as a new key player in tumor lung metastasis by affecting the host microenvironment. These findings could provide a sound theoretical basis for clinical treatment.

Topics: Animals; Disease Models, Animal; Endothelial Cells; Lung; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor-beta (TGF-β) signalling pathway has been reported to be involved in metastasis and at the same time has been considered compellingly an important mediator of epithelial-to-mesenchymal transition (EMT). Besides, EMT process is maintained by zinc-finger E-box-binding homeobox 1 (ZEB1) gene which is induced by TGF-β pathway. TGF-β has been shown to be associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) phenomenon, which is one of the prognostic biomarkers of colorectal cancer (CRC). This study was conducted to determine the link among ZEB1-induced TGF-β, EMAST status and metastasis.. The expression level of ZEB1 was evaluated using quantitative reverse transcription (qRT) real-time PCR in 122 formalin fixed paraffin-embedded tissues of CRC sample with known EMAST status and TGF-β/Smad-dependent pathways. The association among ZEB1 expression, TGF-β signalling pathway, EMAST status and metastatic behaviour was examined.. Our results validated significant association between activated TGF-β signalling pathway and EMAST

Topics: Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Microsatellite Repeats; Neoplasm Metastasis; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Oesophageal squamous cell carcinoma (ESCC) has a relatively unfavourable prognosis due to metastasis and chemoresistance. Our previous research established a comprehensive ESCC database (GSE53625). After analysing data from TCGA database and GSE53625, we found that PLEK2 predicted poor prognosis in ESCC. Moreover, PLEK2 expression was also related to the overall survival of ESCC patients undergoing chemotherapy. Repression of PLEK2 decreased the proliferation, migration, invasion and chemoresistance of ESCC cells in vitro and decreased tumorigenicity and distant metastasis in vivo. Mechanistically, luciferase reporter assay and chromatin immunoprecipitation assay suggested that TGF-β stimulated the process that Smad2/3 binds to the promoter sequences of PLEK2 and induced its expression. RNA-seq suggested LCN2 might a key molecular regulated by PLEK2. LCN2 overexpression in PLEK2 knockdown ESCC cells reversed the effects of decreased migration and invasion. In addition, TGF-β induced the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. Moreover, AKT was phosphorylated in all regulatory processes. This study detected the major role of PLEK2 in driving metastasis and chemoresistance in ESCC by regulating LCN2, which indicates the potential use of PLEK2 as a biomarker to predict prognosis and as a therapeutic target for ESCC.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lipocalin-2; Membrane Proteins; Mice, Inbred BALB C; Neoplasm Metastasis; Oncogenes; Phenotype; Prognosis; Transforming Growth Factor beta; Wound Healing

Aberrant activation of the TGF-β/SMAD signaling pathway is often observed in hepatocellular carcinoma (HCC). Whether lncRNA regulates the TGF-β/SMAD signaling remains largely unknown. Here, we identified an oncogenic lncRNA that was upregulated in HCC and was transcriptionally induced by TGF-β (named lnc-UTGF, lncRNA upregulated by TGF-β). Upon TGF-β stimulation, SMAD2/3 bound to the lnc-UTGF promoter and activated lnc-UTGF expression. In turn, the TGF-β/SMAD signaling was augmented by overexpressing lnc-UTGF, but was inhibited by silencing lnc-UTGF. Mechanism investigations revealed that lnc-UTGF interacted with the mRNAs of SMAD2 and SMAD4 via complementary base-pairing, resulting in enhanced stability of SMAD2/4 mRNAs. These data suggest a novel TGF-β/SMAD/lnc-UTGF positive feedback circuitry. Subsequent gain- and loss-of-function analyses disclosed that lnc-UTGF promoted the migration and invasion of hepatoma cells, and this effect of lnc-UTGF was attenuated by repressing SMAD2/4 expression or by mutating the SMAD2/4-binding sites in lnc-UTGF. Studies using mouse models further confirmed that in vivo metastasis of hepatoma xenografts was inhibited by silencing lnc-UTGF, but was enhanced by ectopic expression of lnc-UTGF. The lnc-UTGF level was positively correlated with the SMAD2/4 levels in xenografts. Consistently, we detected an association of lnc-UTGF upregulation with increase of SMAD2, SMAD4, and their metastasis effector SNAIL1 in human HCC. And high lnc-UTGF level was also significantly associated with enhanced metastasis potential, advanced TNM stages, and worse recurrence-free survival. Conclusion: there exists a lnc-UTGF-mediated positive feedback loop of the TGF-β signaling and its deregulation promotes hepatoma metastasis. These findings may provide a new therapeutic target for HCC metastasis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; HEK293 Cells; Humans; Liver Neoplasms; Mice; Neoplasm Metastasis; Neoplasm Proteins; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

Aberrant expression of transforming growth factor-β1 (TGF-β1) is associated with renal cell carcinoma (RCC) progression by inducing cancer metastasis. However, the downstream effector(s) in TGF-β signaling pathway is not fully characterized. In the present study, the elevation of secreted protein acidic and rich in cysteine (SPARC) as a TGF-β regulated gene in RCC was identified by applying differentially expressed gene analysis and microarray analysis, we further confirmed this result in several RCC cell lines. Clinically, the expression of these two genes is positively correlated in RCC patient specimens. Furthermore, elevated SPARC expression is found in all the subtypes of RCC and positively correlated with the RCC stage and grade. In contrast, SPARC expression is inversely correlated with overall and disease-free survival of patients with RCC, suggesting SPARC as a potent prognostic marker of RCC patient survival. Knocking down SPARC significantly inhibits RCC cell invasion and metastasis both in vitro and in vivo. Similarly, in vitro cell invasion can be diminished by using a specific monoclonal antibody. Mechanistically, SPARC activates protein kinase B (AKT) pathway leading to elevated expression of matrix metalloproteinase-2 that can facilitate RCC invasion. Altogether, our data support that SPARC is a critical role of TGF-β signaling network underlying RCC progression and a potential therapeutic target as well as a prognostic marker.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Matrix Metalloproteinase 2; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Osteonectin; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Treatment Outcome

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

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.

Topics: Animals; Cell Aggregation; Cell Proliferation; Clone Cells; Fibrosis; Hepatic Stellate Cells; Liver; Mice, Inbred NOD; Neoplasm Metastasis; Neoplasms; Organoids; Phenotype; Spleen; Transforming Growth Factor beta

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

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules.

Topics: Computational Biology; Computer Simulation; Extracellular Traps; Humans; In Vitro Techniques; Interleukin-8; Lung Neoplasms; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neutrophils; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

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

Topics: Cell Line, Tumor; DNA-Binding Proteins; Humans; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; RNA-Binding Proteins; Signal Transduction; Smad Proteins; 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 cellular prion protein (PrP

Topics: Animals; B7-H1 Antigen; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; PrPC Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The mechanisms and key factors involved in tumor environments for lung metastasis of CRC are still unclear. Here, using clinical samples from lung metastases of CRC patients, we found that intestinal immune network for IgA production was significantly dysregulated in lung metastases of CRC. Single-cell RNA sequencing discovered a subtype of B cells positive for Erbin, one member of the leucine-rich repeat and PDZ domain (LAP) family, was involved in the lung metastases. Erbin deletion in B cells suppressed lung metastasis of CRC in vivo. And, deletion of Erbin in B cells enhanced the killing effects of CD8

Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Antibodies, Neutralizing; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Immunoglobulin A; Intestines; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Metastasis; Programmed Cell Death 1 Receptor; Receptors, CXCR5; RNA-Seq; Single-Cell Analysis; STAT6 Transcription Factor; Transforming Growth Factor beta

Gastric cancer (GC) metastasis determines the prognosis of patients, and exploring the molecular mechanism of GC metastasis is expected to provide a theoretical basis for clinical treatment. Recent studies have shown that extracellular matrix protein is closely related to GC metastasis. The present study aimed to explore the expression profile and role of COL5A2, as an extracellular matrix protein, in GC.. The expression, overall survival, and progression-free survival data of COL5 family members were extracted from The Cancer Genome Atlas (TCGA) database, respectively. Weighted gene co-expression network analysis of the GSE62229 database was performed out to identify modules and associated genes.. COL5A2 was selected as our research target in the TCGA database, and was also verified in the GSE62229 and GSE15459 datasets. COL5A2 was up-regulated in GC tissues by paraffin immunohistochemistry and RT-qPCR. The prognosis of patients with low COL5A2 expression was better than that of patients with high COL5A2 expression. Scratch and migration experiments showed that knockdown of COL5A2 decreased the migration ability of gastric cancer cells compared with the control group. In vivo, mice with tail vein injection COL5A2 knockdown had fewer and smaller metastatic nodules in liver. GSEA results showed that the TCGA and GSE62229 samples were significantly enriched in several well-known cancer-related pathways, such as the TGF-β, MAPK, and JAK2 signaling pathways.. COL5A2 was most closely related to advanced GC among COL5 family members. High COL5A2 expression is associated with a poor prognosis, and may be a novel therapeutic target for GC.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Collagen Type V; Female; Humans; Janus Kinase 2; Male; MAP Kinase Signaling System; Mice; Middle Aged; Neoplasm Metastasis; Stomach Neoplasms; Survival Analysis; Transforming Growth Factor beta; Up-Regulation

Esophageal adenocarcinoma (EAC) develops from its precursor Barrett's esophagus through intermediate stages of low- and high-grade dysplasia. However, knowledge of genetic drivers and molecular mechanisms implicated in disease progression is limited. Herein, we investigated the effect of Mothers against decapentaplegic homolog 4 (SMAD4) loss on transforming growth factor β (TGF-β) signaling functionality and in vivo tumorigenicity in high-grade dysplastic Barrett's cells.. An in vivo xenograft model was used to test tumorigenicity of SMAD4 knockdown or knockout in CP-B high-grade dysplastic Barrett's cells. RT. We found that SMAD4 knockout significantly alters the TGF-β pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2, and CDC6, whereas the CDKN2A/B tumor-suppressor locus was regulated negatively. We verified that SMAD4 in combination with CDC6-CDKN2A/B or CRYAB genetic alterations in patient tumors have significant predictive value for poor prognosis. Importantly, we investigated the effect of SMAD4 inactivation in Barrett's tumorigenesis. We found that genetic knockdown or knockout of SMAD4 was sufficient to promote tumorigenesis in dysplastic Barrett's esophagus cells in vivo. Progression to invasive EAC was accompanied by distinctive and consistent copy number alterations in SMAD4 knockdown or knockout xenografts.. Altogether, up-regulation of oncogenes, down-regulation of tumor-suppressor genes, and chromosomal instability within the tumors after SMAD4 loss implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett's toward EAC.

Topics: Animals; Barrett Esophagus; Base Sequence; Carcinogenesis; Cell Line; Down-Regulation; Gene Dosage; Genes, Tumor Suppressor; Humans; Mice; Neoplasm Metastasis; Oncogenes; Principal Component Analysis; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

Poorly differentiated colorectal cancer (CRC) is characterized by aggressive invasion and stromal fibroblast activation, which results in rapid progression and poor therapeutic consequences. However, the regulatory mechanism involved remains unclear. Here, we showed that ZNF37A, a member of KRAB-ZFP family, was upregulated in poorly differentiated CRCs and associated with tumor metastasis. ZNF37A enhanced the metastatic potential of multiple CRC cell lines and promoted distant metastasis in an orthotopic CRC model. Further investigation attributed the ZNF37A-exacerbated metastasis to increased extracellular TGF-β and the consequent activation of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME). Mechanistically, ZNF37A formed a complex with KAP1 and bound to the promoter of THSD4, a TME modulator, to suppress its transcription, which is required for ZNF37A-mediated TGF-β activation and CRC metastasis. Collectively, our study indicates that ZNF37A promotes TGF-β signaling in CRC cells and activates CAFs by transcriptionally repressing THSD4 to drive CRC metastasis, implicating ZNF37A as a potential biomarker for CRC differentiation and progression.

Topics: ADAM Proteins; Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Progression; Heterografts; Humans; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Serine Endopeptidases; Survival Rate; 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

Nuclear pore membrane protein 121 (POM121) is a novel biomarker involved in tumorigenesis and metastasis. However, little is known about the role of POM121 in non-small-cell lung cancer (NSCLC).. The aim of this study was to detect the expression of POM121 in NSCLC and its relationship with clinicopathologic feature and cell biological behavior, and explore the underlying mechanisms.. The expression of POM121 in NSCLC tissues and para-carcinoma tissues was compared by quantitative real-time PCR and immunohistochemistry analysis. The relationship between POM121 protein and clinicopathological characteristics in NSCLC was investigated. Roles of POM121 in NSCLC cells were investigated by CCK-8 assay, clone formation assay, transwell migration and invasion assay, and in vivo experiments. Variations of signaling pathways were determined by qRT-PCR and Western blot.. The POM121 expression in NSCLC tissues was significantly higher than that in para-carcinoma tissues, both at the mRNA and protein level. The POM121 expression was related to sex, advanced differentiation, tumor diameter, lymph node metastases, distant metastases, American Joint Committee on Cancer (AJCC) stage, venous invasion, and perineural invasion in NSCLC. Kaplan-Meier analysis indicated that NSCLC patients with high POM121 expression had poor overall survival. Downregulation of POM121 inhibited cell proliferation, clone formation, migration and invasion. TGF-β/SMAD and PI3K/AKT pathways were involved in POM121-induced functional changes in NSCLC cells.. POM121 plays an oncogenic role in NSCLC through TGF-β/SMAD and PI3K/AKT pathways. POM121 expression is a potential independent prognostic factor for NSCLC.

Topics: Aged; Animals; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Smad Proteins; Transfection; Transforming Growth Factor beta

Cancer is a second leading cause of death worldwide, and metastasis is the major cause of cancer-related mortality. The epithelial-mesenchymal transition (EMT), known as phenotypic change from epithelial cells to mesenchymal cells, is a crucial biological process during development. However, inappropriate activation of EMT contributes to tumor progression and promoting metastasis; therefore, inhibiting EMT is considered a promising strategy for developing drugs that can treat or prevent cancer. In the present study, we investigated the anti-cancer effect of bakuchiol (BC), a main component of Ulmus davidiana var. japonica, in human cancer cells using A549, HT29 and MCF7 cells. In MTT and colony forming assay, BC exerted cytotoxicity activity against cancer cells and inhibited proliferation of these cells. Anti-metastatic effects by BC were further confirmed by observing decreased migration and invasion in TGF-β-induced cancer cells after BC treatment. Furthermore, BC treatment resulted in increase of E-cadherin expression and decrease of Snail level in Western blotting and immunofluorescence analysis, supporting its anti-metastatic activity. In addition, BC inhibited lung metastasis of tail vein injected human cancer cells in animal model. These findings suggest that BC inhibits migration and invasion of cancers by suppressing EMT and in vivo metastasis, thereby may be a potential therapeutic agent for treating cancers.

Topics: Animals; Antineoplastic Agents; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Mice, SCID; Neoplasm Metastasis; Neoplasms; Phenols; Plant Bark; Plant Extracts; Plant Roots; Snail Family Transcription Factors; Transforming Growth Factor beta; Ulmus; Xenograft Model Antitumor Assays

Immune checkpoint inhibitors (ICIs) have changed the treatment paradigm of metastatic urothelial carcinoma (mUC), a dominant type of bladder cancer (BC). Previous studies have shown an association between gene mutations in the DNA damage response (DDR) pathway and the immunotherapy response in mUC but have neglected the effect of the activation level of the DDR pathway on the ICI response in mUC. A published immunotherapy cohort with genome, transcriptome and survival data for 348 mUC patients was used. An external cohort (The Cancer Genome Atlas Bladder Cancer) and the GSE78220 cohort were used for validation. The activation level of the DDR pathway was quantified using single-sample gene set enrichment analysis (ssGSEA). Further analysis on the genome, immunogenicity, and the immune microenvironment was conducted using the DDR ssGSEA enrichment score-high (DSSH) group and the DDR ssGSEA enrichment score-low (DSSL) group. In the mUC cohorts, the DSSH group was associated with longer overall survival times (P=0.026; Hazard ratio=0.67; 95%CI: 0.46-0.95). The DSSH group was also associated with higher tumor mutation burden, neoantigen load, immune-activated cell patterns, and immune-related gene expression levels. The GSEA results indicated an immune activation state in DSSH group, which correlated with a down-regulation in the transforming growth factor β receptor signaling pathway. Our study suggests that the activation level of the DDR pathway may be a novel predictive marker for immunotherapy efficacy in patients with mUC.

Topics: Carcinoma; Databases, Genetic; DNA Damage; DNA Repair; Gene Expression Regulation, Neoplastic; Humans; Immune Checkpoint Inhibitors; Neoplasm Metastasis; Signal Transduction; Transcriptome; Transforming Growth Factor beta; Treatment Outcome; Tumor Microenvironment; Urinary Bladder Neoplasms; Urothelium

Bladder cancer (BC) is known as a common and lethal urinary malignancy worldwide. Circular RNAs (circRNAs), an emerging non-coding RNA, participate in carcinogenesis process of several cancers including BC. In this study, high-throughput sequencing and RT-qPCR were applied to discover and validate abnormal high expression of circUBE2K in BC tissues. Fluorescence in situ hybridization (FISH) was used to detect hsa_circ_0009154 (circUBE2K) expression and subcellular localization in BC tissues. High circUBE2K predicted unfavorable prognoses in BCs, as well as correlated with clinical features. CCK8, transwell, EdU and wound healing assays demonstrated down-regulating circUBE2K decreased BC cell phenotype as proliferation, invasion, and migration, respectively. Further studies showed that circUBE2K promoted BC progression via sponging miR-516b-5p and enhancing ARHGAP5 expression through regulating RhoA activity. Dual-luciferase reporter, FISH and RNA pulldown assays were employed to verify the relationships among circUBE2K/miR-516b-5p/ARHGAP5/RhoA axis. Down-regulating miR-516b-5p or overexpressing ARHGAP5 restored RhoA activity mediated BC cell properties after silencing circUBE2K. Subcutaneous xenograft and metastasis model identified circUBE2K significantly increased BC cell metastasis and proliferation in-vivo. Taken together, we found that circUBE2K is a tumor-promoting circRNA in BC that functions as a ceRNA to regulate ARHGAP5 expression via sponging miR-516b-5p.

Topics: Base Sequence; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Silencing; GTPase-Activating Proteins; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; RNA, Circular; Signal Transduction; Transforming Growth Factor beta; Tumor Burden; Up-Regulation; Urinary Bladder Neoplasms

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-β-induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin-dependent transcription in malignant tumor progression of breast cancer.

Topics: Animals; Apoptosis; beta Catenin; Cell Adhesion; Cell Cycle; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Transcriptome; Transforming Growth Factor beta; Wnt3A Protein

The primary cause of colorectal cancer (CRC) recurrence is increased distant metastasis after radiotherapy, so there is a need for targeted therapeutic approaches to reduce the metastatic-relapse risk. Dysregulation of the cell-surface glycoprotein podocalyxin-like protein (PODXL) plays an important role in promoting cancer-cell motility and is associated with poor prognoses for many malignancy types. We found that CRC cells exposed to radiation demonstrated increased TGFβ and PODXL expressions, resulting in increased migration and invasiveness due to increased extracellular matrix deposition. In addition, both TGFβ and PODXL were highly expressed in tissue samples from radiotherapy-treated CRC patients compared to those from patients without this treatment. However, it is unclear whether TGFβ and PODXL interactions are involved in cancer-progression resistance after radiation exposure in CRC. Here, using CRC cells, we showed that silencing PODXL blocked radiation-induced cell migration and invasiveness. Cell treatment with galunisertib (a TGFβ-pathway inhibitor) also led to reduced viability and migration, suggesting that its clinical use may enhance the cytotoxic effects of radiation and lead to the effective inhibition of CRC progression. Overall, the results demonstrate that downregulation of TGFβ and its-mediated PODXL may provide potential therapeutic targets for patients with radiotherapy-resistant CRC.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Cell Survival; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Prognosis; Pyrazoles; Quinolines; Radiation, Ionizing; RNA Interference; RNA, Small Interfering; Sialoglycoproteins; Transforming Growth Factor beta; Up-Regulation; Vimentin

Early findings that PLK1 is highly expressed in cancer have driven an exploration of its functions in metastasis. However, whether PLK1 induces metastasis in vivo and its underlying mechanisms in NSCLC have not yet been determined. Here, we show that the expression of active PLK1 phosphorylated at T210, abundant in TGF-β-treated lung cells, potently induced metastasis in a tail-vein injection model. Active PLK1 with intact polo-box and ATP-binding domains accelerated cell motility and invasiveness by triggering EMT reprogramming, whereas a phosphomimetic version of p-S137-PLK1 did not, indicating that the phosphorylation status of PLK1 may determine the cell traits. Active PLK1-driven invasiveness upregulated TGF-β signaling and TSG6 encoded by TNFAIP6. Loss of TNFAIP6 disturbed the metastatic activity induced by active PLK1 or TGF-β. Clinical relevance shows that PLK1 and TNFAIP6 are strong predictors of poor survival rates in metastatic NSCLC patients. Therefore, we suggest that active PLK1 promotes metastasis by upregulating TGF-β signaling, which amplifies its metastatic properties by forming a positive feedback loop and that the PLK1/TGF-β-driven metastasis is effectively blocked by targeting PLK1 and TSG6, providing PLK1 and TSG6 as negative markers for prognostics and therapeutic targets in metastatic NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Databases, Genetic; Epithelial-Mesenchymal Transition; Feedback, Physiological; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Phosphorylation; Polo-Like Kinase 1; Prognosis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Signal Transduction; Survival Rate; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer cells undergo significant lipid metabolic reprogramming to ensure sufficient energy supply for survival and progression. However, how cancer cells integrate lipid metabolic signaling with cancer progression is not well understood. In the present study, we demonstrated that C/EBPδ, a critical lipid metabolic regulator, is a TGF-β1 downstream gene and promotes lung adenocarcinoma metastasis. Importantly, C/EBPδ caused significant oscillations in both lipid metabolic and epithelial to mesenchymal transition (EMT) gene networks. Mechanistically, we demonstrated that C/EBPδ recruited oncogene NCOA3 to transcriptionally activate Slug, a canonical EMT transcription factor, which in turn induced oxLDL receptor-1 (Lox1) expression and enhanced oxLDL uptake to promote cancer metastasis, which could be blocked with LOX1 neutralizing antibody. In summary, our results unveiled a previously unappreciated interplay between lipid metabolic and metastatic program, as well as the existence of a pivotal C/EBPδ-Slug-Lox1 transcription axis to promote oxLDL levels and cancer metastasis.

Topics: Adenocarcinoma of Lung; Animals; CCAAT-Enhancer-Binding Protein-delta; Cell Line, Tumor; Cell Movement; Cell Survival; Databases, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lipoproteins, LDL; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplastic Cells, Circulating; Nuclear Receptor Coactivator 3; Scavenger Receptors, Class E; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Inflammatory microenvironment is an important factor for promoting cancer invasion and metastasis, but the underlying molecular mechanisms remain unclear. Here, we mimicked an inflammatory microenvironment both in vitro and in vivo and investigated its effects on the invasion and metastasis of colon cancer. Moreover, colon cancer patient samples were also analyzed statistically. Conditioned medium from the differentiated macrophages induced invasion and migration of colon cancer cells in vitro, which could be reversed by the treatment of a neutralizing anti-growth differentiation factor 15 (GDF15) antibody, indicating GDF15 involvement in inflammation-induced invasiveness. Also, we observed similar effects of human recombinant GDF15 on colon cancer cells. Mechanistically, GDF15 activated c-Fos by separating it from Lamin A/C, increasing transcriptional activity of c-Fos and regulating EMT gene expressions. However, c-Fos knockdown using lentivirus shRNA plasmid inhibited GDF15-triggered invasion and migration in vitro. In vivo, inflammation caused by lipopolysaccharides obviously increased GDF15 secretion, and c-Fos knockdown reduced the lung metastasis of colon cancer cells in mice model. In addition, c-Fos expressions in patient samples were found to be associated with colon cancer metastasis and TNM stages. Taken together, GDF15 in inflammatory microenvironment induces colon cancer invasion and metastasis by regulating EMT genes by activating c-Fos, which might be a potential therapeutic target for metastatic colon cancer.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Humans; Inflammation; Lamin Type A; Macrophages; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Proto-Oncogene Proteins c-fos; Transforming Growth Factor beta; Tumor Microenvironment

MTA1, SOX4, EZH2, and TGF-β are all potent inducers of epithelial-mesenchymal transition (EMT) in cancer; however, the signaling relationship among these molecules in EMT is poorly understood. Here, we investigated the function of MTA1 in cancer cells and demonstrated that MTA1 overexpression efficiently activates EMT. This activation resulted in a significant increase in the migratory and invasive properties of three different cancer cell lines through a common mechanism involving SOX4 activation, screened from a gene expression profiling analysis. We showed that both SOX4 and MTA1 are induced by TGF-β and both are indispensable for TGF-β-mediated EMT. Further investigation identified that MTA1 acts upstream of SOX4 in the TGF-β pathway, emphasizing a TGF-β-MTA1-SOX4 signaling axis in EMT induction. The histone methyltransferase EZH2, a component of the polycomb (PcG) repressive complex 2 (PRC2), was identified as a critical responsive gene of the TGF-β-MTA1-SOX4 signaling in three different epithelial cancer cell lines, suggesting that this signaling acts broadly in cancer cells in vitro. The MTA1-SOX4-EZH2 signaling cascade was further verified in TCGA pan-cancer patient samples and in a colon cancer cDNA microarray, and activation of genes in this signaling pathway predicted an unfavorable prognosis in colon cancer patients. Collectively, our data uncover a SOX4-dependent EMT-inducing mechanism underlying MTA1-driven cancer metastasis and suggest a widespread TGF-β-MTA1-SOX4-EZH2 signaling axis that drives EMT in various cancers. We propose that this signaling may be used as a common therapeutic target to control epithelial cancer metastasis.

Topics: Cell Line, Tumor; Colonic Neoplasms; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasms; Prognosis; Repressor Proteins; Signal Transduction; SOXC Transcription Factors; Trans-Activators; Transforming Growth Factor beta

Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies.. Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings. Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling-induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression.. Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Deubiquitinating Enzymes; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Ubiquitin Thiolesterase; Xenograft Model Antitumor Assays; Zebrafish

Colorectal cancer (CRC) is the second deadliest cancer in the US due to its propensity to metastasize. Stromal cells and especially cancer-associated fibroblasts (CAF) play a critical biophysical role in cancer progression, but the precise pro-metastatic mechanisms are not clear. Activin A, a TGF-β family member, is a strong pro-metastatic cytokine in the context of CRC. Here, we assessed the link between biophysical forces and pro-metastatic signaling by testing the hypothesis that CAF-generated mechanical forces lead to activin A release and associated downstream effects. Consistent with our hypothesis, we first determined that stromal activin A secretion increased with increasing substrate stiffness. Then we found that stromally-secreted activin A induced ligand-dependent CRC epithelial cell migration and epithelial to mesenchymal transition (EMT). In addition, serum activin A levels are significantly increased in metastatic (stage IV) CRC patients (1.558 ng/ml versus 0.4179 ng/ml, p < 0.05). We propose that increased tumor microenvironment stiffness leads to stromal cell-mediated TGF-β family signaling relying on the induction and utilization of activin A signaling.

Topics: Activins; Aged; Aged, 80 and over; Cadherins; Cancer-Associated Fibroblasts; Case-Control Studies; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Tumor Microenvironment

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

We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process.. We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-β (TGFβ). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure.. We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFβ-induced cellular plasticity of transformed cells.. HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells.

Topics: Animals; Benzamides; Cell Plasticity; Cisplatin; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxyurea; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasms; Pyridines; Transforming Growth Factor beta; Valproic Acid

Pancreatic ductal adenocarcinoma (PDAC) is intractable due to its strong invasiveness and metastatic ability. Epithelial-mesenchymal transition (EMT) is the pivotal driver of tumor invasion and metastasis. The four-and-a-half LIM domain (FHL) family is involved in regulating transforming growth factor (TGF)-β and Ras signaling, which might control the EMT process. In this study, we found that higher expression of four-and-a-half LIM domains 3 (FHL3) predicted poor prognosis in PDAC. The decreasing of FHL3 changed the EMT phenotype by blocking the TGFβ/Atk/GSK3β/ubiquitin pathways. Interestingly, the GSK3β inhibitor could abrogate the role of FHL3 in the regulation of snail1 and twist1 expression, which implied that GSK3β plays a pivotal role in the FHL3-mediated EMT process. Furthermore, we found that FHL3 can directly bind to GSK3β, which weakened the interaction between GSK3β and snail1/twist1. We also found that the LIM-3 domain of FHL3 was required for the binding of FHL3 to GSK3β. Collectively, our study implied that FHL3, as a binding partner of GSK3β, promoted tumor metastasis in PDAC through inhibiting the ubiquitin-degradation of snail1 and twist1.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Models, Biological; Neoplasm Metastasis; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Ubiquitination

NAD+-dependent SIRT7 deacylase plays essential roles in ribosome biogenesis, stress response, genome integrity, metabolism and aging, while how it is transcriptionally regulated is still largely unclear. TGF-β signaling is highly conserved in multicellular organisms, regulating cell growth, cancer stemness, migration and invasion. Here, we demonstrate that histone deacetylase HDAC8 forms complex with SMAD3/4 heterotrimer and occupies SIRT7 promoter, wherein it deacetylates H4 and thus suppresses SIRT7 transcription. Treatment with HDAC8 inhibitor compromises TGF-β signaling via SIRT7-SMAD4 axis and consequently, inhibits lung metastasis and improves chemotherapy efficacy in breast cancer. Our data establish a regulatory feedback loop of TGF-β signaling, wherein HDAC8 as a novel cofactor of SMAD3/4 complex, transcriptionally suppresses SIRT7 via local chromatin remodeling and thus further activates TGF-β signaling. Targeting HDAC8 exhibits therapeutic potential for TGF-β signaling related diseases.

Topics: Cell Line, Tumor; Cell Movement; Cell Survival; Chromatin Assembly and Disassembly; Drug Resistance, Neoplasm; HEK293 Cells; Histone Deacetylases; Humans; Neoplasm Metastasis; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; Signal Transduction; Sirtuins; Smad3 Protein; Smad4 Protein; Transcription, Genetic; Transforming Growth Factor beta

Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Genetically Modified; Cell Line, Tumor; Extracellular Matrix Proteins; Fibrosarcoma; HCT116 Cells; HEK293 Cells; Hippo Signaling Pathway; Humans; Hyaluronan Receptors; Mice; Mice, Nude; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Sarcoma; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins; Zebrafish

We previously revealed that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates the metastatic capacity of tumors in an autocrine/paracrine manner by activating tumor cell motility and invasiveness and the epithelial-mesenchymal transition. However, the effects of ANGPTL2 on cancer cell glycolytic metabolism, which is a hallmark of tumor cells, are unknown. Here we report evidence supporting a role for tumor cell-derived ANGPTL2 in establishing a preference for glycolytic metabolism. We report that a highly metastatic lung cancer cell subline expressing abundant ANGPTL2 showed upregulated expression of the glucose transporter GLUT3 as well as enhanced glycolytic metabolism relative to a less metastatic parental line. Most notably, ANGPTL2 overexpression in the less metastatic line activated glycolytic metabolism by increasing GLUT3 expression. Moreover, ANGPTL2 signaling through integrin α5β1 increased GLUT3 expression by increasing transforming growth factor-β (TGF-β) signaling and expression of the downstream transcription factor zinc finger E-box binding homeobox 1 (ZEB1). Conversely, ANGPTL2 knockdown in the highly metastatic subline decreased TGF-β1, ZEB1, and GLUT3 expression and antagonized glycolytic metabolism. In primary tumor cells from patients with lung cancer, ANGPTL2 expression levels correlated with GLUT3 expression. Overall, this work suggests that tumor cell-derived ANGPTL2 accelerates activities associated with glycolytic metabolism in lung cancer cells by activating TGF-β-ZEB1-GLUT3 signaling.

Topics: Angiopoietin-Like Protein 2; Angiopoietin-like Proteins; Autocrine Communication; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 3; Glycolysis; Humans; Integrin alpha5beta1; Lung Neoplasms; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Paracrine Communication; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Transforming growth factor β (TGFβ) is a prominent cytokine that promotes tumor progression by activating epithelial-to-mesenchymal transition (EMT). This study indicated that TGFβ exerted metastasis by inducing zinc finger E-box binding homeobox 1 (ZEB1) and a long noncoding RNA, LINC00273, expressions in A549 cells. Knocking down LINC00273 diminished TGFβ induced ZEB1 expression as well as metastasis. Mechanistically, LINC00273 acted as a molecular sponge of microRNA (miR)-200a-3p which liberate ZEB1 to perform its prometastatic functions. LINC00273 knockdown and miR200a3p mimic transfection of A549 cells were used for validating the link between TGFβ and LINC00273 induced metastasis. RNA pulldown and luciferase assay were performed to establish mir200a-3p-LINC00273 interaction. High expressions of LINC00273, TGFβ, and ZEB1 with concurrent low miR200a-3p expression had been verified in vivo and in patient samples. Overall, LINC00273 promoted TGFβ-induced lung cancer EMT through miR-200a-3p/ZEB1 feedback loop and may serve as a potential target for therapeutic intervention in lung cancer metastasis.

Topics: A549 Cells; Animals; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

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

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC.

Topics: Aged; Colorectal Neoplasms; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Retrospective Studies; Transforming Growth Factor beta; Tumor Microenvironment

Long noncoding RNA (lncRNA) AC026904.1 has been confirmed to be necessary for breast cancer metastasis. This work aims to investigate the effects of lncRNA AC026904.1 on lung cancer metastasis. We found that lncRNA AC026904.1 displayed a higher level in metastatic lung cancer tissues than adjacent tissues and nonmetastatic lung cancer tissues, and lung cancer cells treated with TGF-β. The expression of AC026904.1 was increased by the non-canonical TGF-β signaling. Additionally, AC026904.1 acts as an enhancer of the key metastatic factor Slug in the nucleus. This AC026904.1/Slug axis is necessary for TGF-β-mediated migration and epithelial-mesenchymal transition in lung cancer cells. This work firstly uncovers that AC026904.1 increases Slug expression at transcriptional level and subsequently plays critical effects in lung cancer metastasis, providing novel evidences that AC026904.1 holds great potential to be used as a marker for metastatic lung cancer.

Topics: A549 Cells; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Neoplasm Metastasis; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Tumor progression and metastasis are the major causes of death among cancer associated mortality. Metastatic cells acquire features of migration and invasion and usually undergo epithelia-mesenchymal transition (EMT). Acquirement of these various hallmarks rely on different cellular pathways, including TGF-β and Wnt signaling. Recently, we reported that WW domain-containing oxidoreductase (WWOX) acts as a tumor suppressor and has anti-metastatic activities involving regulation of several key microRNAs (miRNAs) in triple-negative breast cancer (TNBC). Here, we report that WWOX restoration in highly metastatic MDA-MB435S cancer cells alters mRNA expression profiles; further, WWOX interacts with various proteins to exert its tumor suppressor function. Careful alignment and analysis of gene and miRNA expression in these cells revealed profound changes in cellular pathways mediating adhesion, invasion and motility. We further demonstrate that WWOX, through regulation of miR-146a levels, regulates SMAD3, which is a member of the TGF-β signaling pathway. Moreover, proteomic analysis of WWOX partners revealed regulation of the Wnt-signaling activation through physical interaction with Disheveled. Altogether, these findings underscore a significant role for WWOX in antagonizing metastasis, further highlighting its role and therapeutic potential in suppressing tumor progression.

Topics: Cell Movement; Cell Proliferation; Dishevelled Proteins; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genetic Pleiotropy; Humans; MicroRNAs; Neoplasm Metastasis; Protein Interaction Maps; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Tumor Suppressor Proteins; Wnt Signaling Pathway; WW Domain-Containing Oxidoreductase

Epithelial mesenchymal transition (EMT) is a well-known and important step in metastasis and thus can be a key target in cancer treatment. Here, we tested the EMT inhibitory actions of Selaginella tamariscina and its active component, amentoflavone (AF). EMT was examined in vitro using wound-healing and invasion assays and by monitoring changes in the expression of the EMT-related proteins, E-cadherin, Snail, and Twist. Metastasis was examined in vivo using SCID mice injected with luciferase-labeled A549 cells. We confirmed that aqueous extracts of S. tamariscina (STE) and AF inhibited EMT in human cancer cell lines. We found that STE and AF at nontoxic concentrations exerted remarkable inhibitory effects on migration (wound healing assay) and invasion (Transwell assay) in tumor necrosis factor (TGF)-β-treated cancer cells. Western blotting and immunofluorescence imaging show that AF treatment also restored E-cadherin expression in these cells compared to cells treated with TGF-β only. Suppression of metastasis by AF was investigated by monitoring migration of tail-vein-injected, circulating A549-luc cells to the lungs in mice. After 3 wk, fewer nodules were observed in mice co-treated with AF compared with those treated with TGF-β only. Our findings indicate that STE and AF are promising EMT inhibitors and, ultimately, potentially potent antitumor agents.

Topics: A549 Cells; Animals; Antigens, CD; Antineoplastic Agents; Biflavonoids; Cadherins; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Mice, SCID; Neoplasm Metastasis; Nuclear Proteins; Plant Extracts; Selaginellaceae; Snail Family Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

Hepatocellular carcinoma (HCC) metastasis is largely responsible for HCC-associated recurrence and mortality. We aimed to identify metastasis-related long non-coding RNAs (lncRNAs) to understand the molecular mechanism of HCC metastasis. We first identified that miR-1258 was downregulated in HCC tissues both in The Cancer Genome Atlas (TCGA) and Sun Yat-sen University Cancer Center (SYSUCC) dataset. MiR-1258 expression negatively correlated with recurrence-free survival and overall survival of HCC patients. MiR-1258 overexpression inhibited migration and invasion of HCC cells both in vitro and in vivo, whereas miR-1258 downregulation promoted cell metastasis. Luciferase assays verified direct binding of miR-1258 to Smad2 and Smad3, thereby attenuating TGF-β/Smad signaling. We further established that lncRNA LINC01278 was a negative regulator of miR-1258. In vivo and in vitro assays demonstrated that LINC01278-mediated HCC metastasis was dependent on miR-1258 expression. Furthermore, miR-1258 downregulation in turn increased LINC01278 expression. We also observed that TCF-4 could bind to the LINC01278 promoter site. In addition, LINC01278 downregulation decreased migration and invasion of HCC cells induced by β-catenin and TGF-β1 both in vitro and in vivo. We uncovered a novel mechanism for β-catenin/TCF-4-LINC01278-miR-1258-Smad2/3 feedback loop activation in HCC metastasis, and the study indicated that LINC01278 could serve as a therapeutic target for HCC metastasis.

Topics: Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Long Noncoding; Smad2 Protein; Smad3 Protein; Transcription Factor 4; Transforming Growth Factor beta; Transplantation, Heterologous; Wnt Signaling Pathway; Wnt1 Protein

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer of the digestive tract that has a high potential for metastasis and a poor prognosis. Girdin was first reported in 2005 as an actin‑binding protein and was designated as Akt‑phosphorylation enhancer (APE); thus, Girdin has been revealed to have an important role in regulating cancer development. There is additional evidence indicating that Girdin is associated with cell proliferation, migration, invasion and survival in certain cancers. However, the potential mechanisms involving Girdin and mobility in pancreatic cancer have not been elucidated. In the present study, it was revealed that Girdin was highly expressed in pancreatic cancer tissue and was associated with tumor grade. The present study, to the best of our knowledge, is the first aimed at investigating the unknown role of Girdin in PDAC metastasis. A short hairpin RNA for Girdin (sh‑Girdin) was successfully constructed with recombinant adenoviral vectors to suppress the expression of Girdin in pancreatic cancer cell lines (PANC‑1 and BXPC‑3). The silencing efficiency of the Girdin shRNA was determined by RT‑qPCR and western blot analysis, and decreased Girdin expression in the cytoplasm was revealed by immunofluorescence detection. Then, sulforhodamine B (SRB) and colony formation assays were used to confirm that the knockdown of Girdin inhibited proliferation in vitro, and Transwell assays were used to examine the influence of Girdin knockdown on cellular mobility. Animal experiments also confirmed that silencing the expression of Girdin in pancreatic cancer cells inhibited the growth and metastasis of pancreatic cancer in vivo. Transforming growth factor‑β (TGF‑β) is a common inducer of epithelial‑mesenchymal transition (EMT) and can effectively induce EMT in PDAC. Notably, TGF‑β‑treated cells exhibited changes in the classic biological markers of EMT. The expression of E‑cadherin, a marker of the epithelial phenotype, increased, and the expression of N‑cadherin and vimentin, markers of the interstitial phenotype, decreased in response to sh‑Girdin. According to these experiments, Girdin may affect pancreatic cancer progression and development by interacting with vimentin. Therefore, there is evidence indicating that Girdin could be designated as a prognostic biological indicator and a candidate therapeutic target for pancreatic cancer.

Topics: Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Microfilament Proteins; Neoplasm Grading; Neoplasm Metastasis; Pancreatic Neoplasms; Prognosis; Transforming Growth Factor beta; Up-Regulation; Vesicular Transport Proteins; Vimentin

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

Transcription growth factor β (TGF-β) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-β-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-β-induced EMT process. Enhancers gained upon TGF-β treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-β stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-β signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-β signaling-associated cancer metastasis.

Topics: A549 Cells; Animals; Carcinogenesis; Cellular Reprogramming; DNA-Binding Proteins; Enhancer Elements, Genetic; Epithelial-Mesenchymal Transition; HEK293 Cells; Hepatocyte Nuclear Factor 3-beta; Hepatocytes; Histone Deacetylase 1; Histones; Humans; Mice; Muscle Proteins; Neoplasm Metastasis; Signal Transduction; Smad2 Protein; Smad3 Protein; TEA Domain Transcription Factors; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

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

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 risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards

Topics: Adult; Aged; Aging; Animals; Cell Line, Tumor; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Methylmalonic Acid; Mice; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Signal Transduction; SOXC Transcription Factors; Transcriptome; Transforming Growth Factor beta

The human Obg-like ATPase 1 (OLA1) protein has been reported to play an important role in cancer cell proliferation. The molecular mechanism underlying OLA1 regulated oral metastasis is still unknown. We investigated in this study the regulatory role of OLA1 playing in oral squamous cell metastasis.. A series of in vitro assays were performed in the cells with RNAi-mediated knockdown or overexpression to expound the regulatory function of OLA1 in oral cancer. We found that the endogenous level of OLA1 in a highly metastatic oral squamous cell line was significantly lower than that in low metastatic oral cells as well as in normal oral cells. Escalated expression of OLA1 resulted in a reduced ability of metastasis in highly metastatic cells, and enhanced its sensitivity to the paclitaxel treatment. Further analysis of the EMT markers showed that Snail, Slug, N-cadherin were up-expressed significantly. Meanwhile, E-cadherin was significantly down-regulated in the oral cancer cells with OLA1-knocked down, suggesting that OLA1 inactivated EMT process. Furthermore, we found that OLA1 suppressed oral squamous cell metastasis by suppressing the activity of a TGFβ/SMAD2/EMT pathway.. Our data suggests that OLA1 may be developed as a potential target for the treatment of oral cancer metastasis.

Topics: Adenosine Triphosphatases; Cadherins; Carcinoma; Cell Line, Tumor; Down-Regulation; GTP-Binding Proteins; Humans; Mouth Neoplasms; Neoplasm Metastasis; Paclitaxel; Signal Transduction; Smad2 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta; Up-Regulation

An effective host immune system prevents the growth of most cancer cells. However, as intestinal nematodes are able to induce both immunotolerance and immunosuppression in the host, it is possible that their presence could allow co-occurring cancer cells to proliferate and metastasize. Our findings indicate that previous, subsequent or concurrent intestinal nematode infection affects the formation of lung metastatic nodules in mice experimentally infected with Heligmosomoides polygyrus. In addition, pre-infection with nematodes renders mice resistant to metastasis development in lungs, with the inoculated EL4 cancer cells being located mainly in mesenteric lymph nodes. The present paper discusses the nematode-induced mechanisms which may influence the metastatic process.

Topics: Animals; Disease Models, Animal; Helminthiasis; Immunomodulation; Intestinal Diseases, Parasitic; Lung Neoplasms; Lymphoma; Male; Mice; Nematode Infections; Nematospiroides dubius; Neoplasm Metastasis; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) signaling is considered to be a key player in gastric cancer metastasis, and the inhibition of the TGF-β/SMAD4 signaling pathway may be a novel strategy for therapeutic interventions in cancer. Here, the anti-metastatic activity of two phytochemicals, eugenol and capsaicin, has been studied, and their potential to antagonize TGF-β has been investigated in gastric cancer cells. Both the phytochemicals exhibited anti-metastatic activity by inhibiting the TGF-β signaling pathway independent of P21 or P53, with capsaicin proving to be more potent than eugenol. However, unlike eugenol, the inhibitory effect of capsaicin on the TGF-β signaling pathway and metastasis was found to be dependent on SMAD4, which was validated in SMAD4-knocked down AGS cell and SMAD4-null SW620 cell line. Furthermore, the use of recombinant TGF-β and TGF-β receptor inhibitor LY2109761 confirmed that the anti-metastatic activity of eugenol is partially and that of capsaicin is principally mediated through the TGF-β signaling pathway. Identifying phytochemicals with the potential to inhibit cancer metastasis by targeting the TGF-β signaling pathway has immense scope for developing a therapeutic strategy against cancer metastasis.

Topics: Antineoplastic Agents; Capsaicin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Eugenol; Humans; Neoplasm Metastasis; Signal Transduction; Smad4 Protein; Stomach Neoplasms; Transforming Growth Factor beta

Breast cancer metastasis is a complex process that depends not only on intrinsic characteristics of metastatic stem cells, but also on the particular microenvironment that supports their growth and modulates the plasticity of the system. In search for microenvironmental factors supporting cancer stem cell (CSC) growth and tumour progression to metastasis, we here investigated the role of the matricellular protein transforming growth factor beta induced (TGFBI) in breast cancer. We crossed the MMTV-PyMT model of mammary gland tumorigenesis with a Tgfbi

Topics: Animals; Extracellular Matrix Proteins; Female; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Neovascularization, Pathologic; Prognosis; Transforming Growth Factor beta; Tumor Hypoxia

Oral squamous cell carcinoma (OSCC) is a prevalent cancer that develops in the head and neck area and has high annual mortality despite optimal treatment. microRNA-218 (miR-218) is a tumour inhibiting non-coding RNA that has been reported to suppress the cell proliferation and invasion in various cancers. Thus, our study aims to determine the mechanism underlying the inhibitory role of miR-218 in OSCC. We conducted a bioinformatics analysis to screen differentially expressed genes in OSCC and their potential upstream miRNAs. After collection of surgical OSCC tissues, we detected GREM1 expression by immunohistochemistry, RT-qPCR and Western blot analysis, and miR-218 expression by RT-qPCR. The target relationship between miR-218 and GREM1 was assessed by dual-luciferase reporter gene assay. After loss- and gain-of-function experiments, OSCC cell proliferation, migration and invasion were determined by MTT assay, scratch test and Transwell assay, respectively. Expression of TGF-β1, Smad4, p21, E-cadherin, Vimentin and Snail was measured by RT-qPCR and Western blot analysis. Finally, effects of miR-218 and GREM1 on tumour formation and liver metastasis were evaluated in xenograft tumour-bearing nude mice. GREM1 was up-regulated, and miR-218 was down-regulated in OSCC tissues, and GREM1 was confirmed to be the target gene of miR-218. Furthermore, after up-regulating miR-218 or silencing GREM1, OSCC cell proliferation, migration and invasion were reduced. In addition, expression of TGF-β signalling pathway-related genes was diminished by overexpressing miR-218 or down-regulating GREM1. Finally, up-regulated miR-218 or down-regulated GREM1 reduced tumour growth and liver metastasis in vivo. Taken together, our findings suggest that the overexpression of miR-218 may inhibit OSCC progression by inactivating the GREM1-dependent TGF-β signalling pathway.

Topics: Adult; Aged; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Computational Biology; Databases, Genetic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Heterografts; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; MicroRNAs; Middle Aged; Models, Biological; Mouth Neoplasms; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; RNA Interference; Signal Transduction; 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

Introduction of α-cyano α,β-unsaturated carbonyl moiety into natural cyclic compounds markedly improves their bioactivities, including inhibitory potential against tumor growth and metastasis. Previously, we showed that cyano enone-bearing derivatives of 18βH-glycyrrhetinic (GA) and deoxycholic acids displayed marked cytotoxicity in different tumor cell lines. Moreover, GA derivative soloxolone methyl (SM) was found to induce ER stress and apoptosis in tumor cells in vitro and inhibit growth of carcinoma Krebs-2 in vivo. In this work, we studied the effects of these compounds used in non-toxic dosage on the processes associated with metastatic potential of tumor cells. Performed screening revealed SM as a hit compound, which inhibits motility of murine melanoma B16 and human lung adenocarcinoma A549 cells and significantly suppresses colony formation of A549 cells. Further study showed that SM effectively blocked transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) of A549 cells: namely, inhibited TGF-β-stimulated motility and invasion of tumor cells as well as loss of their epithelial characteristics, such as, an acquisition of spindle-like phenotype, up- and down-regulation of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin, zona occludens-1 (ZO-1)) markers, respectively. Network pharmacology analysis with subsequent verification by molecular modeling revealed that matrix metalloproteinases MMP-2/-9 and c-Jun N-terminal protein kinase 1 (JNK1) can be considered as hypothetical primary targets of SM, mediating its marked anti-EMT activity. The inhibitory effect of SM on EMT revealed in vitro was further confirmed in a metastatic model of murine B16 melanoma: SM was found to effectively block metastatic dissemination of melanoma B16 cells in vivo, increase expression of E-cadherin and suppress expression of MMP-9 in lung metastatic foci. Altogether, our data provided valuable information for a better understanding of the antitumor activity of cyano enone-bearing semisynthetic compounds and revealed SM as a promising anti-metastatic drug candidate.

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Antineoplastic Agents; Binding Sites; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 8; Molecular Docking Simulation; Neoplasm Metastasis; Neoplasms, Experimental; Protein Binding; Protein Conformation, beta-Strand; Skin Neoplasms; Transforming Growth Factor beta; Triterpenes

Capsaicin is an active alkaloid /principal component of red pepper responsible for the pungency of chili pepper. Capsaicin by changing the intracellular redox homeostasis regulate a variety of signaling pathways ultimately producing a divergent cellular outcome. Several reports showed the potential of capsaicin against cancer metastasis, however unexplored molecular mechanism is still an active part of the research. Several growth factors have a critical role during cancer metastasis among them TGF- β signaling play a vital role.. The present study aimed at analyzing capsaicin modulation of TGF-β signaling using network pharmacology approach. The chemical and protein interaction data of capsaicin was curated and abstracted using STITCH4.0, PubChem and ChEMBL database. Further, the compiled data set was subjected to the pathway and functional enrichment analysis using Protein Analysis THrough Evolutionary Relationship (PANTHER) and, Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. Meanwhile, the pattern of amino acid composition across the capsaicin targets was analyzed using the EMBOSS Pepstat tool. Capsaicin targets involved in TGF- β were identified and their Protein-Protein Interaction (PPI) network constructed using STRING v10 and Cytoscape (v 3.2.1). From the above-constructed network, the clusters were mined using the MCODE clustering algorithm and finally binding affinity of capsaicin with its targets involved in TGF-β signaling pathway was analyzed using Autodock Vina.. The analysis explored capsaicin targets and, their associated functional and pathway annotations. Besides, the analysis also provides a detailed distinct pattern of amino acid composition across the capsaicin targets. The capsaicin targets described as MAPK14, JUN, SMAD3, MAPK3, MAPK1 and MYC involved in TGF-β signaling pathway through pathway enrichment analysis. The binding mode analysis of capsaicin with its targets has shown high affinity with MAPK3, MAPK1, JUN and MYC.. The study explores the potential of capsaicin as a potent modulator of TGF-β signaling pathway during cancer metastasis and proposes new methodology and mechanism of action of capsaicin against TGF- β signaling pathway.

Topics: Capsaicin; Drug Discovery; Humans; Molecular Docking Simulation; Neoplasm Metastasis; Protein Interaction Mapping; Protein Interaction Maps; Signal Transduction; Transforming Growth Factor beta

Metastatic bladder cancer (BLCA) is a lethal disease with an unmet need for study. Transgelin (TAGLN) is an actin-binding protein that affects the dynamics of the actin cytoskeleton indicating its robust potential as a metastasis initiator. Here, we sought to explore the expression pattern of TAGLN and elucidate its specific functioning and mechanisms in BLCA.. A comprehensive assessment of TAGLN expression in BLCA was performed in three cohorts with a total of 847 patients. The potential effects of TAGLN on BLCA were further determined using clinical genomic analyses that guided the subsequent functional and mechanistic studies. In vitro migration, invasion assays and in vivo metastatic mouse model were performed to explore the biological functions of TAGLN in BLCA cells. Immunofluorescence, western blot and correlation analysis were used to investigate the molecular mechanisms of TAGLN.. TAGLN was highly expressed in BLCA and correlated with advanced prognostic features. TAGLN promoted cell colony formation and cell migration and invasion both in vitro and in vivo by inducing invadopodia formation and epithelial-mesenchymal transition, during which a significant correlation between TAGLN and Slug was observed. The progression-dependent correlation between TGF-β and TAGLN was analysed at both the cellular and tissue levels, while TGF-β-mediated migration was abolished by the suppression of TAGLN.. Overall, TAGLN is a promising novel prognosis biomarker of BLCA, and its metastatic mechanisms indicate that TAGLN may represent a novel target agent that can be utilized for the clinical management of invasive and metastatic BLCA. FUND: This work was supported by the National Natural Science Foundation of China [81772703, 81672546, 81602253]; the Natural Science Foundation of Beijing [71772219, 7152146]. and Innovative Fund for Doctoral Students of Peking University Health Science Center (BUM2018BSS002). Funders had no role in the design of the study, data collection, data analysis, interpretation, or the writing of this report.

Topics: Aged; Aged, 80 and over; Animals; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Male; Mice; Microfilament Proteins; Middle Aged; Muscle Proteins; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Podosomes; Prognosis; Transforming Growth Factor beta; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Functional E-cadherin loss, a hallmark of epithelial-mesenchymal transition (EMT), is important for metastasis. However, the mechanism of Snail2 in hepatocellular carcinoma (HCC) EMT and metastasis remains unclear. Here, we showed that Snail2 was upregulated in primary HCC, and significantly increased during transforming growth factor-β-induced liver cell EMT. Snail2-overexpressing and knockdown cell lines have been established to determine its function in EMT in HCC. H3K9 methylation was upregulated and H3K4 and H3K56 acetylation were downregulated at the E-cadherin promoter in Snail2-overexpressing cancer cells. Furthermore, Snail2 interacted with G9a and histone deacetylases (HDACs) to form a complex to suppress E-cadherin transcription. Snail2 overexpression enhanced migration and invasion in HCC cells, whereas G9a and HDAC inhibition significantly reversed this effect. Moreover, Snail2 overexpression in cancer cells increased tumor metastasis and shortened survival time in mice, whereas G9a and HDAC inhibitors extended survival. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also suggests novel treatment strategies for HCC.

Topics: Acetylation; Animals; Azepines; Cadherins; Carcinoma, Hepatocellular; Cell Movement; Disease Progression; Down-Regulation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Histocompatibility Antigens; Histone Deacetylase Inhibitors; Histone Deacetylases; Histone-Lysine N-Methyltransferase; Humans; Hydroxamic Acids; Liver; Liver Neoplasms; Methylation; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Quinazolines; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

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

The molecular mechanisms revealing cervical cancer progression remain unclear. NSD2 belongs to the NSD family of histone lysine methyltransferases (HMTases), and is a histone methyltransferase that regulates dimethylation of histone 3 lysine 36 (H3K36me2). In this study, we explored the effects of NSD2 on the tumorigenesis and metastasis in cervical cancer. We found that NSD2 exhibited a pattern of gradual up-regulation from normal cervix (NC) to cervical carcinoma in situ (CIS) and then to invasive cervical cancer (ICC). NSD2 knockdown markedly reduced the cervical cancer cell proliferation. Loss of function assay in vitro suggested that NSD2 deletion markedly prevented the cervical cancer cell migration and invasion. Consistently, the in vivo results demonstrated that NSD2 knockdown not only reduced tumor growth, but also prevented the development of tumor metastasis. In addition, NSD2 knockdown clearly reduced the expression levels of transforming growth factor-β1 (TGF-β1), TGF-βRI, phosphorylated SMAD2 and SMAD3 in cervical cancer cells, accompanied with the decreased expression of genes that promoted tumor metastasis. Importantly, we found that NSD2 knockdown-regulated expression levels of metastasis-associated genes were reversed by TGF-β1 incubation. Therefore, our findings demonstrated that NSD2-modulated activation of TGF-β1/TGF-βRI/SMADs signaling pathway was crucial for cervical cancer progression, which might be a promising therapeutic strategy to overcome metastasis in cervical cancer.

Topics: Animals; Cell Line; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone-Lysine N-Methyltransferase; Humans; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Receptor, Transforming Growth Factor-beta Type I; Repressor Proteins; RNA Interference; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transplantation, Heterologous; Uterine Cervical Neoplasms

Accumulating evidence indicates that CD36 initiates metastasis and correlates with an unfavorable prognosis in cancers. However, there are few reports regarding the roles of CD36 in initiation and metastasis of cervical cancer.. Using immunohistochemistry, we analyzed 133 cervical cancer samples for CD36 protein expression levels, and then investigated the correlation between changes in its expression and clinicopathologic parameters. The effect of CD36 expression on the epithelial-mesenchymal transition (EMT) in cervical cancer cells was evaluated by Western immunoblotting analysis. In vitro invasion and in vivo metastasis assays were also used to evaluate the role of CD36 in cervical cancer metastasis.. In the present study, we confirmed that CD36 was highly expressed in cervical cancer samples relative to normal cervical tissues. Moreover, overexpression of CD36 promoted invasiveness and metastasis of cervical cancer cells in vitro and in vivo, while CD36 knockdown suppressed proliferation, migration, and invasiveness. We demonstrated that TGF-β treatment attenuated E-cadherin expression and enhanced the expression levels of CD36, vimentin, slug, snail, and twist in si-SiHa, si-HeLa, and C33a-CD36 cells, suggesting that TGF-β synergized with CD36 on EMT via active CD36 expression. We also observed that the expression levels of TGF-β in si-SiHa cells and si-HeLa cells were down-regulated, whereas the expression levels of TGF-β were up-regulated in C33a-CD36 cells. These results imply that CD36 and TGF-β interact with each other to promote the EMT in cervical cancer.. Our findings suggest that CD36 is likely to be an effective target for guiding individualized clinical therapy of cervical cancer.

Topics: Animals; Apoptosis; CD36 Antigens; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Knockdown Techniques; HeLa Cells; Humans; Immunohistochemistry; Kangai-1 Protein; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Transforming Growth Factor beta; Translational Research, Biomedical; Uterine Cervical Neoplasms

Alcohol intake is a well-known lifestyle risk factor for CRC, and an increasing number of studies have revealed that alcohol intake is also tightly associated with CRC metastasis. However, the effect of alcohol on CRC metastasis and its underlying mechanism remain unclear.. A retrospective cohort study was performed to investigate the characteristics of patients with alcohol-related CRC. The effects of ethanol on the biological behaviours of CRC cells were assessed through in vivo and in vitro assays using the Lieber-DeCarli ethanol liquid diet and ethanol, respectively. The ethanol-mediated signalling pathway and downstream factors were screened through ELISA, western blot, immunofluorescence and co-immunoprecipitation.. Most patients with alcohol-related CRC, particularly those with tumour metastasis, were characterized by a notably higher circulating ethanol level and a lower systemic acetaldehyde level. Moreover, CRC cells accumulated in ethanol, but not acetaldehyde, to notably higher levels compared with adjacent normal cells. Alcohol intake significantly promoted CRC metastasis via the ethanol-mediated TGF-β/Smad/Snail axis, and ethanol induced the cytoplasmic mislocalization of RUNX3 and further promoted the aggressiveness of CRC by targeting Snail. Pirfenidone (PFD) significantly eliminated the effects of ethanol on CRC metastasis by specifically blocking TGF-β signalling.. Alcohol intake plays a vital role in CRC metastasis via the ethanol-mediated TGF-β/RUNX3/Snail axis, and PFD might be a novel therapeutic management strategy for CRC.

Topics: Alcohol Drinking; Animals; Biomarkers; Cell Line, Tumor; Colorectal Neoplasms; Core Binding Factor Alpha 3 Subunit; Disease Models, Animal; Epithelial-Mesenchymal Transition; Humans; Incidence; Mice; Neoplasm Metastasis; Neurophysins; Protein Binding; Protein Precursors; Protein Transport; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vasopressins; Xenograft Model Antitumor Assays

Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis.. Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo.. Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2β1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin β1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis.. We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin β1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

Topics: Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Disease Models, Animal; Extracellular Matrix; Extracellular Vesicles; Gene Expression Profiling; Humans; Integrin alpha2beta1; Lung Neoplasms; Mice; Models, Biological; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Non-small cell lung cancer (NSCLC) remains a major cause of death worldwide. As metastatic disease is primarily responsible for the poor clinical outcome of NSCLC, it is important to understand the process, and its underlying molecular mechanism as well, via which NSCLC cells disseminate. In this study, we identified a new competing endogenous RNA (ceRNA), namely, the MYEOV transcript, and found that it is upregulated in NSCLC and associated with a poor prognosis of the disease. We further uncovered that the MYEOV ceRNA plays a critical role in the invasion and metastasis of NSCLC cells. Intriguingly, the MYEOV ceRNA exerted its pro-metastatic function independent of its protein-coding capacity, but in a miR-30c-2-3p binding-dependent manner. Further experiments demonstrated that the MYEOV ceRNA sequestered miR-30c-2-3p from binding its targets TGFBR2 and USP15 mRNAs, which in turn leading to constitutive activation of TGF-β signaling and tumor progression in NSCLC. By identifying a new layer of regulatory modality for TGF-β signaling, our findings extend the current understanding on the molecular mechanism mediating NSCLC progression and highlight a potential role of MYEOV transcript to serve as the therapeutic target.

Topics: A549 Cells; Animals; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Proto-Oncogene Proteins; RNA, Neoplasm; 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 metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-β (TGF-β) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and β-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and β-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/β and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-β signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential.

Topics: Antigens, CD; Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Humans; Intracellular Signaling Peptides and Proteins; Neoplasm Metastasis; NF-kappa B; Pancreatic Neoplasms; RNA, Messenger; Signal Transduction; Thiosemicarbazones; 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

Topics: Adenocarcinoma; Animals; Carcinogenesis; Disease Models, Animal; Endometrial Neoplasms; Endometrium; Female; Humans; Lung Neoplasms; Mice; Neoplasm Metastasis; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Transforming Growth Factor beta; Uterus

Topics: Animals; Colorectal Neoplasms; Disease Models, Animal; Immunotherapy; Neoplasm Metastasis; Neoplasms, Experimental; Organoids; Prognosis; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) is compulsory for metastatic dissemination and is stimulated by TGF-β. Although targeting EMT has significant therapeutic potential, very few pharmacological agents have been shown to exert anti-metastatic effects. BI-69A11, a competitive Akt inhibitor, displays anti-tumor activity toward melanoma and colon carcinoma. This study provides molecular and biochemical insights into the effects of BI-69A11 on EMT in colon carcinoma cells in vitro and in vivo. BI-69A11 inhibited metastasis-associated cellular migration, invasion and adhesion by inhibiting the Akt-β-catenin pathway. The underlying mechanism of BI-69A11-mediated inhibition of EMT included suppression of nuclear transport of β-catenin and diminished phosphorylation of β-catenin, which was accompanied by enhanced E-cadherin-β-catenin complex formation at the plasma membrane. Additionally, BI-69A11 caused increased accumulation of vinculin in the plasma membrane, which fortified focal adhesion junctions leading to inhibition of metastasis. BI-69A11 downregulated activation of the TGF-β-induced non-canonical Akt/NF-κB pathway and blocked TGF-β-induced enhanced expression of Snail causing restoration of E-cadherin. Overall, this study enhances our understanding of the molecular mechanism of BI-69A11-induced reversal of EMT in colorectal carcinoma cells in vitro, in vivo and in TGF-β-induced model systems.

Topics: Antigens, CD; Antineoplastic Agents; Benzimidazoles; beta Catenin; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Quinolones; Transforming Growth Factor beta; Vinculin

Tumour cell immune evasion is a principal hallmark of successful metastasis. Tumour cells in the vasculature adopt a platelet cloak that efficiently suppresses the innate immune system by directly inhibiting Natural Killer (NK) cells, which normally function to neutralise spreading cancers. Here we describe two novel mechanisms of tumour cell evasion of NK cell anti-tumour functions. The first, an 'immune decoy' mechanism in which platelets induce the release of soluble NKG2D ligands from the tumour cell to mask detection and actively suppress NK cell degranulation and inflammatory cytokine (IFNγ) production, concomitantly. This represents a double-hit to immune clearance of malignant cells during metastasis. The second mechanism, a platelet-derived TGFβ-mediated suppression of the CD226/CD96-CD112/CD155 axis, is a novel pathway with poorly understood anti-cancer functions. We have demonstrated that platelets robustly suppress surface expression of CD226 and CD96 on the NK cell surface and their associated ligands on the tumour cell to further enhance NK cell suppression. These highly evolved mechanisms promote successful tumour immune evasion during metastasis and provide a unique opportunity for studying the complexity of cellular interactions in the metastatic cascade and thus novel targets for cancer immunotherapy.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Blood Platelets; Cell Line, Tumor; Histocompatibility Antigens Class I; Humans; Immunity, Innate; Interferon-gamma; Killer Cells, Natural; Nectins; Neoplasm Metastasis; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Virus; Signal Transduction; Transforming Growth Factor beta; Tumor Escape

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

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer-related death worldwide. Increasing evidence indicates that the deregulation of long noncoding RNAs (lncRNAs) contributes to tumor initiation and progression; however, little is known about the biological role of cancer susceptibility candidate 9 (CASC9) in CRC.. Novel lncRNAs potentially involved in CRC tumorigenesis were identified from datasets downloaded from The Cancer LncRNome Atlas and The Atlas of Noncoding RNAs in Cancer. The CRC cell lines HCT-116, HCT-116 p53. CASC9 was frequently upregulated in CRC, which was correlated with advanced TNM stage, and higher CASC9 levels were associated with poor patient outcomes. Knockdown of CASC9 inhibited growth and promoted apoptosis in CRC cells, whereas ectopic CASC9 expression promoted cell growth in vitro and in vivo. We demonstrated that CPSF3 is a CASC9-interacting protein, and knockdown of CPSF3 mimicked the effects of CASC9 knockdown in CRC cells. Furthermore, we found that CASC9 exerts its oncogenic activity by modulating TGFβ2 mRNA stability and upregulating the levels of TGFβ2 and TERT, resulting in an increase in phosphorylated SMAD3 and activation of TGF-β signaling, and enhanced TERT complex function in CRC cells. Finally, CPSF3 was significantly upregulated in CRC tissues as compared with adjacent or non-adjacent normal colon tissues, and CASC9, CPSF3, and TGFβ2 levels in human CRC tissues were positively correlated.. CASC9 is a promising prognostic predictor for patients with CRC and the CASC9-CPSF3-TGFβ2 axis is a potential therapeutic target for CRC treatment.

Topics: Aged; Aged, 80 and over; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cleavage And Polyadenylation Specificity Factor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; RNA Interference; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Burden

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Nerve Tissue Proteins; Rats; Receptors, Immunologic; Roundabout Proteins; Signal Transduction; Smad Proteins; 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

Esophageal squamous cell carcinoma (ESCC) is the most difficult subtype of esophageal cancer to treat due to the paucity of effective targeted therapy. ESCC is believed to arise from cancer stem cells (CSCs) that contribute to metastasis and chemoresistance. Despite advances in diagnosis and treatment, the prognosis of ESCC patients remains poor.. In this study, we applied western blot, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, RNA-Seq analysis, luciferase reporter assay, Chip-qPCR, bioinformatics analysis, and a series of functional assays to show the potential role of LEF1 in regulating esophageal CSCs.. We found that the overexpression of LEF1 was associated with aberrant clinicopathological characteristics and the poor prognosis of ESCC patients. In addition, the elevated expression of LEF1 and OV6 was significantly associated with aberrant clinicopathological features, and poor patient prognosis. Moreover, the overexpression of LEF1 was observed in esophageal CSCs purified by the magnetic sorting of adherent and spheroidal ESCC cells. The increased level of LEF1 in CSCs facilitated the expression of CSC markers, stem cell-like properties, resistance to chemotherapy, and tumorigenicity and increased the percentage of CSCs in ESCC samples. Conversely, the knockdown of LEF1 significantly diminished the self-renewal properties of ESCC. We showed that LEF1 played an important mechanical role in activating the TGF-β signaling pathway by directly binding to the ID1 gene promoter. A positive association between LEF1 and ID1 expression was also observed in clinical ESCC samples.. Our results indicate that the overexpression of LEF1 promotes a CSC-like phenotype in and the tumorigenicity of ESCC by activating the TGF-β signaling pathway. The inhibition of LEF1 might therefore be a novel therapeutic target to inactivate CSCs and inhibit tumor progression.

Topics: Aged; Aged, 80 and over; Animals; Antigens, Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lymphoid Enhancer-Binding Factor 1; Male; Mice; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Neoplastic Stem Cells; Prognosis; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Exosomes from cancer cells or immune cells, carrying bio-macromolecules or microRNAs (miRNAs), participate in tumor pathogenesis and progression by modulating microenvironment. Our study aims to investigate the role of these microRNA-501-3p (miR-501-3p) containing exosomes derived from tumor-associated macrophage (TAM) in the progression of pancreatic ductal adenocarcinoma (PDAC).. Firstly, the function of TAM recruitment in PDAC tissues was assessed, followed by identification of the effects of M2 macrophage-derived exosomes on PDAC cell activities and tumor formation and metastasis in mice. In silico analysis was conducted to predict differentially expressed genes and regulatory miRNAs related to PDAC treated with macrophages, which determined miR-501-3p and TGFBR3 for subsequent experiments. Next, gain- and loss-of-function experiments were performed to examine their role in PDAC progression with the involvement of the TGF-β signaling pathway.. TAM recruitment in PDAC tissues was associated with metastasis. Highly expressed miR-501-3p was observed in PDAC tissues and TAM-derived exosomes. Both M2 macrophage-derived exosomes and miR-501-3p promoted PDAC cell migration and invasion, as well as tumor formation and metastasis in nude mice. MiR-501-3p was verified to target TGFBR3. PDAC cells presented with down-regulated TGFBR3, which was further decreased in response to M2 macrophage treatment. TGF-β signaling pathway activation was implicated in the promotion of miR-501-3p in PDAC development. The suppression of macrophage-derived exosomal miR-501-3p resulted in the inhibition of tumor formation and metastasis in vivo.. M2 macrophage-derived exosomal miR-501-3p inhibits tumor suppressor TGFBR3 gene and facilitates the development of PDAC by activating the TGF-β signaling pathway, which provides novel targets for the molecular treatment of PDAC.

Topics: Adult; Aged; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Pancreatic Neoplasms; Proteoglycans; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Valproic acid (VPA), one of the histone deacetylase inhibitors, can suppress prostate cancer (PCa) cells epithelial mesenchymal transition (EMT). Transcriptional intermediary factor 1γ (TIF1γ) which is a vital protein molecule that possesses ubiquitination enzyme activity, can mediate TGF-β induced EMT. We aimed to investigate the detailed mechanism between VPA and EMT occurrence in PCa cells to clarify the potential mechanism of TIF1γ involved. In our vitro experiments, we first investigated the effect of VPA on the expression TIF1γ. After TIF1γ was knockdown or overexpressed by related lentivirus, EMT of PCa cells were assessed. When TIF1γ knockdown or overexpress stable cell line were established, cells were treated with additional VPA, EMT index were detected and functional experiments were also conducted to confirm whether VPA inhibited EMT of PCa cells via TIF1γ. The mono-ubiquitination of Smad4 was analyzed simultaneously. In vivo, mice were facilitated with PC3 cells or TIF1γ related knockdown or overexpress virus transfected PC3 cells with or without VPA administration. Results showed that in vitro VPA can increase the expression of TIF1γ and also induce the increase expression of E-cadherin, and the decrease of N-cadherin and vimentin. Knocking down of TIF1γ can effectively block the effect of VPA on EMT and metastasis while overexpression of TIF1γ can strengthen its role. In vivo VPA also showed its anti-growth effect including tumor growth and EMT mediated by TIF1γ coincide with in vitro experiments. In conclusion, VPA inhibits the EMT in PCa cells via up-regulating the expression of TIF1γ and the mono-ubiquitination Smad4. VPA could serve as a promising agent in PCa treatment, with new strategies based on its diverse effects on posttranscriptional regulation.

Topics: Animals; Cell Proliferation; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Neoplasm Metastasis; PC-3 Cells; Prostatic Neoplasms; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Valproic Acid; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β) superfamily members are critical signals in tissue homeostasis and pathogenesis. Melanoma grows in the epidermis and invades the dermis before metastasizing. This disease progression is accompanied by increased sensitivity to microenvironmental TGF-β. Here, we found that skin fat cells (adipocytes) promoted metastatic initiation by sensitizing melanoma cells to TGF-β. Analysis of melanoma clinical samples revealed that adipocytes, usually located in the deeper hypodermis layer, were present in the upper dermis layer within proximity to in situ melanoma cells, an observation that correlated with disease aggressiveness. In a coculture system, adipocytes secreted the cytokines IL-6 and TNF-α, which induced a proliferative-to-invasive phenotypic switch in melanoma cells by repressing the expression of the microRNA miR-211. In a xenograft model, miR-211 exhibited a dual role in melanoma progression, promoting cell proliferation while inhibiting metastatic spread. Bioinformatics and molecular analyses indicated that miR-211 directly targeted and repressed the translation of

Topics: Adipocytes; Animals; Cell Proliferation; Coculture Techniques; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Ligands; Melanoma; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasm Transplantation; NIH 3T3 Cells; Phenotype; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-β-induced (TGFΒI) is considered to be a vital gene in several carcinomas. In this study we determined the effect of TGFBI on the proliferative and metastatic potential of human urothelial carcinoma (UC) cells as well as its mRNA and protein expression, which were detected by RT-PCR and western blot, respectively. UC cell proliferation was analyzed by WST-1 assay and Hoechst 33258 staining. The effect of TGFBI on UC cell metastasis was analyzed using adhesion, migration and invasion assays. We found that TGFBI increased the proliferation of UC cells. Moreover, TGFBI enhanced the adhesion, migration and invasion of UC cells by upregulating MMP-2, MMP-9 and calpain-2 expression. We evaluated the effect of Epirubicin (EPI) on the regulation of TGFBI expression and found that TGFBI acts as a downstream target of EPI and is suppressed by EPI in UC cells. EPI is more effective in inhibiting the proliferation and metastasis of UC cells with high TGFBI expression. This study demonstrates that TGFBI might lead to tumorigenesis and progression of UC and those cells with high TGFBI expression may be vulnerable to relapse. EPI could prove to be a therapeutic option in patients with high TGFBI expressing UC cells.

Topics: Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epirubicin; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Recurrence, Local; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation; Urologic Neoplasms; Urothelium

Metastatic castration-resistant prostate cancer (mCRPC) is one of the main contributors to the death of prostate cancer patients. To date, the detailed molecular mechanisms underlying mCRPC are unclear. Given the crucial role of epithelial-mesenchymal transition (EMT) in cancer metastasis, we aimed to analyse the expression and function of Transforming growth factor-beta (TGF-β) signal-associated protein named Sox5 in mCRPC.. The protein expression levels were analysed by western blot, immunohistochemistry and immunofluorescence. Luciferase reporter assays and chromatin immunoprecipitation were employed to validate the target of Sox5. The effect of Smad3/Sox5/Twist1 on PCa progression was investigated in vitro and in vivo.. Here, we found that TGF-β-induced EMT was accompanied by increased Sox5 expression. Interestingly, knockdown of Sox5 expression attenuated EMT induced by TGF-β signalling. Furthermore, we demonstrated that Smad3 could bind to the promoter of Sox5 and regulate its expression. Mechanistically, Sox5 could bind to Twist1 promoter and active Twist1, which initiated EMT. Importantly, knockdown of Sox5 in prostate cancer cells resulted in less of the mesenchymal phenotype and cell migration ability. Furthermore, targeting Sox5 could inhibit prostate cancer progression in a xenograft mouse model. In clinic, patients with high Sox5 expression were more likely to suffer from metastases, and high Sox5 expression also has a lower progression-free survival and cancer specific-survival in clinic database.. Therefore, we propose a new mechanism in which Smad3/Sox5/Twist1 promotes EMT and contributes to PCa progression.

Topics: Animals; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Nuclear Proteins; Promoter Regions, Genetic; Prostatic Neoplasms, Castration-Resistant; Signal Transduction; Smad3 Protein; SOXD Transcription Factors; Survival Analysis; Transforming Growth Factor beta; Twist-Related Protein 1; Up-Regulation

TGF-β regulates both the tumor-forming and migratory abilities of various types of cancer cells. However, it is unclear how the loss of TGF-β signaling components affects these abilities in clear-cell renal cell carcinoma (ccRCC). In this study, we investigated the role of TGFBR3 (TGF-β type III receptor, also known as betaglycan) in ccRCC. Database analysis revealed decreased expression of TGFBR3 in ccRCC tissues, which correlated with poor prognosis in patients. Orthotopic inoculation experiments using immunocompromised mice indicated that low TGFBR3 expression in ccRCC cells enhanced primary tumor formation and lung metastasis. In the presence of TGFBR3, TGF-β2 decreased the aldehyde dehydrogenase (ALDH)-positive ccRCC cell population, in which renal cancer-initiating cells are enriched. Loss of TGFBR3 also enhanced cell migration in cell culture and induced expression of several mesenchymal markers in a TGF-β-independent manner. Increased lamellipodium formation by FAK-PI3K signaling was observed with TGFBR3 downregulation, and this contributed to TGF-β-independent cell migration in ccRCC cells. Taken together, our findings reveal that loss of TGFBR3 endows ccRCC cells with multiple metastatic abilities through TGF-β-dependent and independent pathways.

Topics: Animals; Carcinoma, Renal Cell; Cell Movement; Cells, Cultured; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; HEK293 Cells; Humans; Kidney Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Proteoglycans; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Increasing evidence suggests that perioperative factors including anaesthetics influence cancer recurrence and metastasis after surgery. This study investigated the influence of sevoflurane on the response of lung and renal cancer cells to cisplatin, with focus on transforming growth factor-beta (TGF-β) and osteopontin (OPN) that are both closely associated with cancer tumorigenesis and metastasis.. Non-small cell lung adenocarcinoma (A549) and renal cell carcinoma (RCC4) cells were exposed to 3.6% sevoflurane for two hrs. Malignant potential represented by cell viability, migration, chemosensitivity to cisplatin was evaluated. Expression of OPN, TGF-β1, TGF-β receptor type II (TGF-βRII) and the canonical downstream effector Smad3 was assessed. SiRNA knockdown of TGF-β1 and OPN and chemical inhibition of TGF-βRI/II was performed.. Sevoflurane reduced cell viability (0.394) versus control (0.459) (P < 0.01), enhanced chemosensitivity but had no effect on migration of A549 cells. It enhanced viability (0.467) versus control (0.347) (P < 0.001), chemoresistance and migration of RCC4. In A549, there was enhanced nuclear Smad3. In RCC4, TGF-βRII and OPN were upregulated, while TGF-β1 was over- expressed with reduced nuclear Smad3. TGF-βRII inhibition and OPN knockdown abolished sevoflurane-mediated viability, and migration, respectively, in RCC4.. Sevoflurane promotes the metastatic potential of renal carcinoma, but not of non-small cell lung cancer. This may be associated with its differential effect on cellular signalling including TGF-β. Our findings indicate that sevoflurane may have different effects on the metastatic potential and chemosensitivity of different tumour types.

Topics: A549 Cells; Anesthetics, Inhalation; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Neoplasm Metastasis; Osteopontin; Sevoflurane; Smad3 Protein; Transforming Growth Factor beta; Wound Healing

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, Neoplasm; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Cell Cycle Checkpoints; Cohort Studies; Collagen; Disease Models, Animal; Drug Resistance, Neoplasm; Fibroblasts; Humans; Immunotherapy; Mice; Mutation; Neoplasm Metastasis; Phenotype; Signal Transduction; Transforming Growth Factor beta; Treatment Outcome; Tumor Microenvironment; Urologic Neoplasms; Urothelium

Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (T

Topics: Alleles; Animals; Cell Differentiation; Colonic Neoplasms; Disease Models, Animal; Drug Synergism; Female; Humans; Immune Evasion; Immunotherapy; Intestinal Mucosa; Intestines; Liver Neoplasms; Male; Mice; Mutation; Neoplasm Metastasis; Programmed Cell Death 1 Receptor; Stem Cells; T-Lymphocytes, Cytotoxic; Th1 Cells; Transforming Growth Factor beta; Tumor Microenvironment

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Topics: Adolescent; Animals; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cerebellar Neoplasms; Child; Child, Preschool; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Infant; Male; Medulloblastoma; Mice; Mice, Inbred BALB C; Models, Molecular; Neoplasm Metastasis; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Pyrimidinones; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

TGF-β has been demonstrated to promote tumor metastasis, and the regulatory mechanisms are poorly understood. Here, we report the role of USP2a in promoting metastasis by facilitating TGF-β-triggered signaling. USP2a interacts with TGFBR1 and TGFBR2 upon TGF-β stimulation and removes K33-linked polyubiquitin chains from Lys502 of TGFBR1, promoting the recruitment of SMAD2/3. Simultaneously, TGFBR2 phosphorylates Ser207/Ser225 of USP2a, leading to the disassociation of SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively correlated in human tumor biopsies, and USP2a is hyper-phosphorylated in lung adenocarcinomas with lymph node invasion. Depletion or pharmacologic inhibition of USP2a dampens TGF-β-triggered signaling and metastasis. Our findings have characterized an essential role of USP2a as a potential target for treatment of metastatic cancers.

Topics: Amino Acid Sequence; Animals; Endopeptidases; Female; HEK293 Cells; Humans; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Metastasis; Phosphorylation; Polyubiquitin; Promoter Regions, Genetic; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases; Ubiquitination

The role of transforming growth factorβ (TGF-β)-induced tumor progression in advanced malignancy is well established, but the involvement of long non-coding RNAs (lncRNAs) in TGF-β signaling remains unclear. This study aimed to identify TGF-β-associated lncRNAs in head and neck squamous cell carcinoma (HNSCC).. Expression profiling of lncRNAs was obtained using Gene Expression Omnibus and The Cancer Genome Atlas. Real-time quantitative PCR was used to analyze the expression of EPB41L4A-AS2 in HNSCC cell line. We used bioinformatics resources (DAvID) to conduct Gene Ontology biological processes and KEGG pathways at the significant level. Wound healing assay, cell migration and invasion assays, were used to examine the effects of EPB41L4A-AS2 on tumor cell metastasis in vivo. Protein levels of EPB41L4A-AS2 targets were determined by western blot.. A novel TGF-β-associated lncRNA, EPB41L4A-AS2, was found downregulated by TGF-β and associated with invasion and metastasis. The relationship of EPB41L4A-AS2 with the clinicopathological features and prognosis of HNSCC patients was evaluated. Bioinformatic analyses revealed that EPB41L4A-AS2 may be involved in processes associated with the tumor-associated signaling pathway, especially the TGF-β signaling pathway. Furthermore, a TGF-β-induced epithelial-to-mesenchymal transition (EMT) model was established. Low EPB41L4A-AS2 expression was determined, and overexpression of this gene inhibited cell migration and invasion in the EMT model. Moreover, EPB41L4A-AS2 suppressed TGFBR1 expression.. EPB41L4A-AS2 might serve as a negative regulator of TGF-β signaling and as an effective prognostic biomarker and important target in anti-metastasis therapies of HNSCC patients.

Topics: Cell Line, Tumor; Cell Movement; Computational Biology; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Models, Genetic; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Long Noncoding; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Survival Analysis; Transforming Growth Factor beta; Up-Regulation

Sauchinone, one of the active lignan isolated from the roots of Saururus chinensis, was reported to possess diverse pharmacological properties, such as hepatoprotective, anti-inflammatory and anti-tumor effects. However, the possible role of sauchinone in the epithelial-mesenchymal transition (EMT) remains unclear. Thus, the aim of this study was to investigate the effect of sauchinone on the EMT in gastric cancer cells. Our results demonstrated that sauchinone significantly inhibited transforming growth factor-β1 (TGF-β1)-induced migration and invasion in gastric cancer cells. In addition, sauchinone efficiently suppressed TGF-β1-induced EMT process in gastric cancer cells. Furthermore, pretreatment with sauchinone dramatically inhibited the activation of PI3K/Akt and Smad2/3 signaling pathways in TGF-β1-stimulated gastric cancer cells. In conclusion, our findings revealed that sauchinone inhibits the TGF-β1-induced EMT in gastric cancer cells via down-regulation of PI3K/Akt and Smad2/3 signaling pathways. Thus, sauchinone may be a therapeutic agent for treatment of gastric cancer.

Topics: Benzopyrans; Cell Line, Tumor; Cell Movement; Dioxoles; Epithelial-Mesenchymal Transition; Gastric Mucosa; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad2 Protein; Smad3 Protein; Stomach; Stomach Neoplasms; Transforming Growth Factor beta

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

Inhibin is a heterodimeric TGFβ family ligand that is expressed in many cancers and is a selective biomarker for ovarian cancers; however, its tumor-specific functions remain unknown. Here, we demonstrate that the α subunit of inhibin (INHA), which is critical for the functionality of dimeric inhibin A/B, correlates with microvessel density in human ovarian tissues and is predictive of poor clinical outcomes in multiple cancers. We demonstrate that inhibin-regulated angiogenesis is necessary for metastasis. Although inhibin had no direct impact on tumor cell signaling, both tumor cell-derived and recombinant inhibin elicit a strong paracrine response from endothelial cells by triggering SMAD1/5 activation and angiogenesis i

Topics: Activin Receptors, Type II; Animals; Biomarkers, Tumor; Cell Line, Tumor; Female; Human Umbilical Vein Endothelial Cells; Humans; Inhibins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; Ovarian Neoplasms; Signal Transduction; Transforming Growth Factor beta

The precise role of tumor associated macrophages remains unclear in pancreatic ductal adenocarcinoma (PDAC) while TGF-ß has an unclear role in metastases formation. In order to understand the role of IL23, an interleukin associated with macrophage polarization, we investigated IL23 in the context of TGF-ß expression in PDAC. We hypothesized that IL23 expression is associated with metastatic development and survival in PDAC. We investigated IL23 and TGF-ß protein expression on resected PDAC patient tumor sections who were divided into short-term (<12 months) survivors and long-term (>30 months) survivors. Panc-1 cells treated with IL23, TGF-ß, macrophages, or combinations thereof, were orthotopically implanted into NSG mice. Patients in the long-term survivor group had higher IL23 protein expression (P = 0.01). IL23 expression was linearly correlated with TGF-ß expression in patients in the short-term survivor group (P = 0.038). Macrophages induce a higher rate of PDAC metastasis in the mouse model (P = 0.02), which is abrogated by IL23 and TGF-ß treatment (P < 0.001). Macrophages serve a critical role in PDAC tumor growth and metastasis. TGF-ß contributes to a less tumorigenic TME through regulation of macrophages. Macrophages increases PDAC primary tumor growth and metastases formation while combined IL23 and TGF-ß pre-treatment diminishes these processes.

Topics: Aged; Animals; Carcinoma, Pancreatic Ductal; Disease Models, Animal; Humans; Immunohistochemistry; Interleukin-23 Subunit p19; Macrophages; Mice; Middle Aged; Neoplasm Metastasis; Survival Analysis; Transforming Growth Factor beta

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

Colorectal cancer (CRC) is a malignancy that has high morbidity and mortality and is initiated from accumulative genetic events. Although much effort has been made to elucidate the genetic mechanism underlying this disease, it still remains unknown. Here, we discovered a novel role for multiple epidermal growth factor-like domains protein 6 (MEGF6) in CRC, namely, that it induces the epithelial-to-mesenchymal transition (EMT) to promote CRC metastasis via the transforming growth factor beta (TGFβ)/SMAD signaling pathway.. RNA sequencing data from the Gene Expression Omnibus database were analyzed using R software. Based on The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort, the clinical significance of MEGF6 was investigated. HCT8R, HCT116, and LoVo CRC cells were transfected with small interfering RNA against MEGF6, and their proliferation and sensitivity to fluorouracil were evaluated with the MTT cell proliferation and colony formation assays. Proteins associated with cell growth were detected by western blot analysis. The apoptosis of cells was evaluated by Annexin V/propidium iodide staining, and transwell assays were performed to assess the involvement of MEGF6 in cell migration. Markers of EMT and TGFβ/SMAD signaling were evaluated by quantitative PCR and western blotting, and the correlation between MEGF6 and these markers was assessed in the TCGA colon and renal adenocarcinoma cohort.. The results showed that MEGF6 was upregulated in HCT8R cells. In addition, MEGF6 was significantly overexpressed in tumor tissue and predicted a poor survival in the TCGA-COAD cohort. Moreover, MEGF6 accelerated CRC cell growth and inhibited apoptosis, and promoted CRC metastasis by inducing the EMT. Finally, we found that TGFβ/SMAD signaling triggered the expression of Slug, which regulates the MEGF6-mediated EMT.. MEGF6 may serve as an oncogene to promote cell proliferation and inhibit apoptosis. MEGF6 can also accelerate cell migration via TGFβ/SMAD signaling-mediated EMT.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colon; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Intercellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Mice; Neoplasm Metastasis; Rectum; Signal Transduction; Smad Proteins; 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

Cancer metastatic spread to serous cavity causes malignant pleural effusions (MPEs), indicating dismal prognosis. Tumor microenvironment can implement suppressive activity on host immune responses. Thus, we investigated the prevalence of Tregs and the relationship between them and TGF-

Topics: Cells, Cultured; CTLA-4 Antigen; Forkhead Transcription Factors; Gene Expression Regulation; Glucocorticoid-Induced TNFR-Related Protein; Humans; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Staging; Pleural Effusion, Malignant; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor β (TGF-β) is critical for embryonic development, adult tissue homeostasis, and tumor progression. TGF-β suppresses tumors at early stage, but promotes metastasis at later stage through oncogenes such as Twist1. Gamma-synuclein (SNCG) is overexpressed in a variety of invasive and metastatic cancer. Here, we show that TGF-β induces SNCG expression by Smad-Twist1 axis, thus promoting TGF-β- and Twist1-induced cancer cell migration and invasion. We identify multiple Twist1-binding sites (E-boxes) in SNCG promoter. Chromatin immunoprecipitation and luciferase assays confirm the binding of Twist1 to the E-boxes of SNCG promoter sequence (-129/-1026 bp). Importantly, the Twist1-binding site close to the transcription initiation site is critical for the upregulation of SNCG expression by TGF-β and Twist1. Mutations of Twist1 motif on the SNCG promoter constructs markedly reduces the promoter activity. We further show that TGF-β induces Twist1 expression through Smad thereby enhancing the binding of Twist1 to SNCG promoter, upregulating SNCG promoter activity and increasing SNCG expression. SNCG knockdown abrogates TGF-β- or Twist1-induced cancer cell migration and invasion. Finally, SNCG knockdown inhibits the promotion of cancer metastasis by Twist1. Together, our data demonstrate that SNCG is a novel target of TGF-β-Smad-Twist1 axis and a mediator of Twist1-induced cancer metastasis.

Topics: Cell Movement; E-Box Elements; gamma-Synuclein; Gene Expression Regulation, Neoplastic; HeLa Cells; Hep G2 Cells; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Smad Proteins; Transforming Growth Factor beta; Twist-Related Protein 1; 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

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and metastasis and thus accelerates cancer progression. p21-activated kinase 4 (PAK4) is a critical regulator of prostate cancer (PC) progression. Here, we report that PAK4 activation promotes PC progression through the EMT regulator Slug. We find that phosphorylated PAK4

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Male; Mice; Neoplasm Metastasis; p21-Activated Kinases; Phosphorylation; Prognosis; Prostatic Neoplasms; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

BACKGROUND Malignant glioma is intractable primary brain carcinoma that has a poor survival rate. Natural diterpenoid isoferritin A (IsoA) presents antitumor effects by regulating signal pathways in tumor cells. In the present study we investigated the inhibitory effects of IsoA on glioma cells. MATERIAL AND METHODS The potential molecular mechanism of IsoA-mediated glioma cell growth and metastasis were investigated using Western blot, gene knockdown, immunofluorescence, and immunohistochemistry. RESULTS Results showed that IsoA significantly inhibits growth and metastasis of glioma cells in multiple preclinical settings. In vitro assay showed that IsoA (4 mg/ml) treatment significantly induced apoptosis of glioma cells. Mechanism analysis demonstrated that IsoA (4 mg/ml) treatment decreased TGFβ and regulated EMT markers expression in glioma cells. Reduced expression of TGFβ in glioma cells was closely correlated with inhibitory effects of IsoA on growth and metastasis of glioma cells. TGFβ overexpression promoted glioma cell growth and invasion. Results also showed that IsoA treatment significantly decreased Fibronectin and Vimentin and increased E-cadherin, while TGFβ overexpression abolished the regulation mediated by IsoA in glioma cells. In vivo assay showed that IsoA treatment inhibited tumor growth in a glioma-bearing mouse model. CONCLUSIONS Results indicate that IsoA could be regarded as a potential anti-cancer agent by regulating TGFβ-induced EMT signal pathway.

Topics: Animals; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Diterpenes; Epithelial-Mesenchymal Transition; Ferritins; Gene Expression Regulation, Neoplastic; Glioma; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Topics: Animals; Cachexia; Cation Transport Proteins; Cell Differentiation; Cell Line; Cytokines; Disease Models, Animal; Humans; Mice, Inbred C57BL; Muscle, Skeletal; Myosin Heavy Chains; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation; Zinc

Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis.. CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed. CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival.. Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.

Topics: Animals; Biomarkers; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Cell Survival; Colorectal Neoplasms; Disease Models, Animal; Endoglin; Fibroblasts; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ligands; Male; Mice; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Zebrafish

Topics: Animals; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta; TRPC6 Cation Channel

Peritoneal metastasis is a primary metastatic route for gastric cancers, and the mechanisms underlying this process are still unclear. Peritoneal mesothelial cells (PMCs) undergo mesothelial-to-mesenchymal transition (MMT) to provide a favorable environment for metastatic cancer cells. In this study, we investigated how the exosomal miR-21-5p induces MMT and promotes peritoneal metastasis. Gastric cancer (GC)-derived exosomes were identified by transmission electron microscopy and western blot analysis, then the uptake of exosomes was confirmed by PKH-67 staining. The expression of miR-21-5p and SMAD7 were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and the interactions between miR-21-5p and its target genes SMAD7 were confirmed by Luciferase reporter assays. The MMT of PMCs was determined by invasion assays, adhesion assays, immunofluorescent assay, and western blot. Meanwhile, mouse model of tumor peritoneal dissemination model was performed to investigate the role of exosomal miR-21-5p in peritoneal metastasis in vivo. We found that PMCs could internalize GC-derived exosomal miR-21-5p and led to increased levels of miR-21-5p in PMCs. Through various types of in vitro and in vivo assays, we confirmed that exosomal miR-21-5p was able to induce MMT of PMCs and promote tumor peritoneal metastasis. Moreover, our study revealed that this process was promoted by exosomal miR-21-5p through activating TGF-β/Smad pathway via targeting SMAD7. Altogether, our data suggest that exosomal miR-21-5p induces MMT of PMCs and promote cancer peritoneal dissemination by targeting SMAD7. The exosomal miR-21-5p may be a novel therapeutic target for GC peritoneal metastasis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Epithelium; Exosomes; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Peritoneal Neoplasms; Peritoneum; Signal Transduction; Smad7 Protein; Stomach Neoplasms; Transforming Growth Factor beta

We previously reported that miR-203 functions as a tumor suppressor in ovarian cancer cells by directly targeting transcription factor Snai2 and inhibiting epithelial to mesenchymal transition (EMT), whereas BIRC5/survivin promotes EMT. In this study, we tested our hypothesis that miR-203 inhibits ovarian tumor metastasis by suppressing EMT through targeting BIRC5, using an orthotopic ovarian cancer mouse model.. We overexpressed miR-203 in ovarian cancer SKOV3 and OVCAR3 cells using a lentiviral vector and examined cell migration and invasion using transwell plates. The small molecule inhibitor, YM155, was used to inhibit survivin expression. miR-203-expressing and control SKOV3 cells were intrabursally injected into immunocompromised NSG female mice. Primary tumors in ovaries and metastatic tumors were collected to determine the expression of survivin and EMT markers using Western blot and immunostaining.. Overexpression of miR-203 inhibits EMT by targeting BIRC5 in ovarian cancer SKOV3 and OVCAR3 cells. miR-203 expression enhances the ability of the survivin inhibitor YM155 to reduce tumor cell migration and invasion in vitro. We further showed that miR-203 expression attenuated the TGFβ pathway in both SKOV3 and OVCAR3 cells. miR-203 expression also inhibited primary tumor growth in ovaries and metastatic tumors in multiple peritoneal organs including liver and spleen.. miR-203 inhibits ovarian tumor metastasis by targeting BIRC5/survivin and attenuating the TGFβ pathway.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Inhibitor of Apoptosis Proteins; Lentivirus; Mice; MicroRNAs; Neoplasm Metastasis; Ovarian Neoplasms; Signal Transduction; Survivin; Transforming Growth Factor beta

Previously, we reported a molecular mechanism by which Ahnak potentiates transforming growth factor-β (TGFβ) signaling during cell growth. Here, we show that Ahnak induces epithelial-mesenchymal transition (EMT) in response to TGFβ. EMT phenotypes, including altered in cell morphology, and expression patterns of various EMT marker genes were detected in HaCaT keratinocytes transfected with Ahnak-specific siRNA. Knockdown of Ahnak expression in HaCaT keratinocytes resulted in attenuated cell migration and invasion. We found that Ahnak activates TGFβ signaling via Smad3 phosphorylation, leading to enhanced Smad3 transcriptional activity. To validate function of Ahnak in EMT of B16F10 cells having high metastatic and tumorigenic properties, we established B16F10 cells with stable knockdown of Ahnak. N-cadherin expression and Smad3 phosphorylation were significantly decreased in B16F10-shAhnak cells, compared to B16F10-shControl cells after treatment of TGFβ. Moreover, TGFβ failed to induce cell migration and cell invasion in B16F10-shAhnak cells. To determine whether Ahnak regulates the metastatic activity of B16F10 cells, we established a lung metastasis model in C57BL/6 mice via tail vein injection of B16F10-shAhnak cells. Lung metastasis was significantly suppressed in mice injected with B16F10-shAhnak cells, compared to those injected with B16F10-shControl cells. Taken together, we propose that TGFβ-Ahnak signaling axis regulates EMT during tumor metastasis.

Topics: Animals; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lung; Lung Neoplasms; Male; Melanoma, Experimental; Membrane Proteins; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Proteins; Transforming Growth Factor beta

TGF-β promotes tumor invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in human cancers. However, whether and how circRNAs contribute to TGF-β-induced EMT and metastasis in NSCLC remain vague. Here, we investigated the regulation and function of Circular RNA hsa_circ_0008305 (circPTK2) in TGF-β-induced EMT and tumor metastasis, as well as a link between circPTK2 and transcriptional intermediary factor 1 γ (TIF1γ) in NSCLC.. Circular RNAs were determined by human circRNA Array analysis, real-time quantitative reverse transcriptase PCR and northern blot. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were employed to test the interaction between circPTK2 and miR-429/miR-200b-3p. Ectopic overexpression and siRNA-mediated knockdown of circPTK2, TGF-β-induced EMT, Transwell migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPTK2. Transcription and prognosis analyses were done in public databases.. CircPTK2 and TIF1γ were significantly down-regulated in NSCLC cells undergoing EMT induced by TGF-β. CircPTK2 overexpression augmented TIF1γ expression, inhibited TGF-β-induced EMT and NSCLC cell invasion, whereas circPTK2 knockdown had the opposite effects. CircPTK2 functions as a sponge of miR-429/miR-200b-3p, and miR-429/miR-200b-3p promote TGF-β-induced EMT and NSCLC cell invasion by targeting TIF1γ. CircPTK2 overexpression inhibited the invasion-promoting phenotype of endogenous miR-429/miR-200b-3p in NSCLC cells in response to TGF-β. CircPTK2 overexpression significantly decreased the expression of Snail, an important downstream transcriptional activator of TGF-β/Smad signaling. In an in vivo experiment of metastasis, circPTK2 overexpression suppressed NSCLC cell metastasis. Moreover, circPTK2 expression was dramatically down-regulated and positively correlated with TIF1γ expression in human NSCLC tissues. Especially, circPTK2 was significantly lower in metastatic NSCLC tissues than non-metastatic counterparts.. Our findings show that circPTK2 (hsa_circ_0008305) inhibits TGF-β-induced EMT and metastasis by controlling TIF1γ in NSCLC, revealing a novel mechanism by which circRNA regulates TGF-β-induced EMT and tumor metastasis, and suggesting that circPTK2 overexpression could provide a therapeutic strategy for advanced NSCLC.

Topics: 3' Untranslated Regions; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; MicroRNAs; Neoplasm Metastasis; RNA; RNA Interference; RNA, Circular; Transcription Factors; Transforming Growth Factor beta

Pancreatic cancer remains a challenging disease with an overall cumulative 5-year survival rate around 6%. Though significant progress has been made in the availability of diagnostic techniques and treatment strategies, pancreatic cancer remains a disease of high mortality rate. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that governs the oncogenesis and metastasis process of pancreatic cancer. In the present study, by using the Cancer Genome Atlas (TCGA) dataset analysis, we demonstrated that sorting nexin 6 (SNX6) serves as a biomarker for predicting prognosis of pancreatic cancer. In vitro studies demonstrated that silencing of SNX6 expression reduced cell proliferation, colony formation, invasion, and metastasis. Higher level of SNX6 helps maintain the mesenchymal properties, which renders migration and invasive capacities to pancreatic cancer cells. Moreover, in the process of TGF-β-induced epithelial to mesenchymal transition (EMT), the expression level of SNX6 was increased, and silencing of SNX6 expression could inhibit the TGF-β-induced EMT program. These results collectively uncovered a novel predictive marker for pancreatic cancer and provided the possible underlying molecular mechanism.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Metastasis; Pancreatic Neoplasms; Prognosis; RNA Interference; Sorting Nexins; Survival Analysis; Transforming Growth Factor beta

Helicase‑like transcription factor (HLTF) has been identified as a tumor suppressor gene. The hypermethylation of HTLF is frequently observed in various types of cancer, including colorectal cancer (CRC). However, the mechanisms through which HLTF suppresses CRC progression remain unclear. Thus, the aim of the present study was to explore the biological function of HLTF in CRC cells and the underlying mechanisms. CRC tissues and cells were used to detect the expression of HLTF. Wound‑healing and Transwell assays were performed to assess the motility of CRC cells. The results revealed that HLTF expression was significantly associated with the differentiation status, invasion depth, lymph node metastasis and distant metastasis. A low HLTF expression was significantly associated with a poor survival. Furthermore, HTLF knockdown or ectopic overexpression significantly promoted or suppressed the motility of CRC cells, respectively. With regard to the underlying molecular mechanisms, the protein expression of HTLF was upregulated when the CRC cells were stimulated with transforming growth factor (TGF)‑β, and HLTF upregulation induced an increase in SMAD4 and p‑SMAD2/3 expression and a decrease in levels of the TGF‑β/SMAD pathway downstream genes, Vimentin and zinc finger e‑box binding homeobox 1 (ZEB1). On the whole, the findings of this study suggest that HLTF is negatively associated with the progression of CRC, and its overexpression suppresses the migration and invasion of CRC cells by targeting the TGF‑β/SMAD pathway.

Topics: Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Progression; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; HT29 Cells; Humans; In Vitro Techniques; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Phosphorylation; Prognosis; Signal Transduction; Smad Proteins; Survival Analysis; Transforming Growth Factor beta

Alpinia nantoensis (Zingiberaceae) is an aromatic plant endemic to Taiwan, which is used as food flavoring and traditional herbal medicine. The biological activities of compounds isolated from this plant are rarely investigated.. The present study was aimed to investigate the anti-metastatic potential of trans-3‑methoxy‑5-hydroxystilbene (MHS) a major stilbene isolated from the rhizomes of A. nantonensis.. We investigated the anti-metastatic potential of MHS on human non-small cell lung carcinoma (A549) cell line using wound healing, trans-well, western blot, zymography and immunofluorescence assays.. Initial cytotoxicity assay showed that treatment with MHS did not exhibit cytotoxicity to A549 cells up to the concentration of 40 µM. Further in vitro wound healing and transwell chamber assays revealed that MHS significantly inhibited tumor cell migration in a dose-dependent manner, which is associated with inhibition of matrix mettalloprotinase-2 (MMP-2) and matrix mettalloprotinase-9 (MMP-9) at both enzyme and protein levels. The inhibition of MMPs activity by MHS was reasoned by suppression of their corresponding transcription factor, β-catenin as indicated by reduced levels of β-catenin in the nucleus. MHS also regulates epithelial-to-mesenchymal transition (EMT) by increasing E-cadherin and occludin as well as decreasing N-cadherin levels in A549 cells. Furthermore, pre-treatment with MHS significantly inhibited A549 cells migration and EMT in TGF-β induced A549 cells.. To the best of our knowledge, this is the first report demonstrating that MHS, a plant-derived stilbene has a promising ability to inhibit lung cancer cell metastasis in vitro.

Topics: A549 Cells; Alpinia; Antigens, CD; Antineoplastic Agents, Phytogenic; beta Catenin; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Metastasis; Rhizome; Stilbenes; Taiwan; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) have been extensively characterized in development and cancer, and its dynamics have been modeled as a non-linear process. However, less is known about how such dynamics may affect its biological impact. Here, we use mathematical modeling and experimental analysis of the TGF-β-induced EMT to reveal a non-linear hysteretic response of E-cadherin repression tightly controlled by the strength of the miR-200s/ZEBs negative feedback loop. Hysteretic EMT conveys memory state, ensures rapid and robust cellular response and enables EMT to persist long after withdrawal of stimuli. Importantly, while both hysteretic and non-hysteretic EMT confer similar morphological changes and invasive potential of cancer cells, only hysteretic EMT enhances lung metastatic colonization efficiency. Cells that undergo hysteretic EMT differentially express subsets of stem cell and extracellular matrix related genes with significant clinical prognosis value. These findings illustrate distinct biological impact of EMT depending on the dynamics of the transition.

Topics: Animals; Cadherins; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Feedback, Physiological; Female; Mice, Inbred BALB C; MicroRNAs; Models, Biological; Neoplasm Metastasis; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Tumor-associated macrophages (TAMs) were reported to be involved in colorectal cancer (CRC) progression. However, its biological role and underlying mechanism in CRC remained to be elucidated. In this study, the expressions of the macrophage marker CD68 and transforming growth factor β1 (TGF-β1) in CRC tumor tissues and adjacent tissues were detected by immunohistochemistry. The expression levels of miR-34a, TGF-β1 and vascular endothelial growth factor (VEGF) in CRC tumor tissues and peripheral blood macrophages were measured by quantitative real-time PCR (qRT-PCR) and western blot. TGF-β1 levels in culture supernatant were detected by ELISA. The cell proliferation and invasion of human CRC cell lines CL187 and HCT116 were determined by MTT assay and Transwell assay, respectively. The results showed that the expression of miR-34a was downregulated whereas TGF-β1 and VEGF were upregulated in CRC tumor tissues and peripheral blood macrophages. TGF-β1 secreted by TAMs promoted the proliferation and invasion of CRC cells. TGF-β1-mediated miR-34a downregulation contributed to the proliferation and invasion of CRC cells via upregulating VEGF. MiR-34a in vivo exerted anti-tumor effect in CRC via inhibiting VEGF expression. In conclusion, TGF-β1 secreted by TAMs promoted CRC proliferation and invasion through regulating miR-34a/VEGF axis.

Topics: Animals; Antagomirs; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Paradoxically, during early tumor development in many cancer types, TGF-β acts as a tumor suppressor, whereas in the advanced stages of these cancers, increased TGF-β expression is linked to high metastasis and poor prognosis. These findings suggest that unidentified mechanisms may function to rewire TGF-β signaling toward its prometastatic role in cancer cells. Our current study using non-small-cell lung carcinoma (NSCLC) cell lines, animal models, and clinical specimens demonstrates that suppression of SMAD2, with SMAD3 function intact, switches TGF-β-induced transcriptional responses to a prometastatic state. Importantly, we identified chaperonin containing TCP1 subunit 6A (CCT6A) as an inhibitor and direct binding protein of SMAD2 and found that CCT6A suppresses SMAD2 function in NSCLC cells and promotes metastasis. Furthermore, selective inhibition of SMAD3 or CCT6A efficiently suppresses TGF-β-mediated metastasis. Our findings provide a mechanism that directs TGF-β signaling toward its prometastatic arm and may contribute to the development of therapeutic strategies targeting TGF-β for NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chaperonin Containing TCP-1; Humans; Lung Neoplasms; Mice; Neoplasm Metastasis; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

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

Osteosarcoma is the most common type of bone cancer, especially in children and young adults. Recently, long noncoding RNAs (lncRNAs) have emerged as new prognostic markers and gene regulators in several cancers, including osteosarcoma. In this study, we investigated the contributions of the lncRNA MALAT1 in osteosarcoma with a specific focus on its transcriptional regulation and its interaction with EZH2. Our results showed that MALAT1 was significantly increased in osteosarcoma specimens and cell lines. ROC curve analysis showed that MALAT1 had a higher area under the curve than alkaline phosphatase, and Kaplan-Meier survival analysis indicated that patients with high serum levels of MALAT1 showed reduced survival rate. Knockdown of MALAT1 decreased osteosarcoma cell invasion and promoted E-cadherin expression. Mechanistic investigations showed that MALAT1 was transcriptionally activated by TGF-β. Additionally, EZH2 is highly expressed and associated with the 3' end region of lncRNA MALAT1 in osteosarcoma, and this association finally suppressed the expression of E-cadherin. Subsequently, our gain and loss function assay showed that MALAT1 overexpression promoted cell metastasis and decreased E-cadherin level, however, this effect was partially reversed by EZH2 knockdown. In conclusion, our work illuminates that lncRNA MALAT1 is a potential diagnostic and prognostic factor in osteosarcoma and further demonstrates how MALAT1 confers an oncogenic function. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for osteosarcoma patients.

Topics: 3' Untranslated Regions; Adult; Bone Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Osteosarcoma; Prognosis; RNA, Long Noncoding; ROC Curve; Transforming Growth Factor beta; Tumor Burden; Young Adult

Angiostasis mediated by interferon (IFN)-γ is a key mechanism of anti-tumour immunity; however, the effect of IFN-γ on host vascular endothelial growth factor A (VEGFA)-expressing cells during tumour progression is still elusive. Here, we developed transgenic mice with IFN-γ receptor (IFNγR) expression under control of the Vegfa promoter (V-γR). In these mice, the IFN-γ responsiveness of VEGFA-expressing cells led to dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumour metastasis were observed in the tumour-bearing V-γR mice, in comparison with the control wild-type and IFNγR-deficient mice. Further study showed that perivascular cells were VEGFA-expressing cells and potential IFN-γ targets. In vivo, tumour vascular perfusion and pericyte association with blood vessels were massively disrupted in V-γR mice. In vitro, IFN-γ inhibited transforming growth factor-β signalling by upregulating SMAD7, and therefore downregulated N-cadherin expression in pericytes. Importantly, IFN-γ neutralization in vivo with a monoclonal antibody reduced tumour metastasis. Together, the results suggest that IFNγR-mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumour metastasis. Thus, the inhibition of tumour growth via IFN-γ-induced angiostasis might also accelerate tumour metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Cadherins; Cell Line, Tumor; Down-Regulation; Fibroblasts; Interferon gamma Receptor; Interferon-gamma; Lung Neoplasms; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Pericytes; Receptors, Interferon; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P=.005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial-mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Regulatory Networks; HCT116 Cells; Humans; Kallikreins; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

Osteosarcoma is suggested to be caused by genetic and molecular alterations that disrupt osteoblast differentiation. Recent studies have reported that transmembrane protein 119 (TMEM119) contributes to osteoblast differentiation and bone development. However, the level of TMEM119 expression and its roles in osteosarcoma have not yet been elucidated. In the present study, TMEM119 mRNA and protein expression was found to be up-regulated in osteosarcoma compared with normal bone cyst tissues. The level of TMEM119 protein expression was strongly associated with tumor size, clinical stage, distant metastasis and overall survival time. Moreover, gene set enrichment analysis (GSEA) of the Gene Expression Omnibus (GEO) GSE42352 dataset revealed TMEM119 expression in osteosarcoma tissues to be positively correlated with cell cycle, apoptosis, metastasis and TGF-β signaling. We then knocked down TMEM119 expression in U2OS and MG63 cells using small interfering RNA, which revealed that downregulation of TMEM119 could inhibit the proliferation of osteosarcoma cells by inducing cell cycle arrest in G0/G1 phase and apoptosis. We also found that TMEM119 knockdown significantly inhibited cell migration and invasion, and decreased the expression of TGF-β pathway-related factors (BMP2, BMP7 and TGF-β). TGF-β application rescued the inhibitory effects of TMEM119 knockdown on osteosarcoma cell migration and invasion. Further in vitro experiments with a TGF-β inhibitor (SB431542) or BMP inhibitor (dorsomorphin) suggested that TMEM119 significantly promotes cell migration and invasion, partly through TGF-β/BMP signaling. In conclusion, our data support the notion that TMEM119 contributes to the proliferation, migration and invasion of osteosarcoma cells, and functions as an oncogene in osteosarcoma.

Topics: Adolescent; Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Female; HEK293 Cells; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Osteosarcoma; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The growth and invasion of cancer cells are very complex processes, which can be regulated by the cross-talk between various signalling pathways, or by single signalling pathways that can control multiple aspects of cell behaviour. TGF-β is one of the most investigated signalling pathways in oncology, since it can regulate multiple aspects of cell behaviour: cell proliferation and apoptosis, cell-cell adhesion and epithelial-to-mesenchimal transition via loss of cell adhesion. In this study, we use a mathematical modelling approach to investigate the complex roles of TGF-β signalling pathways on the inhibition and growth of tumours, as well as on the epithelial-to-mesenchimal transition involved in the metastasis of tumour cells. We show that the nonlocal mathematical model derived here to describe repulsive and adhesive cell-cell interactions can explain the formation of new tumour cell aggregations at positions in space that are further away from the main aggregation. Moreover, we show that the increase in cell-cell adhesion leads to fewer but larger aggregations, and the increase in TGF-β molecules - whose late-stage effect is to decrease cell adhesion - leads to many small cellular aggregations. Finally, we perform a sensitivity analysis on some parameters associated with TGF-β dynamics, and use it to investigate the relation between the tumour size and its metastatic spread.

Topics: Animals; Cell Adhesion; Cell Aggregation; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms; Pattern Recognition, Automated; Signal Transduction; Transforming Growth Factor beta; Tumor Burden

Autophagy is a vital process controlling the lysosomal degradation of cellular organelles and thereby regulating tissue homeostasis in an environment-dependent fashion. Recent studies have unveiled the critical role of tumor cell-derived autophagy in regulating pro-tumor and anti-tumor processes depending on different stages and tumor microenvironments. However, the precise mechanism whereby autophagy regulates tumor progression remains largely unclear. Since myeloid cells contribute to tumor progression and metastasis, we evaluated the role of myeloid cell-specific autophagy in the regulation of tumor progression. We found that the number and size of metastatic lesions were smaller in myeloid cell-specific autophagy-deficient mice. Furthermore, autophagy-mediated regulation of TGF-β in myeloid cells was associated with the induction of epithelial-mesenchymal transition (EMT), which increases the invasive and metastatic potentials of tumor cells. Myeloid-derived autophagy also plays a critical role in impairing antitumor immune responses and promoting the survival and accumulation of M2 macrophages in tumor tissues in a CSF-1 and TGF-β-dependent manner. Taken together, our findings elucidate previously unrecognized mechanisms by which myeloid cells promote tumor progression through autophagy-mediated regulation of malignancy and immune tolerance.

Topics: Animals; Autophagy; Epithelial-Mesenchymal Transition; Humans; Lysosomes; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Myeloid Cells; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

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

Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether β3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-β induction of β3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-β exposure. Invadopodia formation and degradation activity is dependent on β3 integrin expression since β3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in β3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for β3 integrin in invadopodia. Our results suggest that β3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. β3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Extracellular Matrix; Humans; Integrin beta3; Lung Neoplasms; Neoplasm Metastasis; Podosomes; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide owing to its high rates of metastasis and recurrence. The oncogene IQ motif-containing GTPase activating protein 3 (IQGAP3) is ubiquitously overexpressed in several human cancers, including liver, ovary, lung, large intestine, gastric, bone marrow, and breast malignancies and is involved in the invasion and metastasis of cancer cells. Therefore, we aimed to determine the biological role and molecular mechanism of IQGAP3 in HCC.. We used 120 archived clinical HCC samples, 9 snap-frozen HCC tumor tissues, and 4 normal liver tissues. Expression of IQGAP3 mRNA and protein in HCC cell lines (Hep3B, SMMC-7721, HCCC-9810, HepG2, BEL-7404, HCCLM3, QGY-7701, Huh7, and MHCC97H) and normal liver epithelial cells LO2 was examined by western blot, quantitative polymerase chain reaction, and immunohistochemistry. In addition, wound-healing and transwell matrix penetration assays were used to assess the migratory and invasive abilities of HCC cells, respectively.. Expression of the IQGAP3 was robustly upregulated in HCC cells and tissues. High expression of IQGAP3 in HCC correlated with aggressive clinicopathological features and was an independent poor prognostic factor for overall survival. Furthermore, ectopic expression of IQGAP3 markedly enhanced HCC cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) in vitro and promoted metastasis of orthotopic hepatic tumors in nude mice. Conversely, silencing endogenous IQGAP3 showed an opposite effect. Mechanistically, IQGAP3 promoted EMT and metastasis by activating TGF-β signaling.. IQGAP3 functions as an important regulator of metastasis and EMT by constitutively activating the TGF-β signaling pathway in HCC. Our findings present new evidence of the role of IQGAP3 in EMT and metastasis, indicating its potential as a prognostic biomarker candidate and a therapeutic target against HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; RNA, Messenger; Signal Transduction; Statistics, Nonparametric; Transforming Growth Factor beta; Up-Regulation

Metastasis accounts for the most lethal reason for the death of ovarian cancer patients, but remains largely untreated. Epithelial-mesenchymal transition (EMT) is critical for the conversion of early-stage ovarian tumours into metastatic malignancies. Thus the exploration of the signalling pathways promoting EMT would open potential opportunities for the treatment of metastatic ovarian cancer. Herein, the putative role of MDM2 in regulating EMT and metastasis of ovarian cancer SKOV3 cells was investigated.. The regulatory effects by MDM2 on cell motility was emulated by wound-healing and transwell assays. The effects on EMT transition and Smad pathway were studied by depicting the expression levels of epithelial marker E-cadherin as well as key components of Smad pathway. To evaluate the clinical relevance of our findings, the correlation of MDM2 expression levels with the stages of 104 ovarian cancer patients was investigated by immunohistochemistry assay.. We demonstrate that MDM2 functions as a key factor to drive EMT and motility of ovarian SKOV3 cells, by facilitating the activation of TGF-β-Smad pathway, which results in the increased transcription of snail/slug and the subsequent loss of E-cadherin levels. Such induction of EMT is sustained in either E3 ligase-depleted MDM2 or E3 ligase inhibitor HLI-373-treated cells, while being impaired by the N-terminal deletion of MDM2, which is also reflected by the inhibitory effects against EMT by Nutlin-3a, the N-terminal targeting agent. The expression levels of MDM2 is highly correlated with the stages of the ovarian cancer patients, and the higher expression of MDM2 together with TGFB are closely correlated with poor prognosis and predict a high risk of ovarian cancer patients.. This study suggests that MDM2 activates Smad pathway to promote EMT in ovarian cancer metastasis, and targeting the N-terminal of MDM2 can reprogram EMT and impede the mobility of cancer cells.

Topics: Aminoquinolines; Antigens, CD; Blotting, Western; Cadherins; Carcinoma; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Gene Knockdown Techniques; Humans; Immunohistochemistry; Neoplasm Metastasis; Neoplasm Staging; Ovarian Neoplasms; Proto-Oncogene Proteins c-mdm2; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Thymine; Transforming Growth Factor beta

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, is known to exhibit tumor suppressor activity in colon cancer, the mechanism of which is not understood. Here we show that R-spondin 1 (RSPO1)/LGR5 directly activates TGFβ signaling cooperatively with TGFβ type II receptor in colon cancer cells, enhancing TGFβ-mediated growth inhibition and stress-induced apoptosis. Knockdown of LGR5 attenuated downstream TGFβ signaling and increased cell proliferation, survival, and metastasis in an orthotopic model of colon cancer

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; HCT116 Cells; Humans; Neoplasm Metastasis; Receptors, G-Protein-Coupled; Receptors, Transforming Growth Factor beta; Thrombospondins; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

Hypermethylation-induced epigenetic silencing of tumour suppressor genes (TSGs) are frequent events during carcinogenesis. MicroRNA-142 (miR-142) is found to be dysregulated in cancer patients to participate into tumour growth, metastasis and angiogenesis. However, the tumour suppressive role of miR-142 and the status of methylation are not fully understood in hepatocellular carcinoma (HCC).. Hepatocellular carcinoma tissues and corresponding non-neoplastic tissues were collected. The expression and function of miR-142 and TGF-β in two HCC cell lines were determined. The miRNA-mRNA network of miR-142 was analysed in HCC cell lines.. We found that the miR-142 expression was reduced in tumour tissues and two HCC cell lines HepG2 and SMMC7721, which correlated to higher TNM stage, metastasis and differentiation. Moreover, miR-142 was identified to directly target and inhibit transforming growth factor β (TGF-β), leading to decreased cell vitality, proliferation, EMT and the ability of pro-angiogenesis in TGF-β-dependent manner. Interestingly, the status of methylation of miR-142 was analysed and the results found the hypermethylated miR-142 in tumour patients and cell lines. The treatment of methylation inhibitor 5-Aza could restore the expression of miR-142 to suppress the TGF-β expression, which impaired TGF-β-induced tumour growth.. These findings implicated that miR-142 was a tumour suppressor gene in HCC and often hyermethylated to increase TGF-β-induced development of hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta

Thymoquinone, a major ingredient of black seed oil (Nigella sativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-β (TGF-β) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-β, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-β/Smad2/3 signaling pathway, which may be partially reversed by TGF-β overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-β/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-β.

Topics: Benzoquinones; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Epithelial tumour cells can gain invasive and metastatic capabilities by undergoing an epithelial-mesenchymal transition. Transcriptional regulators and post-transcriptional effectors like microRNAs orchestrate this process of high cellular plasticity and its malignant consequences. Here, using microRNA sequencing in a time-resolved manner and functional validation, we have identified microRNAs that are critical for the regulation of an epithelial-mesenchymal transition and of mesenchymal tumour cell migration. We report that miR-1199-5p is downregulated in its expression during an epithelial-mesenchymal transition, while its forced expression prevents an epithelial-mesenchymal transition, tumour cell migration and invasion in vitro, and lung metastasis in vivo. Mechanistically, miR-1199-5p acts in a reciprocal double-negative feedback loop with the epithelial-mesenchymal transition transcription factor Zeb1. This function resembles the activities of miR-200 family members, guardians of an epithelial cell phenotype. However, miR-1199-5p and miR-200 family members share only six target genes, indicating that, besides regulating Zeb1 expression, they exert distinct functions during an epithelial-mesenchymal transition.

Topics: Animals; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; MicroRNAs; Neoplasm Metastasis; Phenotype; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

This study aimed to investigate the role of long non-coding RNA MEG3 (lncRNA MEG3) in osteosarcoma (OS) and further explore the underlying molecular mechanism.. The expression profiles of MEG3 in OS cell lines and normal osteoblast cell line were detected by qRT-PCR. MEG3 was over-expressed in OS cell line by using LV-MEG3. MTT and colony-formation assays were applied for cell proliferation analysis. Cell migration assay was applied to investigate the cell migration ability. In addition, the expression levels of cell growth and metastasis related factors (Notch1, Hes1, TGF-β, N-cadheren and E-cadheren) were determined to illustrate the mechanisms.. We found that compared with normal osteoblast hFOB1.19 cell line, MEG3 was significantly down-regulated in MG63 and U2OS cell lines, particularly in MG-63 cells. MEG3 was significantly up-regulated in MG63 cells by LV-MEG3. Cell proliferation and migration ability were obviously repressed by MEG3 over-expression. In addition, MEG3 over-expression markedly inhibited Notch1, Hes1,TGF-β and N-cadheren expression, and the expression level of E-cadheren was improved.. These results indicated that MEG3 could prevent cell growth and metastasis of OS by repressing Notch and TGF-β signaling pathway, thus providing a potential therapeutic target for OS treatment (Tab. 1, Fig. 4, Ref. 30).

Topics: Antigens, CD; Bone Neoplasms; Cadherins; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Osteoblasts; Osteosarcoma; Receptor, Notch1; RNA, Long Noncoding; Transcription Factor HES-1; Transforming Growth Factor beta; Tumor Stem Cell Assay; Up-Regulation

The anticancer properties of ursolic acid (UA) and metformin (Met) have been well demonstrated. However, whether these compounds can act synergistically to prevent and treat cancer is not known. We present in this study, the synergism between UA and Met, and that of a new codrug made of UA and Met (UA-Met) against several cancer cell lines. The combination of high concentration of UA (25, 50, 75, 100 μM) and Met (5, 10, 20, 40 mM) resulted in synergetic cytotoxicity on MDA-MB-231 and MCF-7 cells (CI < 0.8). Molecular and cellular studies showed that codrug UA-Met significantly inhibited the invasion (∼55.3 ± 2.74%) and migration (∼52.4 ± 1.57%) of TGF-β induced breast cancer MDA-MB-231 and MCF-7 cells in vitro at low concentration of 10 μM. These effects were accompanied by down-regulation of CXCR4, uPA, vimentin, E-cadherin, N-cadherin, and MMP-2/9 proteins expression and regulation of the AMPK/m-TOR signaling pathways as expected from UA and Met. Moreover, UA-Met could reduce the progression of pulmonary metastasis by 4T1 cells (63.4 ± 3.52%) without influencing the glucose blood level in mice. Our study suggests that the codrug UA-Met is safe and effective in preventing cancer metastasis and possibly treatment of cancer.

Topics: Animals; Cadherins; Cell Line, Tumor; Drug Synergism; Female; Humans; Metformin; Mice; Neoplasm Metastasis; Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Triterpenes; Ursolic Acid; Vimentin; Xenograft Model Antitumor Assays

The importance of miRNAs in the progression of prostate cancer (PCa) has further been supported by the finding that miRNAs have been identified as potential oncogenes or tumor suppressors in PCa. Indeed, in eukaryotes, miRNAs have been found to regulate and control gene expression by degrading mRNA at the post-transcriptional level. In this study, we investigated the expression of miR-34 family members, miR-34b and miR-34c, in different PCa cell lines, and discussed the molecular mechanism of miR-34b in the invasion and migration of PCa cells in vitro. The difference analyses of the transcriptome between the DU145 and PC3 cell lines demonstrated that both miR-34b and -34c target critical pathways that are involved in metabolism, such as proliferation, and migration, and invasion. The molecular expression of miR-34b/c were lower in PC3 cells. Moreover, over-expression of miR-34b/c in PC3 cells caused profound phenotypic changes, including decreased cell proliferation, migration and invasion. Moreover, the players that regulate expression levels of transforming growth factor-β (TGF-β), TGF-β receptor 1 (TGF-βR1), and p53 or phosphorylation levels of mothers against decapentaplegic 3 (SMAD3) in the TGF-β/Smad3 signaling pathway have yet to be elucidated, and will provide novel tools for diagnosis and treatment of metastatic PCa.

Topics: Cell Line, Tumor; Cell Movement; Humans; Male; MicroRNAs; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, Transforming Growth Factor beta; Smad3 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Epithelial-to-mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N-cadherin, MMP-9, MMP-2, Twist and Snail) and suppressed TGF-β-induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N-cadherin), MMP-9, MMP-2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E-cadherin) in both A549 and H1299 cells. TGF-β-induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF-β-induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 232: 346-354, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Cell Line, Tumor; Cell Movement; Cell Survival; Down-Regulation; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Salicylates; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) has been implicated in the progression of bladder cancer. To study its contribution to bladder cancer metastasis, we established new xenograft models derived from human bladder cancer cell lines utilizing an orthotopic "recycling" technique that allowed us to isolate and examine the primary tumor and its corresponding circulating tumor cells (CTC's) and metastatic lesions. Using whole genome mRNA expression profiling, we found that a reversible epithelial-to-mesenchymal transition (EMT) characterized by TGFβ pathway activation and SNAIL expression was associated with the accumulation of CTCs. Finally, we observed that conditional silencing of SNAIL completely blocked CTC production and regional/distant metastasis. Using this unique bladder cancer xenograft model, we conclude that metastasis is dependent on a reversible EMT mediated by SNAIL.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplastic Cells, Circulating; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta; Urinary Bladder Neoplasms; Whole Genome Sequencing

c-Myc is a well-characterized proto-oncogene that induces cellular transformation and modulates programmed cell death. While recent studies have demonstrated high expression of c-Myc protein in advanced and metastatic melanoma, the clinical and biological implications remain to be fully elucidated. In this study, we investigated the effect of c-Myc overexpression in melanoma tumorigenesis. Clinicopathological analysis demonstrated that c-Myc expression positively correlated with the formation of vasculogenic mimicry (VM) and linearly patterned programmed cell necrosis (LPPCN). Clinically, high c-Myc expression was significantly associated with distant metastasis and poor prognosis, while biologically, c-Myc overexpression led to significant increases in cell motility, invasiveness and metastasis. Moreover, c-Myc induced the formation of VM and promoted the expression of epithelial-mesenchymal transition (EMT)-associated protein Snail both in vivo and in vitro. High expression of c-Myc increased Bax expression in hypoxic conditions and induced cell apoptosis. Taken together, we conclude that c-Myc overexpression promotes the formation of VM by EMT and LPPCN in melanoma. Our improved understanding of the clinical and biological effects of c-Myc overexpression in melanoma highlights the incomplete understanding of this oncogene, and indicates that c-Myc is a potential therapeutic target of this disease.. High c-Myc expression is associated with tumor metastasis and poor prognosis in human melanoma. c-Myc upregulates Snail expression to promote EMT via the TGF-β/Snail/Ecadherin signal pathway. c-Myc leads to cell death by upregulating Bax expression causing a lower Bcl2/Bax ratio under severe hypoxic conditions. c-Myc promotes vasculogenic mimicry and linearly patterned programmed cell necrosis.

Topics: Adult; Aged; Animals; bcl-2-Associated X Protein; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression; Heterografts; Humans; Immunohistochemistry; Male; Melanoma; Mice; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Recent evidence from a comprehensive genome analysis and functional studies have revealed that FOXM1 is a crucial metastatic regulator that drives cancer progression. However, the regulatory mechanism by which FOXM1 exerts its metastatic functions in cancer cells remains obscure. Here, we report that DLX1 acts as a FOXM1 downstream target, exerting pro-metastatic function in ovarian cancers. Both FOXM1 isoforms (FOXM1B or FOXM1C) could transcriptionally upregulate DLX1 through two conserved binding sites, located at +61 to +69bp downstream (TFBS1) and -675 to -667bp upstream (TFBS2) of the DLX1 promoter, respectively. This regulation was further accentuated by the significant correlation between the nuclear expression of FOXM1 and DLX1 in high-grade serous ovarian cancers. Functionally, the ectopic expression of DLX1 promoted ovarian cancer cell growth, cell migration/invasion and intraperitoneal dissemination of ovarian cancer in mice, whereas small interfering RNA-mediated DLX1 knockdown in FOXM1-overexpressing ovarian cancer cells abrogated these oncogenic capacities. In contrast, depletion of FOXM1 by shRNAi only partially attenuated tumor growth and exerted almost no effect on cell migration/invasion and the intraperitoneal dissemination of DLX1-overexpressing ovarian cancer cells. Furthermore, the mechanistic studies showed that DLX1 positively modulates transforming growth factor-β (TGF-β) signaling by upregulating PAI-1 and JUNB through direct interaction with SMAD4 in the nucleus upon TGF-β1 induction. Taken together, these data strongly suggest that DLX1 has a pivotal role in FOXM1 signaling to promote cancer aggressiveness through intensifying TGF-β/SMAD4 signaling in high-grade serous ovarian cancer cells.

Topics: Animals; Binding Sites; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Disease Progression; Female; Forkhead Box Protein M1; Heterografts; Homeodomain Proteins; Humans; Mice; Neoplasm Grading; Neoplasm Metastasis; Nucleotide Motifs; Ovarian Neoplasms; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Smad4 Protein; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

Topics: Animals; Disease Models, Animal; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neoplasms, Experimental; Pancreas; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion.. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR.

Topics: Animals; Carcinoma, Ovarian Epithelial; Cell Communication; Cell Line, Tumor; Cell Shape; Down-Regulation; Enzyme Induction; Epithelial Cells; Epithelium; Exosomes; Female; Humans; Hyaluronan Receptors; Matrix Metalloproteinases; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Omentum; Ovarian Neoplasms; Peritoneum; Signal Transduction; Transforming Growth Factor beta

Fibronectin is a component of the extracellular matrix that links collagen fibers to integrins on the cell's surface. The splicing isoforms, containing the ED-B domain, are not expressed in adult tissues but only in tumor stroma or during embryonic development. Fibroblasts and endothelial cells express ED-B fibronectin during angiogenesis. Also cancer cells can synthetize ED-B fibronectin, but its function in tumor growth needs to be further elucidated.We evaluated the expression of ED-B fibronectin in prostate cancer cell lines: PC3 and DU145. Using TGF-β, we induced epithelial to mesenchymal transition in culture and observed an increase of ED-B fibronectin expression. Thereafter, we evaluated the expression of ED-B fibronectin in multipotent mesangiogenic progenitor cells, and in mesenchymal stromal cells. The expression of ED-B fibronectin was much higher in mesenchymal than prostate cancer cells even after the epithelial to mesenchymal transition.Epithelial to mesenchymal transition is a key step for tumor progression contributing to the metastatic spread. Therefore, circulating cancer cells could seed into the metastatic niche taking advantage from the ED-B fibronectin that secrete their own.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cytokines; Disease Progression; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Male; Mesenchymal Stem Cells; Neoplasm Metastasis; Prostatic Neoplasms; Transforming Growth Factor beta; Up-Regulation

Our previous research showed that Gankyrin was overexpressed in NSCLC and significantly associated with clinicopathologic features and poor prognosis. In this study, we will explore potential effect of Gankyrin on EMT and metastasis in NSCLC. The ectopic higher expression of Gankyrin markedly increased the migration and invasion in NSCLC cells. In contrast, silencing Gankyrin inhibit this aggressive behavior in NSCLC cells. Further study demonstrated that overexpression of Gankyrin could decrease E-cadherin expression and increase expression of Vimentin and Twist1 at mRNA and protein levels. These data indicated that Gankyrin could facilitate occurrence and development of EMT. Also IHC analysis showed that Gankyrin expression was negatively correlated with E-cadherin expression, while positively correlated with Vimentin and Twist1 expression in NSCLC tissues. The mechanism study finally suggested that the Gankyrin-driven EMT was partially due to IL-6/p-STAT3 and TGF-β/p-SMAD3 pathways activation. Taken together, our data provided a novel mechanism of Gankyrin promoting EMT and metastasis in NSCLC through forming a closed circle with IL-6/p-STAT3 and TGF-β/p-SMAD3 signaling pathway.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; Neoplasm Metastasis; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Transforming Growth Factor beta; Up-Regulation

Despite advancements in the diagnosis and treatment of colorectal cancer (CRC), many patients die because of tumor metastasis or recurrence. Therefore, identifying new prognostic markers and elucidating the mechanisms of CRC metastasis and recurrence will help to improve the prognosis of the disease. As dysregulation of microRNAs is strongly related to cancer progression, the aim of this study was to identify the role of miR-4775 in the prognosis of CRC patients and the underling mechanisms involved in CRC progression.. qPCR and in situ hybridization were used to evaluate the expression of miR-4775 in 544 pairs of paraffin-embedded normal and CRC tissues. Kaplan-Meier analysis with the log-rank test was used for survival analyses. Immunohistochemical staining was applied to investigate the expression of miR-4775-regulated Smad7/TGFβ pathway-associated markers. In vitro and in vivo invasion and metastasis assays were used to explore the function of miR-4775 in the progression of CRC.. miR-4775 was identified as a high-risk factor for CRC metastasis and recurrence, with high levels predicting poor survival among the 544 studied CRC patients. Furthermore, high miR-4775 expression promoted the invasion of CRC cells as well as metastasis and the epithelial to mesenchymal transition (EMT) via Smad7-mediated activation of TGFβ signaling both in vitro and in vivo. Downregulating miR-4775 or overexpressing Smad7 reversed the tumor-promoting roles of miR-4775/Smad7/TGFβ in vitro and in vivo.. miR-4775 promotes CRC metastasis and recurrence in a Smad7/TGFβ signaling-dependent manner, providing a new therapeutic target for inhibiting the metastasis or recurrence of the disease.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Disease Models, Animal; Epithelial-Mesenchymal Transition; Heterografts; Humans; Kaplan-Meier Estimate; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-β signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.

Topics: Adult; Animals; Cell Movement; Colorectal Neoplasms; Disease Models, Animal; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genetic Engineering; Humans; Male; Mice; Mice, Inbred NOD; Middle Aged; Mutation; Neoplasm Metastasis; Neoplastic Processes; Organoids; Signal Transduction; Transforming Growth Factor beta

Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Genotype; Humans; Mice; Mice, Inbred C57BL; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Proto-Oncogene Proteins p21(ras); Transcriptome; Transforming Growth Factor beta

Topics: Adult; Aged; Angiopoietin-Like Protein 2; Angiopoietin-like Proteins; Animals; Bevacizumab; Cell Line, Tumor; Chemokine CXCL1; Colorectal Neoplasms; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Interleukin-8; Male; Mice; Middle Aged; Neoplasm Metastasis; Prognosis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value.

Topics: Animals; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Osteosarcoma; Phosphorylation; Transforming Growth Factor beta

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

Long non-coding RNA-activated by TGF-β (lncRNA-ATB) promotes the invasion-metastasis cascade in hepatocellular carcinoma via downregulating E-cadherin (E-cad) and inducing epithelial-to- mesenchymal transition (EMT) and is clinically significant in human colon cancer. However, its molecular mechanisms in colon cancer progression remain unclear. This study aimed to elucidate the role of lncRNA-ATB and its clinical value in colon cancer.. Expression levels of lncRNA-ATB in colon cancer tissues and colon cancer cell lines were evaluated using quantitative real-time polymerase chain reaction. The clinicopathological significance and prognostic value of lncRNA-ATB were investigated, and roles of lncRNA-ATB in regulating E-cad and other EMT-related markers expression and colon cancer progression were evaluated in vitro. Expression levels of lncRNA-ATB and E-cad in human plasma were evaluated.. Long non-coding RNA-activated by TGF-β was upregulated in colon cancer tissues compared with adjacent mucosa (P < 0.001). LncRNA-ATB levels were also higher in metastatic cancer tissues (P < 0.001). Among the three highly invasive colon cancer cell lines, lncRNA-ATB levels were relatively higher with concurrent low levels of E-cad compared with levels in the three low-invasive cell lines. LncRNA-ATB expression correlated with pN stage (P < 0.01) and American Joint Committee on Cancer stage (P < 0.01). Striking differences were observed in overall survival and disease-free survival in cases with both high lncRNA-ATB expression and low E-cad expression. Reduction of lncRNA-ATB increased expression of epithelial markers E-cad, ZO-1, and decreased expression of mesenchymal markers ZEB1 and N-cadherin (N-cad), and significantly influenced colon cancer cell progression. Plasma lncRNA-ATB was upregulated in colon cancer patients one month after surgery (P < 0.05).. Long non-coding RNA-activated by TGF-β may act on colon tumorigenesis by suppressing E-cad expression and promoting EMT process, and lncRNA-ATB inhibition may provide a promising therapeutic option for suppressing colon cancer progression.

Topics: Cadherins; Carcinogenesis; Colonic Neoplasms; Disease Progression; Down-Regulation; Female; Gene Expression; Humans; Male; Molecular Targeted Therapy; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; RNA, Long Noncoding; Survival Rate; Transforming Growth Factor beta; Up-Regulation

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are associated with a variety of human cancers. Currently, little data are available regarding miRNA expression in vulvar squamous cell carcinoma (VSCC); the mechanism of action of miRNAs in VSCC still requires investigation. The aim of the present study was to investigate the miRNA expression profile in VSCC using a miRCURY™ LNA array. The expression levels of selected miRNAs were quantified by RT-qPCR. The relationship between miR-590-5p expression and clinical pathology was assessed. The expression levels of crucial transforming growth factor-β (TGF-β) and Smad pathway factors were detected. We further investigated the role of miR-590-5p via in vitro studies in the A431 human VSCC cell line. A total of 157 miRNAs showed significantly altered expression in this type of carcinoma. Of particular interest, miR-590-5p, miR-182-5p and miR-183-5p were upregulated, and miR-603, miR-103a-3p and miR-107 were downregulated. A positive relationship was found between miR-590-5p expression and lymph node metastasis. In VSCC, TGFβ1 and TGFβ2 were significantly overexpressed and TGFβRII and Smad4 were significantly underexpressed at both the RNA and protein levels. In A431 cells, overexpression of miR-590-5p promoted proliferation, migration and G1-S phase transition and downregulated TGFβRII. The knockdown of TGFβRII by siRNA promoted malignant behaviours in the A431 cells. In conclusion, we present the miRNA expression profile in VSCC, and our findings suggest that the upregulation of miR-590-5p promotes cellular malignant behaviours via the target gene TGFβRII.

Topics: Aged, 80 and over; Carcinoma, Squamous Cell; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Middle Aged; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad Proteins; Transforming Growth Factor beta; Up-Regulation; Vulvar Neoplasms

Metastatic spread of cancer cells from the primary tumors to distant vital organs, such as lung, liver, brain, and bone, is responsible for the majority of cancer-related deaths. Development of metastatic lesions is critically dependent on the interaction of tumor cells with the stromal microenvironment. As a multifunctional paracrine signaling factor that is abundantly produced by both tumor and stromal cells, TGFβ has been well established as an important mediator of tumor-stromal interaction during cancer metastasis. Imaging the in vivo dynamic of TGFβ signaling activity during cancer metastasis is critical for understanding the pathogenesis of the disease, and for the development of effective anti-metastasis treatments. In this chapter, I describe several xenograft methods to introduce human breast cancer cells into nude mice in order to generate spontaneous and experimental metastases, as well as the luciferase-based bioluminescence imaging method for quantitative imaging analysis of TGFβ signaling in tumor cells during metastasis.

Topics: Animals; Disease Models, Animal; Heterografts; Humans; Luminescent Measurements; Mice; Molecular Imaging; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) is known to regulate a large number of biological processes and is involved in various aspects of tumor development. Recent studies have shown that the biogenesis of miRNAs can be regulated by TGF-β signaling directly via Smad-dependent mechanisms and/or other unknown mechanisms, which may induce autoregulatory feedback loops in response to the activation of TGF-β signaling, influencing the fate of tumor cells. In this chapter, we summarize the currently described mechanisms underlying TGF-β's regulation of miRNA biogenesis, and the functional role of TGF-β-regulated miRNAs in tumor initiation, epithelial-mesenchymal transition, and tumor microenvironment modulation. Finally, we introduce methods to study TGF-β-regulated miRNAs and their functions in tumor progression and metastasis using an example of publication from our lab demonstrating the presence of a TGF-β-miR-34a-CCL22 signaling axis, which serves as a potent etiological pathway for the development of hepatocellular carcinoma venous metastases.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-β-mediated EMT in lung cancer cells. miR-93 binds directly to the 3'-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-β signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-β treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-β-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.

Topics: Carcinogenesis; Cell Line, Tumor; Endosomal Sorting Complexes Required for Transport; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasm Staging; Smad2 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

TGFβ is a known driver of epithelial-mesenchymal transition (EMT) which is associated with tumor aggressiveness and metastasis. However, EMT has not been fully explored in clinical specimens of castration-resistant prostate cancer (CRPC) metastases. To assess EMT in CRPC, gene expression analysis was performed on 149 visceral and bone metastases from 62 CRPC patients and immunohistochemical analysis was performed on 185 CRPC bone and visceral metastases from 42 CRPC patients. In addition, to assess the potential of metastases to seed further metastases the mitochondrial genome was sequenced at different metastatic sites in one patient. TGFβ was increased in bone versus visceral metastases. While primarily cytoplasmic; nuclear and cytoplasmic Twist were significantly higher in bone than in visceral metastases. Slug and Zeb1 were unchanged, with the exception of nuclear Zeb1 being significantly higher in visceral metastases. Importantly, nuclear Twist, Slug, and Zeb1 were only present in a subset of epithelial cells that had an EMT-like phenotype. Underscoring the relevance of EMT-like cells, mitochondrial sequencing revealed that metastases could seed additional metastases in the same patient. In conclusion, while TGFβ expression and EMT-associated protein expression is present in a considerable number of CRPC visceral and bone metastases, nuclear Twist, Slug, and Zeb1 localization and an EMT-like phenotype (elongated nuclei and cytoplasmic compartment) was only present in a small subset of CRPC bone metastases. Mitochondrial sequencing from different metastases in a CRPC patient provided evidence for the seeding of metastases from previously established metastases, highlighting the biological relevance of EMT-like behavior in CRPC metastases.

Topics: Bone Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Homeodomain Proteins; Humans; Immunohistochemistry; Laser Capture Microdissection; Male; Neoplasm Metastasis; Nuclear Proteins; Prostatic Neoplasms, Castration-Resistant; Real-Time Polymerase Chain Reaction; Snail Family Transcription Factors; Tissue Array Analysis; Transcription Factors; Transcriptome; Transforming Growth Factor beta; Twist-Related Protein 1; Zinc Finger E-box-Binding Homeobox 1

Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Mitochondrial function is dysregulated frequently in cancer cells to allow for adaptation to a harsh tumor microenvironment. Targeting mitochondrial biogenesis and bioenergetics is, therefore, an attractive therapeutic strategy. In this study, we performed quantitative proteomic analyses in human parental and metastatic breast cancer cell lines to identify mitochondrial proteins involved in TNBC metastasis. We found that single-strand DNA-binding protein 1 (SSBP1) was downregulated in highly metastatic breast cancer cells. Moreover, SSBP1 downregulation promoted TNBC cell metastasis in vitro and in vivo. Mechanistically, SSBP1 loss decreased mitochondrial DNA copy number, thereby potentiating calcineurin-mediated mitochondrial retrograde signaling that induced c-Rel/p50 nuclear localization, activated TGFβ promoter activity, and TGFβ-driven epithelial-to-mesenchymal transition. Low SSBP1 expression correlated with tumor progression and poor prognosis in patients. Collectively, our findings identified SSBP1 as a novel metastasis suppressor and elucidated the mechanisms by which dysregulated mitochondrial signaling contributes to metastatic potential, providing potential new prognostic indicators for patients with TNBC.

Topics: Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Mitochondria; Mitochondrial Proteins; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

Tripartite Motif Containing 25 (TRIM25), a member of TRIM proteins, has been found abnormally expressed in cancers of female reproductive system. Here, TRIM25 was conspicuously expressed in human gastric cancer (GC) tissues in which its higher expression generally correlated with the poor prognosis of patients. Small interfering RNA (siRNA)-mediated knockdown of TRIM25 expression in MGC-803 and AGS cells had no effects on cell proliferation, whereas reduced cell migration and invasion. Gene set enrichment analysis on The Cancer Genome Atlas stomach adenocarcinoma (STAD) dataset revealed that several signaling pathways, including the migration, E-cadherin and transforming growth factor-β (TGF-β) pathways, were enriched in TRIM25 higher expression patients. Moreover, ectopic expression of TRIM25 in a GC cell line with lower expression of TRIM25 significantly promoted the migration and invasion. Further experiments with TGF-β inhibitor suggested that TRIM25 may exert its function through TGF-β pathway. In summary, our results indicate that TRIM25 acts as an oncogene in GC and thus presents a novel target for the detection and treatment of GC.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Metastasis; RNA Interference; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Transcription Factors; Transfection; Transforming Growth Factor beta; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Up-Regulation

The transforming growth factor-β (TGF-β) is a family of structurally related proteins that comprises of TGF-β, activins/inhibins, and bone morphogenic proteins (BMPs). Members of the TGF-β family control numerous cellular functions including proliferation, apoptosis, differentiation, epithelial-mesenchymal transition (EMT), and migration. The first identified member, TGF-β is implicated in several human diseases, such as vascular diseases, autoimmune disorders, and carcinogenesis. Activation of the TGF-β receptor by its ligands induces the phosphorylation of serine/threonine residues and triggers phosphorylation of the intracellular effectors, SMADs. Upon activation, SMAD proteins translocate to the nucleus and induce transcription of their target genes, regulating several cellular functions. TGF-β dysregulation has been implicated in carcinogenesis. In early stages of cancer, TGF-β exhibits tumor suppressive effects by inhibiting cell cycle progression and promoting apoptosis. However, in late stages TGF-β exerts tumor promoting effects, increasing tumor invasiveness, and metastasis. Furthermore, the TGF-β signaling pathway communicates with other signaling pathways in a synergistic or antagonistic manner and regulates cellular functions. Elevated TGF-β activity has been associated with poor clinical outcome. Given the pivotal role of TGF-β in tumor progression, this pathway is an attractive target for cancer therapy. Several therapeutic tools such as TGF-β antibodies, antisense oligonucleotides, and small molecules inhibitors of TGF-β receptor-1 (TGF-βR1) have shown immense potential to inhibit TGF-β signaling. Finally, in the interest of developing future therapies, further studies are warranted to identify novel points of convergence of TGF-β with other signaling pathways and oncogenic factors in the tumor microenvironment.

Topics: Antineoplastic Agents; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phosphorylation; Prognosis; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Survivors from sepsis are in an immunosuppressed state that is associated with higher long-term mortality and risk of opportunistic infections. Whether these factors contribute to neoplastic proliferation, however, remains unclear. Tumor-associated macrophages (TAM) can support malignant cell proliferation, survival, and angiogenesis. We addressed the relationship between the post-sepsis state, tumor progression and TAM accumulation, and phenotypic and genetic profile, using a mouse model of sepsis resolution and then B16 melanoma in mice. In addition, we measured the serum concentrations of TNFα, TGFβ, CCL2, and CXCL12 and determined the effect of in vivo CXCR4/CXCL12 inhibition in this context. Mice that survived sepsis showed increased tumor progression both in the short and long term, and survival times were shorter. TAM accumulation, TAM local proliferation, and serum concentrations of TGFβ, CXCL12, and TNFα were increased. Naïve mice inoculated with B16 together with macrophages from post-sepsis mice also had faster tumor progression and shorter survival. Post-sepsis TAMs had less expression of MHC-II and leukocyte activation-related genes. Inhibition of CXCR4/CXCL12 prevented the post-sepsis-induced tumor progression, TAM accumulation, and TAM in situ proliferation. Collectively, our data show that the post-sepsis state was associated with TAM accumulation through CXCR4/CXCL12, which contributed to B16 melanoma progression.

Topics: Animals; Chemokine CXCL12; Disease Models, Animal; Disease Progression; Gene Expression; Macrophages; Male; Melanoma, Experimental; Mice; Neoplasm Metastasis; Neoplasms; Receptors, CXCR4; Sepsis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Metastasis is the main cause of mortality in cancer patients. Although there are many anti-cancer drugs targeting tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression; in particular, lymphangiogenesis is pivotal for metastasis in cancer. Here we report that lithium inhibits colon cancer metastasis by blocking lymphangiogenesis. Lithium reduces the expression of transforming growth factor-β-induced protein (TGFBIp) in colon cancer cells by inhibiting Smad3 phosphorylation via GSK3β inactivation. Moreover, lithium inhibits lymphatic endothelial cell migration, which is increased upon TGFBIp expression in tumor cells. Lithium had no significant effect on SW620 tumor growth in vitro and in vivo; however, it inhibited lymphangiogenesis in tumors. In tumor xenografts model, lithium was found to prevent metastasis to the lungs, liver, and lymph nodes by inhibiting TGFBIp-induced tumor lymphangiogenesis. Collectively, our findings demonstrate a novel role of lithium in the inhibition of colon cancer metastasis by blocking TGFBIp expression, and thereby TGFBIp-induced lymphangiogenesis, in primary tumors.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Lithium; Lymphangiogenesis; Mice; Neoplasm Metastasis; Neoplasm Proteins; Phosphorylation; Smad3 Protein; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) is the crucial step that cancer cells must pass before they can undergo metastasis. The transition requires the activity of complex functional networks that downregulate properties of the epithelial phenotype and upregulate characteristics of the mesenchymal phenotype. The networks frequently include reciprocal repressions between transcription factors (TFs) driving the EMT and microRNAs (miRs) inducing the reverse process, termed mesenchymal-epithelial transition (MET). In this work we develop four kinetic models that are based on experimental data and hypotheses describing how autocrine transforming growth factor-β (TGF-β) signal transduction induces and maintains an EMT by upregulating the TFs ZEB1 and ZEB2 which repress the expression of the miR-200b/c family members. After successful model calibration we validate our models by predicting requirements for the maintenance of the mesenchymal steady state which agree with experimental data. Finally, we apply our validated kinetic models for the design of experiments in cancer therapy. We demonstrate how steady state properties of the kinetic models, combined with data from tumor-derived cell lines of individual patients, can predict the minimal amount of an inhibitor to induce a MET.

Topics: Animals; Autocrine Communication; Benzodioxoles; Computer Simulation; Dogs; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Imidazoles; Kinetics; Madin Darby Canine Kidney Cells; MicroRNAs; Models, Biological; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Pyridines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reproducibility of Results; Signal Transduction; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Non-canonical transforming growth factor β (TGFβ) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3' untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained. This study characterizes the binding kinetics of the consensus-binding motif and hnRNP E1, its various K-homology (KH) domains and p-hnRNP E1. Levels of p-hnRNP E1 are highly upregulated in metastatic cancer cells and low in normal epithelial tissue. We show a correlation between this PTM and levels of Akt2 and its activated form, phosphorylated serine-474 (p-Akt2). Using cellular progression models of metastasis, we observed a signature high level of Akt2, p-Akt2 and p-hnRNP E1 protein expression, coupled to a significantly reduced level of total hnRNP E1 in metastatic cells. Genes that are translationally silenced by hnRNP E1 and expressed by its dissociation are highly implicated in the progression of EMT and metastasis. This study provides insight into a non-canonical TGFβ signaling cascade that is responsible for inducing EMT by aberrant expression of hnRNP E1 silenced targets. The relevance of this system in metastatic progression is clearly shown in cellular models by the high abundance of p-hnRNP E1 and low levels of hnRNP E1. New insights provided by the resolution of this molecular mechanism provide targets for therapeutic intervention and give further insight into the role of the TGFβ microenvironment.

Topics: 3' Untranslated Regions; Base Sequence; Binding Sites; Caco-2 Cells; Cell Line, Tumor; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HCT116 Cells; Heterogeneous-Nuclear Ribonucleoproteins; HT29 Cells; Humans; Neoplasm Metastasis; Phosphorylation; Protein Binding; Protein Domains; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA-Binding Proteins; Serine; Signal Transduction; 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

UGT1A1*28/*6 as predictors of severe irinotecan-related diarrhea (SIRD) were duplicated by many studies. However, some patients of lower risk genotype (UGT1A1*1/*1) still suffered SIRD and the extremely low frequency of UGT1A1*6/*6 limited its clinical usage. Previous studies proved that the transforming growth factor (TGFB) family may have some effect on MTX-induced mucositis. However, the associations between TGFB gene variants and SIRD have never been reported so far. Our aim was to improve the predictive value of UGT1A1 gene variants on SIRD.. Six SNPs (TGFB1 rs1800469; TGFBR1 rs10733710, rs334354 and rs6478974; TGFBR2 rs3087465; UGT1A1*6) and UGT1A1*28 were selected for genotyping in 160 metastatic colorectal cancer patients treated with irinotecan in a prospective multicenter trial (NCT01282658).. UGT1A1*6, UGT1A1*28, rs1800469 and rs3087465 were all associated with SIRD (p = 0.026, 0.014, 0.047 and 0.045 respectively). A novel genetic score model (with a cut off value of 1.5) based on them was created to predict SIRD (OR = 11.718; 95 % CI 2.489-55.157, p = 0.002). In patients of gene score > 1.5, the risk of SIRD was much higher (23.5 vs. 2.8 %, p = 2.24E-04) and continued in the first 6 cycles of chemotherapy, while in patients with gene score ≤1.5, the risk was much lower and none of them suffered SIRD after the first cycle of chemotherapy (p = 0.0003).. The novel genetic score model improved the predictive value of UGT1A1 on SIRD. If validated, it will provide valuable information for clinical use of irinotecan.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents, Phytogenic; Camptothecin; Colorectal Neoplasms; Diarrhea; Female; Glucuronosyltransferase; Humans; Irinotecan; Male; Middle Aged; Neoplasm Metastasis; Prospective Studies; Transforming Growth Factor beta; Young Adult

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

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

Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2-/- mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Movement; Cells, Cultured; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Metastasis; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, PAR-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, G-Protein-Coupled; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Claudins; Humans; Lung Neoplasms; Mice; Neoplasm Metastasis; RNA-Binding Proteins; Signal Transduction; Transforming Growth Factor beta

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

Transforming growth factor beta (TGF-β) plays a major role in tumorigenesis. MicroRNA-181b (miRNA-181b) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. This study aimed to confirm the relationship of miRNA-181b and the TGF-β-Smad2/3/4 pathway with the induction of the epithelial-to-mesenchymal transition (EMT) in gastric cancer.. This study investigated the ability of TGF-β to induce migration by wound healing and transwell invasion assays in human gastric cancer cell lines. miRNA expression was altered using miRNA-181b mimic and inhibitor in the same system. Expression of miRNA-181b, the hypothetical target gene Timp3 and EMT-related markers were analyzed by real-time real-time quantitative RT-PCR. Immunoblotting was used to investigate the levels of phospho-Smad2 and Smad4. Dual-luciferase reporter assays were performed to confirm the direct binding of miRNA-181b to Timp3.. miRNA-181b was significantly upregulated in response to TGF-β treatment in gastric cancer cell lines. Overexpression of miR-181b mimic induced an in vitro EMT-like change to a phenotype similar to that following TGF-β treatment alone and was reversed by miRNA-181b inhibitor. Inhibition of TGF-β-Smad2/3 signaling with SD-208 significantly attenuated the upregulation of miRNA-181b. Knockdown of Smad4 in gastric cancer cells strongly attenuated the upregulation of miRNA-181b. Moreover, miR-181b was found to directly target the 3' untranslated region (3'UTR) of Timp3 mRNA affecting TGF-β-induced EMT.. Our results elucidate a novel mechanism through which the TGF-β pathway regulates the EMT of gastric cancer cells by increasing the levels of miRNA-181b to target Timp3 via the Smad2/3/4-dependent pathway. These findings provide insights into the cellular and environmental factors regulating EMT, which may guide future studies on therapeutic strategies targeting these cells.

Topics: 3' Untranslated Regions; Blotting, Western; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; MicroRNAs; Microscopy, Fluorescence; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Stomach Neoplasms; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) induces the expression of Disabled-2 (Dab2), an endocytic adaptor and tumour suppressor, concomitant with the induction of an epithelial-mesenchymal transition (EMT) in mammary epithelial cells. Here we show that following TGF-β-mediated EMT, sustained TGF-β treatment leads to proteolytic degradation of Dab2 by cathepsin B (CTSB), loss of the mesenchymal phenotype and induction of autophagy. CTSB inhibition or expression of a CTSB-resistant Dab2 mutant maintains Dab2 expression and shifts long-term TGF-β-treated cells from autophagy to apoptosis. We further show that Dab2 interacts with Beclin-1 to promote casein-kinase-2-mediated phosphorylation of Beclin-1, preventing Beclin-1-Vps34 interaction and subsequent autophagosome assembly. Thus, CTSB-mediated degradation of Dab2 allows Beclin-1-Vps34 induction of autophagy, whereas sustained Dab2 expression prevents autophagy and promotes apoptosis by stabilizing the pro-apoptotic Bim protein. In vivo studies suggest that Dab2-mediated regulation of autophagy modulates chemotherapeutic resistance and tumour metastasis.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cathepsin B; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Mice; Neoplasm Metastasis; RNA, Small Interfering; Transforming Growth Factor beta; Tumor Suppressor Proteins

Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined.. In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated.. It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system.. Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β.

Topics: Benzamides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Cisplatin; Coculture Techniques; Dioxoles; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinases; Mesenchymal Stem Cells; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Microenvironment

Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-β) or oncogenetic (MYC) factors. Both TGF-β- and MYC-induced EMT required ZEB1, but engaged distinct TGF-β-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-β and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Line; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Female; Humans; Hyaluronan Receptors; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Microcirculation; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Proto-Oncogene Proteins c-myc; Receptors, Calcitriol; RNA-Binding Proteins; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.. We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.. The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines' metastatic ability.. The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.

Topics: A549 Cells; Animals; Cell Adhesion; Cell Line, Tumor; Cell Separation; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Genetic Heterogeneity; Humans; MCF-7 Cells; Mice; Microfluidic Analytical Techniques; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Neoplastic Cells, Circulating; Transforming Growth Factor beta

Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an EMT-independent action of Snail1 on tumor invasion, as it is required for the activation of cancer-associated fibroblasts (CAF). Snail1 expression in fibroblasts requires signals derived from tumor cells, such as TGFβ; reciprocally, in fibroblasts, Snail1 organizes a complex program that stimulates invasion of epithelial cells independent of the expression of Snail1 in these cells. Epithelial cell invasion is stimulated by the secretion by fibroblast of diffusible signaling molecules, such as prostaglandin E

Topics: Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Dinoprostone; Epithelial-Mesenchymal Transition; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Snail Family Transcription Factors; Transforming Growth Factor beta

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Topics: Animals; Caco-2 Cells; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Cytokines; Exocytosis; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Inflammation; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B; Promoter Regions, Genetic; rab27 GTP-Binding Proteins; RNA Interference; Stem Cells; Transcription Factor RelA; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Hypoxia plays a vital role in tumor metabolism, proliferation, apoptosis, invasion and metastasis via hypoxia-inducible factor (HIF). Epithelial to mesenchymal transition (EMT) is a crucial process to metastasis, which could be triggered by hypoxia. EMT could be regulated by HIF via multiple pathways including TGF-β, Notch, and Wnt/β-catenin. It has been shown that anti-HIF drugs combined with anti-EMT therapies could be a promising strategy for tumor therapy.. 缺氧是肿瘤微环境的主要特点，通过缺氧诱导因子(hypoxia-inducible factor，HIF)激活众多靶基因，从而影响肿瘤的代谢、增殖、凋亡、侵袭转移、耐药等多个方面。上皮细胞间质化(epithelial to mesenchymal transition，EMT)是肿瘤发生侵袭转移的重要起始过程，缺氧亦是EMT的诱发因素之一并可通过多条信号转导通路包括TGF-β，Notch，Wnt/β-catenin等调节EMT。研究缺氧诱导EMT的机制并阻断其进程，可成为治疗肿瘤的新靶点。.

Topics: Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate, which is approximately 20% during the first year and decreases to less than 6% within five years of the disease. This is due to premature dissemination accompanied by a lack of disease-specific symptoms during the initial stages. Additionally, to date there are no biomarkers for an early prognosis available.A growing number of studies indicate that epithelial to mesenchymal transition (EMT), triggered by WNT-, TGF-β- and other signaling pathways is crucial for the initiation of the metastatic process in PDAC. Here we show, that BCL9L is up-regulated in PDAC cell lines and patient tissue compared to non-cancer controls. RNAi-induced BCL9L knockdown negatively affected proliferation, migration and invasion of pancreatic cancer cells. On a molecular basis, BCL9L depletion provoked an increment of E-cadherin protein levels, with concomitant increase of β-catenin retention at the plasma membrane. This is linked to the induction of a strong epithelial phenotype in pancreatic cancer cells upon BCL9L knockdown even in the presence of the EMT-inducer TGF-β. Finally, xenograft mouse models of pancreatic cancer revealed a highly significant reduction in the number of liver metastases upon BCL9L knockdown. Taken together, our findings underline the key importance of BCL9L for EMT and thus progression and metastasis of pancreatic cancer cells. Direct targeting of this protein might be a valuable approach to effectively antagonize invasion and metastasis of PDAC.

Topics: Adherens Junctions; Animals; beta Catenin; Cadherins; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Mice; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; Protein Transport; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

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

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

The observation that approximately 15% of women with disseminated breast cancer will develop symptomatic brain metastases combined with treatment guidelines discouraging single-agent chemotherapeutic strategies facilitates the desire for novel strategies aimed at outright brain metastasis prevention. Effective and robust preclinical methods to evaluate early-stage metastatic processes, brain metastases burden, and overall mean survival are lacking. Here, we develop a novel method to quantitate early metastatic events (arresting and extravasation) in addition to traditional end time-point parameters such as tumor burden and survival in an experimental mouse model of brain metastases of breast cancer. Using this method, a reduced number of viable brain-seeking metastatic cells (from 3,331 ± 263 cells/brain to 1,079 ± 495 cells/brain) were arrested in brain one week postinjection after TGFβ knockdown. Treatment with a TGFβ receptor inhibitor, galunisertib, reduced the number of arrested cells in brain to 808 ± 82 cells/brain. Furthermore, we observed a reduction in the percentage of extravasated cells (from 63% to 30%) compared with cells remaining intralumenal when TGFβ is knocked down or inhibited with galunisertib (40%). The observed reduction of extravasated metastatic cells in brain translated to smaller and fewer brain metastases and resulted in prolonged mean survival (from 36 days to 62 days). This method opens up potentially new avenues of metastases prevention research by providing critical data important to early brain metastasis of breast cancer events.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Metastasis; Pyrazoles; Quinolines; Signal Transduction; Transforming Growth Factor beta

Breast cancer metastasis suppressor 1 (BRMS1) is downregulated in non-small cell lung cancer (NSCLC), and its reduction correlates with disease progression. Herein, we investigate the mechanisms through which loss of the BRMS1 gene contributes to epithelial-to-mesenchymal transition (EMT). Using a short hairpin RNA (shRNA) system, we show that loss of BRMS1 promotes basal and transforming growth factor beta-induced EMT in NSCLC cells. NSCLC cells expressing BRMS1 shRNAs (BRMS1 knockdown [BRMS1(KD)]) display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers. Mesenchymal phenotypes observed in BRMS1(KD) cells are dependent on RelA/p65, the transcriptionally active subunit of nuclear factor kappa B (NF-κB). In addition, chromatin immunoprecipitation analysis demonstrates that loss of BRMS1 increases Twist1 promoter occupancy of RelA/p65 K310-a key histone modification associated with increased transcription. Knockdown of Twist1 results in reversal of BRMS1(KD)-mediated EMT phenotypic changes. Moreover, in our animal model, BRMS1(KD)/Twist1(KD) double knockdown cells were less efficient in establishing lung tumors than BRMS1(KD) cells. Collectively, this study demonstrates that loss of BRMS1 promotes malignant phenotypes that are dependent on NF-κB-dependent regulation of Twist1. These observations offer fresh insight into the mechanisms through which BRMS1 regulates the development of metastases in NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histones; Humans; Lung Neoplasms; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B p50 Subunit; Nuclear Proteins; Phenotype; Repressor Proteins; RNA, Small Interfering; Transforming Growth Factor beta; Twist-Related Protein 1; Wound Healing

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFβ/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFβ-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFβ on the expression of epithelial-mesenchymal transition-related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFβ/Smad signaling pathway via TRIM33 in ccRCC.. This study suggests that miR-629 has biomarker potential through its ability to regulate TGFβ/Smad signaling and accelerate ccRCC cell motility and invasion.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis Regulatory Proteins; Carcinoma, Renal Cell; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; MicroRNAs; Middle Aged; Mitochondrial Proteins; Neoplasm Metastasis; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

The contemporary use of nanomedicines for cancer treatment has been largely limited to serving as carriers for existing therapeutic agents. Here, we provide definitive evidence that, the metallofullerenol nanomaterial Gd@C82(OH)22, while essentially not toxic to normal mammary epithelial cells, possesses intrinsic inhibitory activity against triple-negative breast cancer cells. Gd@C82(OH)22 blocks epithelial-to-mesenchymal transition with resultant efficient elimination of breast cancer stem cells (CSCs) resulting in abrogation of tumour initiation and metastasis. In normoxic conditions, Gd@C82(OH)22 mediates these effects by blocking TGF-β signalling. Moreover, under hypoxic conditions found in the tumour microenvironment, cellular uptake of Gd@C82(OH)22 is facilitated where it functions as a bi-potent inhibitor of HIF-1α and TGF-β activities, enhancing CSC elimination. These studies indicate that nanomaterials can be engineered to directly target CSCs. Thus, Gd-metallofullerenol is identified as a kind of non-toxic CSC specific inhibitors with significant therapeutic potential.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Epithelial-Mesenchymal Transition; Female; Fullerenes; Gadolinium; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Nanomedicine; Nanostructures; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

The mechanisms by which TGF-β promotes lung adenocarcinoma (ADC) metastasis are largely unknown. Here, we report that in lung ADC cells, TGF-β potently induces expression of DOCK4, but not other DOCK family members, via the Smad pathway and that DOCK4 induction mediates TGF-β's prometastatic effects by enhancing tumor cell extravasation. TGF-β-induced DOCK4 stimulates lung ADC cell protrusion, motility, and invasion without affecting epithelial-to-mesenchymal transition. These processes, which are fundamental to tumor cell extravasation, are driven by DOCK4-mediated Rac1 activation, unveiling a novel link between TGF-β and Rac1. Thus, our findings uncover the atypical Rac1 activator DOCK4 as a key component of the TGF-β/Smad pathway that promotes lung ADC cell extravasation and metastasis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Humans; Lung Neoplasms; Mice; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

Topics: Antigens, CD; Cadherins; Carcinoma, Squamous Cell; Cell Movement; Epithelial-Mesenchymal Transition; Flow Cytometry; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Histocompatibility Antigens; Histone-Lysine N-Methyltransferase; Histones; Humans; Lymphatic Metastasis; Methylation; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Transplantation; Neoplastic Stem Cells; Prognosis; Squamous Cell Carcinoma of Head and Neck; Stem Cells; Transforming Growth Factor beta; Treatment Outcome; Wound Healing

DACH1 has been found down-regulated in a variety of human cancers, but its clinical significance and functional roles in colorectal cancer (CRC) remain unknown. In this study, we identified DACH1 as a tumor suppressor in CRC. Suppression of DACH1 strikingly increased cell growth, migration and invasion potential of CRC cell line SW480. Expression analysis of a set of epithelial-mesenchymal transition (EMT) markers by RT-qPCR and western blot showed an increase in the expression of mesenchymal markers (vimentin and N-cadherin) and a reduction in the expression of epithelial marker (E-cadherin and γ-catenin). Furthermore, EMT characteristics in DACH1-downregulated CRC cells were abrogated by TGF-β inhibitor SB431542. DACH1 overexpression reduced TGF-β-induced EMT and inhibited SW480 cell invasion which can be reversed in the presence of TGF-β. Thus, our results suggest that DACH1 loss of function results in increased cell growth, motility and invasiveness through TGF-β-mediated EMT, and DACH1 loss of function has important therapeutic implications for targeted therapies of CRC.

Topics: Base Sequence; Biomarkers, Tumor; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; DNA Primers; Down-Regulation; Epithelial-Mesenchymal Transition; Eye Proteins; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Topics: Aged; Animals; Carcinogenesis; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Insulin-Like Growth Factor Binding Proteins; Male; Mice; Middle Aged; Neoplasm Metastasis; Smad Proteins; Transforming Growth Factor beta; Wnt Signaling Pathway

Transforming growth factor-β (TGF-β) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-β acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain-containing mTOR-interacting protein (Deptor), in coupling TGF-β to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α-negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli.

Topics: Animals; Caspases; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Mice; Neoplasm Metastasis; Signal Transduction; Smad3 Protein; Survivin; Transforming Growth Factor beta; Triple Negative Breast Neoplasms

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

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

As patient survival drops precipitously from early-stage cancers to late-stage and metastatic cancers, microRNAs that promote relapse and metastasis can serve as prognostic and predictive markers as well as therapeutic targets for chemoprevention. Here we show that miR-1269a promotes colorectal cancer (CRC) metastasis and forms a positive feedback loop with TGF-β signalling. miR-1269a is upregulated in late-stage CRCs, and long-term monitoring of 100 stage II CRC patients revealed that miR-1269a expression in their surgically removed primary tumours is strongly associated with risk of CRC relapse and metastasis. Consistent with clinical observations, miR-1269a significantly increases the ability of CRC cells to invade and metastasize in vivo. TGF-β activates miR-1269 via Sox4, while miR-1269a enhances TGF-β signalling by targeting Smad7 and HOXD10, hence forming a positive feedback loop. Our findings suggest that miR-1269a is a potential marker to inform adjuvant chemotherapy decisions for CRC patients and a potential therapeutic target to deter metastasis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; Colorectal Neoplasms; Feedback, Physiological; Female; Fluorescent Antibody Technique; HCT116 Cells; Homeodomain Proteins; HT29 Cells; Humans; Male; Mice; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Prognosis; Real-Time Polymerase Chain Reaction; Smad7 Protein; SOXC Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Young Adult

A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive 'macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in response to tumour-derived factors, such as TGFβ, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFβ levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis.

Topics: Animals; Cell Differentiation; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Protein 1; Interferon Regulatory Factors; Leukocytes, Mononuclear; Melanoma, Experimental; Mice, Inbred C57BL; Myeloid Cells; Neoplasm Metastasis; Transforming Growth Factor beta

The VAV proteins VAV1, VAV2 and VAV3 have been identified as important molecules in tumorigenesis, tumor growth and cell migration. In addition to the full-length isoforms, a much shorter family member, VAV3.1, also known as VAV3 isoform 2, is known to be differentially expressed in a broad variety of tissues. Furthermore, VAV3.1 was shown to be down-regulated in cultured keratinocytes by the growth factors epidermal growth factor (EGF) EGF and transforming growth factor beta (TGFβ) TGFβ which in turn play important roles in the pathogenesis of oral squamous cell carcinoma (OSCC). Herein we showed that VAV3.1 is underexpressed in OSCC tissue samples compared to corresponding normal mucosa. We further demonstrated a trend of distinctive down-regulation of mRNA for VAV3.1 in tissues of locally advanced OSCC that have already metastasized to regional lymph nodes, indicating an increased malignant potential of tumors with low VAV3.1 mRNA expression. Moreover, in other studies a correlation between increased VAV3 expression and cancer progression was shown. In the present study, the analyzed OSCC tissue samples showed no significant change of VAV3 mRNA expression. Taken together, our data support the hypothesis that molecular interactions and signaling cascades of VAV3 can be regulated or directed by the competing molecule VAV3.1. Additionally, discrete and different functions of VAV3.1 in metastasis and tumorigenesis are conceivable.

Topics: Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Metastasis; Protein Isoforms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-vav; Signal Transduction; Transforming Growth Factor beta

The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.. The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.. We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.. Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

Topics: Actins; Adult; Aged; Antigens, Neoplasm; Biomarkers; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cohort Studies; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-19; Liver Neoplasms; Male; Microscopy, Fluorescence; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Odds Ratio; Prognosis; Recombinant Proteins; Stem Cells; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We report here that silencing SMURF2 (SMAD specific E3 ubiquitin protein ligase 2) promoted EMT in HPDE6c7 normal pancreas cells and overexpression of SMURF2 inhibited TGF-β-mediated EMT in the cells. Subsequent studies showed that SMURF2 was downregulated in pancreatic cancer tissues and it promoted mesenchymal-epithelial transition (MET) in pancreatic cancer cells as well as its expression negatively associated with gemcitabine-resistance, but it did not alter cell viability, cell cycle and cell senescence. In addition, we demonstrated that miR‑15b degraded SMURF2 and its overexpression promoted EMT in pancreatic cancer, and its expression was associated with metastasis in the disease. Elucidating molecular mechanism of EMT in pancreatic cancer not only will help us to further understand the pathogenesis and progression of the disease, but also offers new targets for effective therapies.

Topics: Cell Line; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Metastasis; Pancreatic Neoplasms; Promoter Regions, Genetic; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Cancer cachexia is a debilitating condition characterized by a combination of anorexia, muscle wasting, weight loss, and malnutrition. This condition affects an overwhelming majority of patients with pancreatic cancer and is a primary cause of cancer-related death. However, few, if any, effective therapies exist for both treatment and prevention of this syndrome. In order to develop novel therapeutic strategies for pancreatic cancer cachexia, appropriate animal models are necessary. In this study, we developed and validated a syngeneic, metastatic, murine model of pancreatic cancer cachexia. Using our model, we investigated the ability of transforming growth factor beta (TGF-β) blockade to mitigate the metabolic changes associated with cachexia. We found that TGF-β inhibition using the anti-TGF-β antibody 1D11.16.8 significantly improved overall mortality, weight loss, fat mass, lean body mass, bone mineral density, and skeletal muscle proteolysis in mice harboring advanced pancreatic cancer. Other immunotherapeutic strategies we employed were not effective. Collectively, we validated a simplified but useful model of pancreatic cancer cachexia to investigate immunologic treatment strategies. In addition, we showed that TGF-β inhibition can decrease the metabolic changes associated with cancer cachexia and improve overall survival.

Topics: Animals; Antibodies; Body Composition; Cachexia; Cell Line, Tumor; Disease Models, Animal; Immunotherapy; Male; Mice; Mice, Inbred C57BL; Muscular Atrophy; Neoplasm Metastasis; Pancreatic Neoplasms; Survival Analysis; 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

Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFβ signaling. In contrast, RNF2's oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms.. The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.

Topics: Animals; Catalysis; Cell Transformation, Neoplastic; Cyclin D2; Disease Progression; E1A-Associated p300 Protein; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Demethylases; Humans; Latent TGF-beta Binding Proteins; MAP Kinase Signaling System; Melanoma; Mice; Neoplasm Metastasis; Nuclear Proteins; Oncogenes; Phosphorylation; Polycomb Repressive Complex 1; Prognosis; Promoter Regions, Genetic; Transforming Growth Factor beta

Cell migration underlies metastatic dissemination of cancer cells, and fast "amoeboid" migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.

Topics: Actomyosin; Animals; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Movement; Cell Shape; Epithelial-Mesenchymal Transition; Humans; Keratinocytes; Melanocytes; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Nuclear Proteins; Smad2 Protein; Trans-Activators; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

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

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a part of the ribonucleoprotein complex which regulates diverse biological events. While overexpression of hnRNP K has been shown to be related to tumorigenesis in several cancers, both the expression patterns and biological mechanisms of hnRNP K in renal cell carcinoma (RCC) cells remain unclear. In this study, we showed that hnRNP K protein was strongly expressed in selected RCC cell lines (ACHN, A498, Caki-1, 786-0), and knock-down of hnRNP K expression by siRNA induced cell growth inhibition and apoptosis. Based on immunohistochemical (IHC) analysis of hnRNP K expression in human clear cell RCC specimens, we demonstrated that there was a significant positive correlation between hnRNP K staining score and tumor aggressiveness (e.g., Fuhrman grade, metastasis). Particularly, the rate of cytoplasmic localization of hnRNP K in primary RCC with distant metastasis was significantly higher than that in RCC without metastasis. Additionally, our results indicated that the cytoplasmic distribution of hnRNP K induced by TGF-β stimulus mainly contributed to TGF-β-triggered tumor cell invasion in RCC cells. Dominant cytoplasmic expression of ectopic hnRNP K markedly suppressed the inhibition of invasion by knock-down of endogenous hnRNP K. The expression level of matrix metalloproteinase protein-2 was decreased by endogenous hnRNP K knock-down, and restored by ectopic hnRNP K. Therefore, hnRNP K may be a key molecule involved in cell motility in RCC cells, and molecular mechanism associated with the subcellular localization of hnRNP K may be a novel target in the treatment of metastatic RCC.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cytoplasm; Down-Regulation; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Kidney Neoplasms; Mutation; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Transport; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) induces epithelial-mesenchymal transition (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, identification of TGF-β-inducible, EMT-specific genes during metastatic progression of lung cancer remains elusive. Here, we functionally validate a previously described post-transcriptional pathway by which TGF-β modulates expression of interleukin-like EMT inducer (ILEI), and EMT itself. We show that poly r(C)-binding protein 1 (PCBP1) binds ILEI transcript and repress its translation. TGF-β activation leads to phosphorylation at serine-43 of PCBP1 by protein kinase Bβ/Akt2, inducing its release from the ILEI transcript and translational activation. Modulation of hnRNP E1 expression modification altered TGF-β-mediated reversal of translational silencing of ILEI transcripts and EMT. Furthermore, ILEI could induce, as well as maintain, CD24(low)CD44(high) subpopulation in A549 cells treated with TGF-β, which might explain its capability to induce metastatic progression. These results thus validate the existence of an evolutionary conserved TGF-β-inducible post-transcriptional regulon that controls EMT and subsequent metastatic progression of lung cancer.

Topics: CD24 Antigen; Cell Line, Tumor; Cytokines; Disease Progression; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Hyaluronan Receptors; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Proteins; Protein Binding; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Transforming Growth Factor beta

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

Topics: Animals; Argonaute Proteins; Binding, Competitive; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; HMGA2 Protein; Humans; Lung Neoplasms; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Receptors, Transforming Growth Factor beta; RNA Isoforms; Transcription, Genetic; Transforming Growth Factor beta

The role for the inhibitors of differentiation (Ids) proteins in melanomagenesis has been poorly explored. In other cell types, Ids have been shown to contribute to cell proliferation, migration and angiogenesis and, along with a number of other genes, are direct downstream targets of the transforming growth factor (TGF)-β pathway. Expression of Smad7, which suppress TGF-β signaling, or synthetic TGF-β inhibitors, was shown to potently suppress melanomagenesis. We found that endogenous Id2, Id3 and Id4 expression was elevated in 1205Lu versus 1205Lu cells constitutively expressing Smad7, indicating Ids may play a role in melanomagenesis. Therefore, the effects of Tet-inducible expression of Id2, Id3 or Id4 along with Smad7 in TGF-β-dependent 1205Lu human melanoma cells were explored in vitro and in vivo. 1205Lu cells formed subcutaneous tumors in athymic mice, whereas cells expressing Smad7 failed to form tumors. However, 1205Lu cells expressing Smad7 along with doxycycline-induced Id2, Id3 or Id4 were able to overcome the potent tumorigenic block mediated by S7, to varying degrees. Conversely, Id small interfering RNA knockdown suppressed anchorage-independent growth of melanoma. Histology of tumors from 1205Lu cells expressing Smad7 + Id4 revealed an average of 31% necrosis, compared with 5.2% in tumors from 1205Lu with vector only. Downstream, Ids suppressed cyclin-dependent kinase inhibitors, and re-upregulated invasion and metastasis-related genes matrix metalloproteinase 2 (MMP2), MMP9, CXCR4 and osteopontin, shown previously to be downregulated in response to Smad7. This study shows that Id2, Id3 and Id4 are each able to overcome TGF-β dependence, and establish a role for Ids as key mediators of TGF-β melanomagenesis.

Topics: Base Sequence; Cell Line, Tumor; Cell Proliferation; DNA Primers; Humans; Inhibitor of Differentiation Protein 2; Inhibitor of Differentiation Proteins; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

Dysregulation in miRNA expression contributes towards the initiation and progression of metastasis by regulating multiple target genes. In this study, variations in miRNA expression profiles were investigated between high and low invasive NSCLC cell lines followed by identification of miRNAs with targets governing NSCLC's metastatic potential.. Two NSCLC sub-cell lines possessing opposing migration and invasion properties were established using serial transwell invasion assays. Global miRNA expression profiles were obtained using microarray followed by RT-qPCR validation. Target prediction and pathway enrichment analyses were conducted on dysregulated miRNAs using DIANA-mirPath, DIANA-microT 4.0 and TargetScan 5.2 softwares. Metastatic effects of dysregulated miRNAs were evaluated using wound healing assay, invasion assay and HUVEC angiogenesis assay following transfection with mimics and inhibitors.. A total of eleven differentially expressed miRNAs were revealed from microarray analyses, with four miRNAs validated through RT-qPCR. Three of these miRNAs were further selected for biological function validations, with only two modulating metastasis. A pathway model describing interactions between miRNAs and metastasis highlighted four major pathways: non-canonical Wnt/PCP, TGF-β, MAPK and integrin-FAK-Src signalling cascade.. These results provide a list of potential candidate metastatic markers during the classification of NSCLCs and a platform for the development of bio-therapeutics targeting these miRNA control elements.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Movement; Computer Simulation; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 2; Gene Expression Profiling; Humans; Integrins; Lung Neoplasms; Microarray Analysis; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Signal Transduction; Software; src-Family Kinases; Transforming Growth Factor beta; Wnt Proteins

A key feature of TGF-β signaling activation in cancer cells is the sustained activation of SMAD complexes in the nucleus; however, the drivers of SMAD activation are poorly defined. Here, using human and mouse breast cancer cell lines, we found that oncogene forkhead box M1 (FOXM1) interacts with SMAD3 to sustain activation of the SMAD3/SMAD4 complex in the nucleus. FOXM1 prevented the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding SMAD3 and monoubiquitinating SMAD4, which stabilized the SMAD3/SMAD4 complex. Loss of FOXM1 abolished TGF-β-induced SMAD3/SMAD4 formation. Moreover, the interaction of FOXM1 and SMAD3 promoted TGF-β/SMAD3-mediated transcriptional activity and target gene expression. We found that FOXM1/SMAD3 interaction was required for TGF-β-induced breast cancer invasion, which was the result of SMAD3/SMAD4-dependent upregulation of the transcription factor SLUG. Importantly, the function of FOXM1 in TGF-β-induced invasion was not dependent on FOXM1's transcriptional activity. Knockdown of SMAD3 diminished FOXM1-induced metastasis. Furthermore, FOXM1 levels correlated with activated TGF-β signaling and metastasis in human breast cancer specimens. Together, our data indicate that FOXM1 promotes breast cancer metastasis by increasing nuclear retention of SMAD3 and identify crosstalk between FOXM1 and TGF-β/SMAD3 pathways. This study highlights the critical interaction of FOXM1 and SMAD3 for controlling TGF-β signaling during metastasis.

Topics: Animals; Cell Line, Tumor; Cell Nucleus; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; HEK293 Cells; Humans; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; NIH 3T3 Cells; Signal Transduction; Smad3 Protein; Smad4 Protein; Transcription Factors; Transforming Growth Factor beta; Ubiquitin; Up-Regulation

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-β signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression.

Topics: ADAM Proteins; ADAM17 Protein; Antigens, CD; Biomarkers, Tumor; Bone and Bones; Caspase 3; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cellular Microenvironment; Disease Progression; Fetal Proteins; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; Transforming Growth Factor beta

The transforming growth factor beta (TGFβ) signaling cascade is considered as one of the pivotal oncogenic pathways in most advanced cancers. Inhibition of the TGFβ signaling pathway by specific antagonists, neutralizing antibodies, or small molecules is considered as an effective strategy for the treatment of tumor growth and metastasis. Here we demonstrated the identification of a series of tetrahydro-β-carboline derivatives from virtual screening which potentially inhibit the TGFβ signaling pathway. Optimization of the initial hit compound 2-benzoyl-1,3,4,9-tetrahydro-β-carboline (8a) through substitution at different positions to define the structure-activity relationship resulted in the discovery of potent inhibitors of the TGFβ signaling pathway. Among them, compound 8d, one of the tested compounds, not only showed potent inhibition of lung cancer cell proliferation and migration in vitro but also strongly suppressed growth of lung cancer and breast cancer in vivo.

Topics: Animals; Antineoplastic Agents; Carbolines; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Screening Assays, Antitumor; Female; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Docking Simulation; Neoplasm Metastasis; Neoplasm Transplantation; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor beta

Decorin (DCN) has been suggested to display an anti-metastatic role by antagonizing bioactive TGF-β in advanced human cancers. However, the epigenetic mechanisms by which defective expression of DCN causes cancer metastasis remain unclear. We focused on non-small cell lung cancer (NSCLC) cell lines with low metastatic potential (95C) and high metastatic potential (95D), which share a similar genetic background. Quantitative PCR and clonal bisulfite sequencing indicated that the methylation levels of the +58CpG site in the DCN 5'-UTR region was significantly higher in 95D cells with low expression of DCN mRNA compared to 95C cells with high expression of DCN mRNA. In silico prediction and ChIP assay showed a correlation between +58CpG site and AhR, which was reported as a transcriptional activator. Importantly, EMSA and luciferase reporter gene assays suggested that +58CpG methylation specifically diminished the recruitment of AhR to DCN 5'-UTR sequence and caused a reduction of approximately 50% in transcriptional activity. At baseline, 95D cells exhibited higher p-Smad3 levels and lower E-cadherin expression when compared with 95C cells. The demethylating agent 5-Aza significantly led to restoration of DCN expression, reduced level of p-Smad3, increased expression of E-cadherin in 95D cells. Taken together, we identified the methylated +58CpG in DCN 5'-UTR associated with reduced expression of DCN mRNA, and revealed that +58CpG methylation may be one of the mechanisms accounting for reduced recruitment of the transcriptional activator AhR to DCN 5'-UTR, and suggest that this mechanism promotes TGF-β/Smad signaling by enhancing the phosphorylation of Smad3, thereby downregulating E-cadherin in NSCLC cells with high metastatic potential.

Topics: Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; CpG Islands; Decorin; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Metastasis; RNA, Messenger; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) plays important roles in tumor metastasis by regulating miRNAs expression. miR-182 is an important molecule in the regulation of cancer progression. The aim of the study is to assess the role of miR-182 in TGF-β-induced cancer metastasis. In the present study, we found that miR-182 levels are significantly upregulated in GBC tissues compared with normal controls, and miR-182 expression is remarkably increased in primary tumors that subsequently metastasized, when compared to those primary tumors that did not metastasize. TGF-β induces miR-182 expression in GBC cells, and overexpression of miR-182 promotes GBC cell migration and invasion, whereas miR-182 inhibition suppresses TGF-β-induced cancer cell migration and invasion. The blockage of miR-182 by a specific inhibitor effectively inhibits pulmonary metastases in vivo. We further identified that the cell adhesion molecule1 (CADM1) is a new target gene of miR-182. miR-182 negatively regulates CADM1 expression in vitro and in vivo. Importantly, re-expression of CADM1 in GBC cells partially abrogates miR-182-induced cell invasion.. miR-182 is an important mediator of GBC metastasis, thus offering a new target for the development of therapeutic agents against GBC.

Topics: Cell Adhesion Molecule-1; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Gallbladder Neoplasms; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunoglobulins; MicroRNAs; Neoplasm Metastasis; Transforming Growth Factor beta

The effects of berberine on the metastatic potential of lung cancer cells and its underlying mechanisms have not been fully elucidated. Since epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis, we attempted to investigate the potential use of berberine as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal in A549 cells.. In this study, we investigated the anticancer activity of berberine against A549 cells in vitro and in vivo. BBR-induced apoptosis of the human lung cancer cells was determined by flow cytometry. The ability of BBR to inhibit TGF-β-induced EMT was examined by QRT-PCR and Western blotting. The impact of BBR on A549 cell migration and invasion was evaluated by transwell assay.. We demonstrated that TGF-β1 induced epithelial-to-mesenchymal to promote lung cancer invasion and metastasis. Berberine inhibited invasion and migration of A549 cells, increased expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker Vimentin, as well as decreased the level of epithelial-to-mesenchymal -inducing transcription factors Snail1 and Slug during the initiation of TGF-β1-induced epithelial-to-mesenchymal. Furthermore, berberine inhibited growth of lung cancer cells in vivo xenograft.. Our findings provided new evidence that berberine is an effective inhibitor of the metastatic potential of A549 cells through suppression of TGF-β1-induced epithelial-to-mesenchymal.

Topics: Animals; Apoptosis; Berberine; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Cell Survival; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Transcription Factors; Transforming Growth Factor beta; Treatment Outcome; Xenograft Model Antitumor Assays

Radiotherapy is an effective treatment method for lung cancer, particularly when the disease is at an advanced stage. However, previous researchers have observed that the majority of patients with conventional radiation therapy develop distant metastases and succumb to the disease. Thus, identifying and understanding novel pathways for the development of new therapeutic targets is a major goal in research on pulmonary neoplasms. Recent studies suggest that epithelial-mesenchymal transition (EMT) is the most important contributor to cancer metastasis. Induction of this complex process requires endogenously produced microRNAs; specifically, downregulation of the miRNA-200c causes an induction of EMT. We recently identified the tank-binding kinase-1 (TBK1) as a downstream effector of the miR-200c-driven pathway, but the biological function of TBK1 in EMT remains unknown. In this study, we tested whether TBK1 has a role in radiation-induced EMT and identified associated potential mechanisms. Human alveolar type II epithelial carcinoma A549 cells were irradiated with (60)Co γ-rays. Western blotting revealed a time- and dose-dependent decrease in E-cadherin with a concomitant increase in vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. TBK1 siRNA significantly inhibited radiation-induced suppression of the epithelial marker E-cadherin and upregulation of the mesenchymal marker vimentin. The invasion and migratory potential of lung cancer cells upon radiation treatment was also reduced by TBK1 knockdown. Furthermore, radiation-induced EMT attenuated by TBK1 depletion was partially dependent on transcriptional factor ZEB1 expression. Finally, we found glycogen synthase kinase-3β (GSK-3β) is involved in regulation of radiation-induced EMT by TBK1. Thus, our findings reveal that TBK1 signaling regulates radiation-induced EMT by controlling GSK-3β phosphorylation and ZEB1 expression. TBK1 may therefore constitute a useful target for treatment of radiotherapy-induced metastasis diseases.

Topics: Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gamma Rays; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Humans; Neoplasm Metastasis; Neoplasms; NF-kappa B; Protein Serine-Threonine Kinases; Radiotherapy; Smad Proteins; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Tumor-produced extracellular matrix (ECM) proteins can be key elements in tumor growth and metastasis. Transforming growth factor beta-inducible (TGFBI) protein is a secreted ECM component that can have dual function in cancer, acting as tumor suppressor or promoter. Although TGFBI is expressed in human melanoma cells, the exact role it might have in melanoma metastasis remains elusive. Assessing the expression and secretion of TGFBI, we show that human metastatic melanomas express and secrete significantly higher amounts of TGFBI, compared with nevus lesions and primary melanoma tumors. Intravenous injection of highly metastatic human melanoma cells expressing shRNA that targets TGFBI assigns a critical role for TGFBI in the formation of melanoma distal metastases in nude mice. In vivo assays demonstrate that TGFBI silencing does not interfere with melanoma cells' dissemination to distal sites but rather with their proliferation and outgrowth within new microenvironment. In line, TGFBI silencing increases melanoma cells motility/invasion/extravasation in vitro but interferes with their progression through the cell cycle, drastically reducing their proliferation. Furthermore, we show that TGFBI is a regulator of cyclins and cyclin-dependent kinases in melanoma. Collectively, our data describe a mechanism of melanoma metastatic outgrowth via promotion of growth/survival by the ECM protein TGFBI.

Topics: Animals; Apoptosis; Biopsy; Cell Adhesion; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Extracellular Matrix Proteins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Small Interfering; Skin Neoplasms; Transforming Growth Factor beta

Cofilin (CFL) is an F-actin-severing protein required for the cytoskeleton reorganization and filopodia formation, which drives cell migration. CFL binding and severing of F-actin is controlled by Ser3 phosphorylation, but the contributions of this step to cell migration during invasion and metastasis of cancer cells are unclear. In this study, we addressed the question in prostate cancer cells, including the response to TGF-β, a critical regulator of migration. In cells expressing wild-type CFL, TGF-β treatment increased LIMK-2 activity and cofilin phosphorylation, decreasing filopodia formation. Conversely, constitutively active CFL (SerAla) promoted filipodia formation and cell migration mediated by TGF-β. Notably, in cocultures of prostate cancer epithelial cells and cancer-associated fibroblasts, active CFL promoted invasive migration in response to TGF-β in the microenvironment. Further, constitutively active CFL elevated the metastatic ability of prostate cancer cells in vivo. We found that levels of active CFL correlated with metastasis in a mouse model of prostate tumor and that in human prostate cancer, CFL expression was increased significantly in metastatic tumors. Our findings show that the actin-severing protein CFL coordinates responses to TGF-β that are needed for invasive cancer migration and metastasis.

Topics: Actin Depolymerizing Factors; Animals; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Heterografts; Humans; Male; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Prostatic Neoplasms; Signal Transduction; Transforming Growth Factor beta

Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the β1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of β1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked β1 integrin function or knockdown of β1 switched the migratory behavior of human and mouse E-cadherin-positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor-β (TGFβ) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFβ receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in β1 integrin-deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that β1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting β1 integrins may have undesirable effects in TNBC.

Topics: Animals; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Extracellular Matrix; Flow Cytometry; Gene Silencing; Homeodomain Proteins; Humans; Immunohistochemistry; Integrin beta1; Luciferases; Lung Neoplasms; Mice; Mice, Knockout; MicroRNAs; Neoplasm Metastasis; Repressor Proteins; Signal Transduction; Time-Lapse Imaging; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Zebrafish; Zinc Finger E-box Binding Homeobox 2

In advanced cancers, the TGF-β pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-β signalling. NR4A1 promotes TGF-β/SMAD signalling by facilitating AXIN2-RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-β-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-β-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-β-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-β signalling in breast cancer.

Topics: Animals; Cells, Cultured; Chromatin Immunoprecipitation; Female; Immunohistochemistry; Mammary Neoplasms, Animal; Mice; Mice, Knockout; Neoplasm Metastasis; Nuclear Receptor Subfamily 4, Group A, Member 1; Reverse Transcriptase Polymerase Chain Reaction; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Ubiquitination; Zebrafish

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

Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Down-Regulation; Enzyme Activation; Extracellular Matrix Proteins; Humans; Immunoprecipitation; Integrin alpha2beta1; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Transforming Growth Factor beta

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

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

The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

Topics: Animals; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Homeodomain Proteins; Humans; Interleukin-11; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Prognosis; Repressor Proteins; RNA, Long Noncoding; RNA, Messenger; STAT3 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

The impact of complement on cancer metastasis has not been well studied. In this report, we demonstrate in a preclinical mouse model of breast cancer that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8(+) and CD4(+) T-cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells to the lungs and regulation of TGFβ and IL10 production in these cells. TGFβ and IL10 favored generation of T regulatory cells (Treg) and Th2-oriented responses that rendered CD8(+) T cells dysfunctional. Importantly, pharmacologic blockade of C5aR or its genetic ablation in C5aR-deficient mice were sufficient to reduce lung metastases. Depletion of CD8(+) T cells abolished this beneficial effect, suggesting that CD8(+) T cells were responsible for the effects of C5aR inhibition. In contrast to previous findings, we observed that C5aR signaling promoted Treg generation and suppressed T-cell responses in organs where metastases arose. Overall, our findings indicated that the immunomodulatory functions of C5aR are highly context dependent. Furthermore, they offered proof-of-concept for complement-based immunotherapies to prevent or reduce cancer metastasis.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Complement C5a; Female; Immunosuppression Therapy; Interleukin-10; Liver Neoplasms; Lung; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Metastasis; Receptor, Anaphylatoxin C5a; Signal Transduction; T-Lymphocytes, Regulatory; 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

Topics: Antineoplastic Agents; Brain Neoplasms; Diagnosis, Differential; Extracellular Matrix Proteins; G1 Phase; Humans; Lung Neoplasms; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Phospholipases A2; S Phase; Skin Neoplasms; Transforming Growth Factor beta

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

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non-tumour tissues were examined. Methylation-specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re-expression was induced by 5-Aza-2'-deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non-tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial-mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)-β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1-expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF-β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.

Topics: Animals; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Movement; Cell Survival; DNA Methylation; Docetaxel; Dose-Response Relationship, Drug; Epigenesis, Genetic; Eye Proteins; Female; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Taxoids; Transcription Factors; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor-suppressor or -promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor-β/ Drosophila mothers against decapentaplegic protein (TGF-β/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF-β-inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis-related TGF-β/Smad downstream c-myc down-regulation, as well as p21/cip1 and p15/ink4b up-regulation in early-stage HCC. Meanwhile, TGF-β inducible epithelial-mesenchymal transition and TGF-β/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down-regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor- and protein kinase B-signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down-regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis.. TIF1γ regulates tumor growth and metastasis through inhibition of TGF-β/Smad signaling and may serve as a novel prognostic biomarker in HCC.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; CpG Islands; DNA Methylation; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Humans; Liver; Liver Neoplasms, Experimental; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; Smad2 Protein; Transcription Factors; 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

Elevated levels of TGFβ are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFβ pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFβ remains challenging because TGFβ has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFβ promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFβ receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFβ signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFβ-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.

Topics: Animals; Antibodies, Monoclonal; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Line, Tumor; Deoxycytidine; Gemcitabine; Humans; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; NIH 3T3 Cells; Pancreatic Neoplasms; Random Allocation; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

SMAD4 has been suggested to inhibit the activity of the WNT/β-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/β-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of β-catenin/T-cell factor (TCF) complex by stabilizing β-catenin through the inhibition of GSK-3β. Here, SMAD4 modulated AURKA in a TGFβ-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the β-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of β-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and β-catenin was independent of TGFβ signaling because blockage of TGFβR1 or restoration of TGFβ signaling did not prevent suppression of AURKA and β-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of β-catenin transcriptional activity via AURKA degradation in a TGFβ-independent manner.. SMAD4 interacts with AURKA and antagonizes its tumor-promoting potential, thus demonstrating a novel mechanism of tumor suppression.

Topics: Animals; Aurora Kinase A; beta Catenin; Cell Line, Tumor; Cell Movement; HeLa Cells; Humans; Mice; Mutation; Neoplasm Metastasis; Neoplasms; NIH 3T3 Cells; Proteasome Endopeptidase Complex; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

The Notch signaling pathway plays an important role in the bone metastasis microenvironment. Although recent evidence suggests that Notch signaling contributes to bone metastasis in breast and prostate cancer, its role and possible mechanisms in non-small cell lung cancer (NSCLC) bone metastasis are not yet clear. Here, we show that Notch3 is overexpressed in NSCLC bone metastases. The inhibition of Notch3 by small interfering RNA transfection decreased the invasion ability of NSCLC cells and transforming growth factor (TGF)-induced interleukin (IL)-6 and parathyroid hormone-related protein (pTHrP) expression in vitro. We also observed that Notch3 induced a strong morphological transformation, promoting the epithelial-mesenchymal transition (EMT). Western blotting and real-time polymerase chain reaction assays revealed that the forced overexpression of Notch3 induced the expression and activity of ZEB-1 and subsequent suppression of E-cadherin and upregulation of fibronectin, contributing to EMT and invasion. Western blotting and immunofluorescence assays showed that RNA interference-mediated ZEB-1 suppression blocked Notch-induced EMT-like transformation and subsequently reversed the observed effects on E-cadherin and downregulated fibronectin. A luciferase reporter system showed that Notch-induced ZEB-1 requires a functional binding site in the ZEB-1 promoter. In vitro invasion assays showed that the inhibition of ZEB-1 can decrease Notch3-promoted invasion and the expression of pTHrP and IL-6. Our results demonstrated that Notch upregulates ZEB-1, which contributes to TGF-β-induced EMT-like transformation and bone metastasis in NSCLC.

Topics: Binding Sites; Bone Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Interleukin-6; Lung Neoplasms; Neoplasm Metastasis; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Protein Binding; Receptor, Notch3; Receptors, Notch; RNA, Small Interfering; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

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

Colon carcinoma is a malignant tumor showing a marked preference to metastasize to distant organs. The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. However, whether circulating cytokines are linked to the circulation of tumor cells, as individual cells or clusters, remain unclear. In this study, we investigated the circulating levels of TGF-beta, CXCL1, VEGF and PAI-1 as potential bioindicators of the presence of CTCs in patients with metastatic colon cancer.. Circulating tumor cells (CTCs) were isolated from peripheral blood by immunomagnetic separation and phenotypically characterized in a cohort of 103 patients with metastatic colon cancer. TGF-beta, CXCL1, VEGF and PAI-1 concentrations were determined by immunoassay in plasma samples from the same patients.. We detected two different populations of CTCs, single cells or clusters in patients with metastatic colon cancer. Importantly, we found that the presence of clustered CTCs is significantly associated with elevated circulating levels of TGF-beta and CXCL1 and with reduced overall survival. Finally, we observed that circulating levels of cytokines are differently associated with the two populations of CTCs.. Taken together, these findings show that detection of clustered CTCs represents a negative prognostic factor in patients with metastatic colon cancer. The presence of clustered CTCs is associated with elevated circulating levels of cytokines such as TGF-beta and CXCL1. This suggests an additional role for circulating cytokines as predictive tool for cancer prognosis and diagnosis of minimal residual disease as well as assessment of tumor sensitivity to anticancer therapy.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chemokine CXCL1; Colorectal Neoplasms; Cytokines; Female; Genetic Predisposition to Disease; Humans; Italy; Male; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Phenotype; Prognosis; Transforming Growth Factor beta

Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; DNA-Binding Proteins; Enhancer Elements, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Introns; Lung Neoplasms; Mice; Models, Biological; Neoplasm Metastasis; Peptide Hydrolases; Pre-B-Cell Leukemia Transcription Factor 1; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Signal Transduction; Smad3 Protein; Survival Analysis; Transcription Factor AP-1; Transcription, Genetic; Transforming Growth Factor beta

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

Tumor metastasis is a prominent cause of treatment failure in cervical carcinoma. Phenethyl isothiocyanate (PEITC) is an active component extracted from cruciferous plants that has exhibited anticancer activity in various types of human cancer; however, its effect on the inhibition of metastasis remains unclear. The current study aimed to explore the effect of PEITC on the suppression of metastasis in HeLa cervical carcinoma cells. Multiple variables were assessed with different methods as follows: Cell viability, with a Vi‑CELL analyzer; cell adhesion, by MTS assay; cell invasion, by Transwell assay; cell cycle, by flow cytometry assay; cytokine concentration, by ELISA assay; metastasis‑related gene and protein expression, by quantitative polymerase chain reaction and western blotting; and transcription factor activity, by gene reporter assay. The results indicated that PEITC exhibited an inhibitory effect on the adhesion and invasion of HeLa cells by induction of G2/M phase arrest, it reduced the expression of CDK1, MMP‑2/9, CD44, ICAM‑1, increased the production of TGF‑β, IL‑6 and IL‑8, and increased the phosphorylation of Smad2. These results suggest that PEITC may be a potential antitumor compound, acting through the TGF‑β/Smad2 pathway; and it has the potential for future use as a therapy for cervical carcinoma subsequent to further studies.

Topics: Antineoplastic Agents; Cell Adhesion; Cell Cycle Checkpoints; Cell Movement; Cell Proliferation; Drug Screening Assays, Antitumor; Female; Gene Expression; HeLa Cells; Humans; Interleukin-6; Interleukin-8; Isothiocyanates; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Uterine Cervical Neoplasms

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell-like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow-derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.

Topics: Animals; Arginase; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Disease Progression; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Inflammation; Interleukin-4; Macrophages; Mice; Neoplasm Metastasis; Pancreatic Neoplasms; Phenotype; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Transforming Growth Factor beta

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

The epithelial-to-mesenchymal transition (EMT) process results in a loss of cell-cell adhesion, increased cell mobility, and is crucial for enabling the metastasis of cancer cells. Recently, the enzyme SIRT1 has been implicated in a variety of physiological processes; however, its role in regulating oral cancer metastasis and EMT is not fully elucidated. Here, we propose a mechanism by which the enzyme sirtuin1 (SIRT1) regulates the EMT process in oral cancer by deacetylating Smad4 and repressing the effect of TGF-β signaling on matrix metalloproteinase-7 (MMP7).. The roles of SIRT1 in tumor cell migration/invasion and metastasis to the lungs were investigated using the Boyden chamber assay and orthotopic injections, respectively. RNA interference was used to knockdown either SIRT1 or Smad4 expression in oral squamous cell carcinoma (OSCC) cell lines. Immunoblotting, zymographic assays, and co-immunoprecipitation were used to examine the effects of SIRT1 overexpression on MMP7 expression and activity, as well as on SIRT1/ Smad4 interaction.. We found that compared with normal human oral keratinocytes (HOKs), SIRT1 was underexpressed in OSCC cells, and also in oral cancer tissues obtained from 14 of 21 OSCC patients compared with expression in their matched normal tissues. Overexpression of SIRT1 inhibited migration of OSCC cells in vitro, as well as their metastasis to the lung in vivo. Furthermore, up-regulation of SIRT1 in metastatic OSCCs significantly inhibited the migration and invasion abilities of OSCC cells, while concomitantly increasing the expression of E-cadherin, and decreasing the expressions of mesenchymal markers. We also identified Smad4, a TGF-β-activated transcription factor, as a direct target protein for SIRT1. Overexpression of SIRT1 in OSCC cells led to decreased levels of acetylated Smad4, and inhibition of TGF-β-induced signaling. By associating and deacetylating Smad4, SIRT1 enzyme can influence MMP7 expression, MMP enzyme activity, and consequently, cell migration, invasion, and tumor metastasis in OSCCs.. These findings provide a valuable insight into the potential role of the SIRT1 enzyme in regulating cell migration and invasion in oral squamous cell carcinoma. Our findings suggest the SIRT1/Smad4/MMP7 pathway as a target for oral cancer driven by EMT.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Male; Matrix Metalloproteinase 7; Mice; Mice, SCID; Mouth Neoplasms; Neoplasm Metastasis; Sirtuin 1; Smad4 Protein; Transforming Growth Factor beta

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

TGF-β can act as a tumor suppressor at early stages of cancer progression and as a tumor promoter at later stages. The E3 ubiquitin ligase Arkadia (RNF111) is a critical component of the TGF-β signaling pathway, being required for a subset of responses, those mediated by Smad3-Smad4 complexes. It acts by mediating ligand-induced degradation of Ski and SnoN (SKIL), which are 2 potent transcriptional repressors. Here, we investigate the role of Arkadia in cancer using model systems to address both potential tumor-suppressive and tumor-promoting roles. Stable reexpression of Arkadia in lung carcinoma NCI-H460 cells, which we show contain a hemizygous nonsense mutation in the Arkadia/RNF111 gene, efficiently restored TGF-β-induced Smad3-dependent transcription, and substantially decreased the ability of these cells to grow in soft agar in vitro. However, it had no effect on tumor growth in vivo in mouse models. Moreover, loss of Arkadia in cancer cell lines and human tumors is rare, arguing against a prominent tumor-suppressive role. In contrast, we have uncovered a potent tumor-promoting function for Arkadia. Using 3 different cancer cell lines whose tumorigenic properties are driven by TGF-β signaling, we show that loss of Arkadia function, either by overexpression of dominant negative Arkadia or by siRNA-induced knockdown, substantially inhibited lung colonization in tail vein injection experiments in immunodeficient mice. Our findings indicate that Arkadia is not critical for regulating tumor growth per se, but is required for the early stages of cancer cell colonization at the sites of metastasis.

Topics: Animals; Biocatalysis; Blotting, Western; Cell Line, Tumor; Humans; Mice; Mutation; Neoplasm Metastasis; Nuclear Proteins; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

A wealth of evidence has now demonstrated that the microenvironment in which a tumorigenic cell evolves is as critical to its evolution as the genetic mutations it accrues. However, there is still relatively little known about how signals from the microenvironment contribute to the early events in the progression to malignancy. To address this question, we used a premalignant mammary model to examine how fibroblasts, and the extracellular matrix (ECM) proteins they secrete, influence progression to malignancy. Their effect on metastatic malignant cells was also assessed for comparison. We found that carcinoma-associated fibroblasts, and the distinct aligned ECM they deposit, can cause both premalignant and malignant mammary epithelial cells to assume a mesenchymal morphology that is associated with increased dissemination and metastasis, while benign reduction mammoplasty fibroblasts favor the maintenance of an epithelial morphology and constrain early dissemination, tumor growth, and metastasis. Our results suggest that normalizing the organization of the ECM could be effective in limiting systemic dissemination and tumor growth.

Topics: Animals; Breast; Cell Line, Tumor; Cell Transformation, Neoplastic; Coculture Techniques; Epithelial Cells; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Phenotype; Proto-Oncogene Proteins c-jun; rho GTP-Binding Proteins; Signal Transduction; Transforming Growth Factor beta

Although the potential roles of endothelial cells in the microvascules of prostate cancer during angiogenesis have been documented, their direct impacts on the prostate cancer metastasis remain unclear. We found that the CD31-positive and CD34-positive endothelial cells are increased in prostate cancer compared with the normal tissues and that these endothelial cells were decreased upon castration, gradually recovered with time, and increased after prostate cancer progressed into the castration-resistant stage, suggesting a potential linkage of these endothelial cells with androgen deprivation therapy. The in vitro invasion assays showed that the coculture of endothelial cells with prostate cancer cells significantly enhanced the invasion ability of the prostate cancer cells. Mechanism dissection found that coculture of prostate cancer cells with endothelial cells led to increased interleukin (IL)-6 secretion from endothelial cells, which may result in downregulation of androgen receptor (AR) signaling in prostate cancer cells and then the activation of TGF-β/matrix metalloproteinase-9 (MMP-9) signaling. The consequences of the IL-6→AR→TGFβ→MMP-9 signaling pathway might then trigger the increased invasion of prostate cancer cells. Blocking the IL-6→AR→TGFβ→MMP-9 signaling pathway either by IL-6 antibody, AR-siRNA, or TGF-β1 inhibitor all interrupted the ability of endothelial cells to influence prostate cancer invasion. These results, for the first time, revealed the important roles of endothelial cells within the prostate cancer microenvironment to promote the prostate cancer metastasis and provide new potential targets of IL-6→AR→TGFβ→MMP-9 signals to battle the prostate cancer metastasis.

Topics: Apoptosis; Cell Line, Tumor; Endothelial Cells; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Male; Matrix Metalloproteinase 9; Neoplasm Metastasis; Neovascularization, Pathologic; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.

Topics: Adenocarcinoma; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; RNA Processing, Post-Transcriptional; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

TGF-β is overexpressed in advanced human cancers. It correlates with metastasis and poor prognosis. However, TGF-β functions as both a tumor suppressor and a tumor promoter. Here, we report for the first time that genetic deletion of Tgfbr2 specifically in myeloid cells (Tgfbr2(MyeKO)) significantly inhibited tumor metastasis. Reconstitution of tumor-bearing mice with Tgfbr2(MyeKO) bone marrow recapitulated the inhibited metastasis phenotype. This effect is mediated through decreased production of type II cytokines, TGF-β1, arginase 1, and inducible nitric oxide synthase, which promoted IFN-γ production and improved systemic immunity. Depletion of CD8 T cells diminished the metastasis defect in the Tgfbr2(MyeKO) mice. Consistent with animal studies, myeloid cells from patients with advanced-stage cancer showed increased TGF-β receptor II expression. Our studies show that myeloid-specific TGF-β signaling is an essential component of the metastasis-promoting puzzle of TGF-β. This is in contrast to the previously reported tumor-suppressing phenotypes in fibroblasts, epithelial cells, and T cells.

Topics: Animals; Arginase; Cell Line, Tumor; Disease Models, Animal; Epithelial Cells; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Knockout; Myeloid Cells; Neoplasm Metastasis; Nitric Oxide Synthase; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment

Cancer metastasis is a major cause for cancer-related death and inhibiting cancer metastasis is an alternative way to treat cancer. Several lines of reported evidence suggest that NADPH oxidase 4 (NOX4) is a potential target for intervention of cancer metastasis, as the reactive oxygen species (ROS) generated by this enzyme plays important roles in TGF-β signaling, an important inducer of cancer metastasis. Here we show (1) that TGF-β induces ROS production in breast cancer 4T1 cells and enhances cell migration and that the effect of TGF-β depends on NOX4 expression, (2) that knockdown of NOX4 via RNAi significantly decreases the migration ability of 4T1 cells in the presence or absence of TGF-β and significantly attenuates distant metastasis of 4T1 cells to lung and bone, (3) that Schisandrin B (Sch B), a naturally occurring dibenzocyclooctadiene lignan with very low toxicity, is a novel NOX inhibitor and its IC50 toward NOX4 is 9.3μM, and (4) that Sch B suppresses TGF-β-induced and NOX4-associated ROS production in 4T1 cells and inhibits TGF-β-enhanced cell migration. Similar to NOX4 knockdown observed in this study, Sch B significantly attenuated 4T1 cells distant metastasis to lung and bone in our recently published study. In line with previous reports, the study suggests that pharmacologically targeting NOX4 may be a potential approach to disrupt cancer metastasis.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cyclooctanes; Female; Humans; Lignans; Mice; Mice, Inbred BALB C; Microscopy, Confocal; NADPH Oxidase 4; NADPH Oxidases; Neoplasm Metastasis; Polycyclic Compounds; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

Despite recent advances, understanding of molecular genetic alterations underlying thyroid carcinogenesis remains unclear. One key question is how dynamic temporal changes in global genomic expression affect carcinogenesis as the disease progresses. To address this question, we used a mouse model that spontaneously develops follicular thyroid cancer similar to human cancer (Thrb (PV/PV) mice). Using complementary DNA microarrays, we compared global gene expression profiles of thyroid tumors of Thrb (PV/PV) mice with the age- and gender-matched thyroids of wild-type mice at 3 weeks and at 2, 4, 6 and 14 months. These time points covered the pathological progression from early hyperplasia to capsular invasion, vascular invasion and eventual metastasis. Microarray data indicated that 462 genes were upregulated (Up-cluster genes) and 110 genes were downregulated (Down-cluster genes). Three major expression patterns (trending up, cyclical and spiking up and then down) and two (trending down and cyclical) were apparent in the Up-cluster and Down-cluster genes, respectively. Functional clustering of tumor-related genes followed by Ingenuity Pathways Analysis identified the transforming growth factor β (TGF β)-mediated network as key signaling pathways. Further functional analyses showed sustained activation of TGFβ receptor-pSMAD2/3 signaling, leading to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, collagens and laminins. These TGFβ-induced changes facilitated epithelial-to-mesenchymal transition, which promotes cancer invasion and migration. Thus, complex temporal changes in gene expression patterns drive thyroid cancer progression, and persistent activation of TGFβ-TGFRβII-pSMAD2/3 signaling leads to EMT, thus promoting metastasis. This study provides new understanding of progression and metastatic spread of human thyroid cancer.

Topics: Animals; Carcinogenesis; Cluster Analysis; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Male; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Thyroid Neoplasms; Transforming Growth Factor beta

The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β's role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.

Topics: Animals; Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The non-histone chromatin-binding protein HMGA2 is expressed predominantly in the mesenchyme before its differentiation, but it is also expressed in tumors of epithelial origin. Ectopic expression of HMGA2 in epithelial cells induces epithelial-mesenchymal transition (EMT), which has been implicated in the acquisition of metastatic characters in tumor cells. However, little is known about in vivo modulation of HMGA2 and its effector functions in tumor metastasis. Here, we report that HMGA2 loss of function in a mouse model of cancer reduces tumor multiplicity. HMGA2-positive cells were identified at the invasive front of human and mouse tumors. In addition, in a mouse allograft model, HMGA2 overexpression converted nonmetastatic 4TO7 breast cancer cells to metastatic cells that homed specifically to liver. Interestingly, expression of HMGA2 enhanced TGFβ signaling by activating expression of the TGFβ type II receptor, which also localized to the invasive front of tumors. Together our results argued that HMGA2 plays a critical role in EMT by activating the TGFβ signaling pathway, thereby inducing invasion and metastasis of human epithelial cancers.

Topics: Animals; Cell Line, Tumor; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; HCT116 Cells; HMGA2 Protein; HT29 Cells; Humans; MCF-7 Cells; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

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

Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.. BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.. Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).. These studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.

Topics: Animals; B7-2 Antigen; CD8-Positive T-Lymphocytes; Cell Count; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Epitopes; Female; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Immunization; Immunoglobulin G; Lymph Nodes; Lymphocyte Depletion; Mice; Neoplasm Metastasis; Neoplasms; Papillomavirus E7 Proteins; Receptors, Transforming Growth Factor beta; Signal Transduction; Solubility; T-Lymphocytes, Cytotoxic; Time Factors; Transforming Growth Factor beta

TP53 (Tumor Protein 53, previously known as p53) is probably the best known of all tumor suppressor genes, and is mutated in nearly all (96%) high-grade serous ovarian cancer (HGS-OvCa), which is the most common histopathological type of epithelial ovarian cancer (EOC). Recently, TP53 is found to involve in regulating cell metabolic pathways besides its classical tumor suppressive functions. In addition, emerging evidence suggests that mutant TP53 is associated with cancer metastasis. Through summarizing and comparing the roles of wild-type TP53 and mutant TP53 in the progression of various types of cancer, we hypothesize that mutant TP53 in HGS-OvCa cells interacts with sterol regulatory element-binding proteins (SREBPs) and guanidinoacetate N-methyltransferase (GAMT), leading to increased gene expression of key enzymes involved in fatty acids (FAs) and cholesterol biosynthesis and the inhibition of fatty acid oxidation (FAO), thus promotes lipid anabolism to accelerate tumor growth and progression. Elevated platelet number in patients' tumor microenvironment results in increased TGF-β production. Then, TGF-β acts in concert with mutant TP53 to promote HGS-OvCa metastasis by assembling a mutant-TP53/p63/Smads protein complex, in which p63's functions as metastasis suppressor are antagonized, and by enhancing the activities of the Slug/Snail and Twist families to drive induce EMT-like transition. Then adipocyte-derived IL-8 facilitates the metastasis of transformative cancer cells to abdominal adipose tissue (e.g., omentum). Once metastasis is established, mutant TP53 together with adipocyte-derived IL-8 upregulates Fatty acid-binding protein 4 (FABP4) expression and then promotes FAs absorption from adipocytes to support rapid tumor growth in adipocyte-rich metastatic environments. In summary, these indicate that mutant TP53 may play determinant roles in the progression of HGS-OvCa.

Topics: Fatty Acid-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Guanidinoacetate N-Methyltransferase; Humans; Interleukin-8; Lipid Metabolism; Mutation; Neoplasm Metastasis; Omentum; Ovarian Neoplasms; Peritoneal Neoplasms; Sterol Regulatory Element Binding Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Tumor-induced immune suppression is a major impediment to many potentially effective cancer therapies. TGF-β has previously been described as having both tumor-promoting and tumor-suppressive characteristics. In this issue of Cancer Discovery, Pang and colleagues show that myeloid-specific TGF-β signaling is a critical mediator in tumor metastasis. These findings point to a more specific means to reduce cancer immunosuppression, prevent metastasis, and minimize treatment-related adverse events.

Topics: Animals; Humans; Myeloid Cells; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

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

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

Overexpressed cysteine-rich protein 61 (Cyr61) is believed to enhance osteosarcoma (OS) cell metastasis, but the mechanism of Cyr61 overexpression in OS is not clear so far. In this study 33 OS samples were analyzed by immunostaining and focused on two parts: the correlation between overexpression of Cyr61 and OS metastasis; the mechanism of regulating Cyr61 expression in OS. Twenty-five out of 33 cases (75.76 %) with metastasis showed high expression of Cyr61. Furthermore, Cyr61 expression in Saos-2 cells was reduced by siRNA, and lower expression of Cyr61 in Saos-2 cell resulted in a cell migration deficiency and had no effect on cell proliferation. Particularly, Cyr61 expression was significantly increased in Saos-2 cells in response to different dosages of transforming growth factor beta (TGF-β), indicating that the expression of Cyr61 is TGF-β dependent. A transwell assay showed that Saos-2 cells stimulated with TGF-β had a greater capacity for migration than the control cells. The p38 MAPK-specific inhibitor SB203580 was able to reduce Cyr61 expression and inhibit the migration of Saos-2 cells stimulated with TGF-β. These results obtained provide new evidence that overexpressed Cyr61 plays a key role in the metastasis of OS cells and Cyr61 is a potential target downstream of TGF-β/p38 MAPK to regulate cell migration.

Topics: Adolescent; Adult; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Child; Cysteine-Rich Protein 61; Enzyme Inhibitors; Female; Humans; Imidazoles; Male; Neoplasm Metastasis; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; 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

Cancer metastasis is one of the most critical events in cancer patients, and the median overall survival of stage IIIb or IV patients with metastatic lung cancer in the TNM classification is only 8 or 5 months, respectively. We previously demonstrated that Juzentaihoto, a Japanese traditional medicine, can inhibit cancer metastasis through the activation of macrophages and T cells in mouse cancer metastatic models; however, the mechanism(s) through which Juzentaihoto directly affects tumor cells during the metastasis process and which herbal components from Juzentaihoto inhibit the metastatic potential have not been elucidated. In this study, we focused on the epithelial-to-mesenchymal transition (EMT), which plays an important role in the formation of cancer metastasis. We newly determined that only the Cinnamomi Cortex (CC) extract, one of 10 herbal components of Juzentaihoto, inhibits TGF-β-induced EMT. Moreover, the contents of catechin trimer in CC extracts were significantly correlated with the efficacy of inhibiting TGF-β-induced EMT. Finally, the structure of the catechin trimer from CC extract was chemically identified as procyanidin C1 and the compound showed inhibitory activity against TGF-β-induced EMT. This illustrates that procyanidin C1 is the main active compound in the CC extract responsible for EMT inhibition and that procyanidin C1 could be useful as a lead compound to develop inhibitors of cancer metastasis and other diseases related to EMT.

Topics: Biflavonoids; Cadherins; Catechin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cinnamomum zeylanicum; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Neoplasm Metastasis; Plant Extracts; Proanthocyanidins; Smad2 Protein; Transforming Growth Factor beta

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS (G12V) expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Ducts; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Survival Analysis; Transforming Growth Factor beta

Leptin, an adipocyte-derived cytokine associated with obesity, has been reported to participate in carcinogenesis. Epithelial-mesenchymal transition (EMT) is also considered as a key event in tumor metastasis. The aim of this study is to investigate the mechanism of leptin in the promotion of EMT leading to metastasis in A549 lung cancer cells. We investigated the effect of leptin on migration of A549 cells using wound healing and transwell assays. The incidence of EMT in A549 cells was examined by real-time PCR and immunofluorescence staining. The expression of TGF-β in A549 cells was detected by real-time PCR, and blocking of TGF-β in A549 cells was achieved by siRNA techniques. Additional work was performed using 100 patient samples, which included samples from 50 patients diagnosed with lung cancer and an additional 50 patients diagnosed with lung cancer with metastatic bone lesions. Leptin expression was measured using immunohistochemistry techniques. We demonstrated that leptin can effectively enhance the metastasis of human lung cancer A549 cell line using both wound healing and transwell assays. We also found the incidence of EMT in A549 cells after leptin exposure. Furthermore, we detected the expression of TGF-β in A549 cells, which had been reported to play an important role in inducing EMT. We showed that leptin can significantly upregulate TGF-β at both the mRNA and protein levels in A549 cells. Using siRNA to block the expression of TGF-β in A549 cells, we confirmed the role of TGF-β in the promotion of metastasis and induction of EMT. Furthermore, we found that in patient samples leptin was present at higher levels in samples associated with diagnosis of lung cancer bone metastases tissue than lung cancer tissue. Our results indicated that leptin promoted the metastasis of A549 human lung cancer cell lines by inducing EMT in a TGF-β-dependent manner.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Leptin; Lung Neoplasms; Neoplasm Metastasis; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Mediator Complex; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Herein, a mathematical model of a molecular control system for the regulation of secondary tumors is formulated and analyzed to explore how secondary tumors can be controlled by a primary tumor with/without a surgery and the microenvironment. This control system is composed of fibroblast growth factor-2 (FGF2), urokinase-type plasminogen activator (uPA), plasmin, transforming growth factor-beta (TGFβ), latent TGFβ (LTGFβ), and tumor density. The control of secondary tumors by primary tumors was first modeled by Boushaba, Nilsen-Hamiton and Levine in [46]. The model is based on the idea that the vascularization of a secondary tumor can be suppressed by inhibitors from a larger primary tumor. The emergence of tumors at secondary sites 5-7 cm from a primary site was observed after surgical removal of the primary tumor in silico. The model supports the notion that the fate of secondary tumors after surgery depends on the distance from the primary tumor and the surrounding microenvironment. As such, the primary tumor did not influence the growth of remote secondary tumors, but it could effectively suppress the growth of the secondary tumors if they were too close to the primary tumor, even after it was removed. Thus, the model predicts the emergence of secondary tumors after the excision of the primary tumor when the distance between these tumors is in the "distance window." It also predicts that the growth behaviors of the secondary tumors depend on the local microenvironment. Based on these findings, we propose several treatment options for better clinical outcomes.

Topics: Animals; Antineoplastic Agents; Cell Count; Cell Death; Computer Simulation; Diffusion; Extracellular Matrix; Fibrinolysin; Fibroblast Growth Factor 2; Half-Life; Humans; Models, Biological; Neoplasm Metastasis; Neoplasms; Plasminogen; Time Factors; Transforming Growth Factor beta; Tumor Microenvironment; Urokinase-Type Plasminogen Activator

Mutations in phosphatase and tensin homologue (PTEN) or genomic alterations in the phosphatidylinositol-3-OH kinase-signalling pathway are the most common genetic alterations reported in human prostate cancer. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire metastatic potential remains poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-β signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression, underscoring that TGF-β signalling might represent a pre-invasive checkpoint to prevent PTEN-mediated prostate tumorigenesis. Here we show that COUP transcription factor II (COUP-TFII, also known as NR2F2), a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-β-dependent checkpoint for PTEN-null indolent tumours. Overexpression of COUP-TFII in the mouse prostate epithelium cooperates with PTEN deletion to augment malignant progression and produce an aggressive metastasis-prone tumour. The functional counteraction between COUP-TFII and SMAD4 is reinforced by genetically engineered mouse models in which conditional loss of SMAD4 diminishes the inhibitory effects elicited by COUP-TFII ablation. The biological significance of COUP-TFII in prostate carcinogenesis is substantiated by patient sample analysis, in which COUP-TFII expression or activity is tightly correlated with tumour recurrence and disease progression, whereas it is inversely associated with TGF-β signalling. These findings reveal that the destruction of the TGF-β-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUP-TFII as a potential drug target for the intervention of metastatic human prostate cancer.

Topics: Animals; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Transformation, Neoplastic; COUP Transcription Factor II; Disease Models, Animal; Disease Progression; Gene Deletion; Humans; Male; Mice; Neoplasm Metastasis; Proportional Hazards Models; Prostate; Prostatic Neoplasms; PTEN Phosphohydrolase; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.

Topics: Androgens; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; p21-Activated Kinases; Prostatic Neoplasms; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-β (TGF-β) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4(+) CD25(high) CD127(-) Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.

Topics: Adult; Aged; Cell Movement; Cell Proliferation; Clinical Trials, Phase II as Topic; Dual Specificity Phosphatase 1; Gene Expression Regulation, Neoplastic; Humans; Interleukin-2; Leukocytes, Mononuclear; Male; Middle Aged; Multicenter Studies as Topic; Neoplasm Metastasis; Prostatic Neoplasms; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Randomized Controlled Trials as Topic; RGS Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation; Young Adult

Metastasis is the rapid proliferation of cancer cells (secondary tumour) at a specific place, generally leading to death. This occurs at anatomical parts providing the necessary environment for vascularity, oxygen and food to hide their actions and trigger the rapid growth of cancer. Prostate and breast cancers, for example, use bone marrow for their proliferation. Bone-supporting cancer cells thus adapt to the environment, mimicking the behaviour of genetic and molecular bone cells. Evidence of this has been given in Cecchini et al. (2005, EAU Update Ser. 3:214-226), providing arguments such as how cancer cell growth is so active during bone reabsorption. This paper simulates metastasis activation in bone marrow. A mathematical model has been developed involving the activation of molecules from bone tissue cells, which are necessary for cancer to proliferate. Here, we simulate two forms of secondary tumour growth depending on the type of metastasis: osteosclerosis and osteolysis.

Topics: Biomedical Engineering; Bone Marrow Neoplasms; Bone Neoplasms; Bone Remodeling; Cell Differentiation; Cell Proliferation; Computer Simulation; Humans; Mathematical Concepts; Models, Biological; Neoplasm Metastasis; Osteolysis; Osteosclerosis; Parathyroid Hormone-Related Protein; Somatomedins; 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

Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-β, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-β-activated kinase 1-mediated activation of the nuclear factor-κB complex. TGF-β-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-β-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44+/CD117+). Furthermore, CD44+/CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-β-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype.

Topics: Cell Line, Tumor; Enzyme Activation; Epithelial-Mesenchymal Transition; Female; GTP-Binding Proteins; Humans; Hyaluronan Receptors; Neoplasm Metastasis; Neoplastic Stem Cells; Ovarian Neoplasms; Protein Glutamine gamma Glutamyltransferase 2; Proto-Oncogene Proteins c-kit; Transforming Growth Factor beta; Transglutaminases

Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+ and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Azabicyclo Compounds; Carcinoma; Disease Progression; Drug Synergism; Female; Immunotherapy; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Receptors, OX40; Signal Transduction; Transforming Growth Factor beta; Tumor Burden

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

The study was aimed to investigate the relationship between plasma transforming growth factor beta 1 (TGF-β(1)) expression and the characteristics of hepatocellular carcinoma (HCC).. Five hundred and seventy-one patients with HCC were subjected. Plasma TGF-β(1) levels were measured by enzyme-linked immunosorbent assay at diagnosis and compared in accordance with clinical and radiological characteristics.. Plasma TGF-β(1) levels were significantly higher in the diffuse infiltrative type (n = 159) than in the nodular type of HCC (n = 412; 3.94 ± 0.34 vs. 3.79 ± 0.29 log(10) pg/ml; p < 0.001). They were much higher in patients with portal vein thrombosis or extrahepatic metastasis than in those without (3.88 ± 0.34 vs. 3.81 ± 0.29 log(10) pg/ml, p = 0.008; 3.94 ± 0.35 vs. 3.82 ± 0.30 log(10) pg/ml, p = 0.013, respectively). Also, plasma TGF-β(1) levels showed a positive correlation with the size of HCC (r = 0.014, p < 0.001). Additionally, plasma TGF-β(1) levels were inversely related to the survival periods (p < 0.001).. TGF-β(1) was overexpressed in invasive types of HCC and it may be involved in the rapid progression of HCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Cell Proliferation; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Transforming Growth Factor beta

Gene chip technology can be used to identify and localize signal transduction genes associated with metastasis. We used the human genome U133A gene chip to detect differences in gene expression profiles among high (H) and low (L) metastatic human ovarian cancer cell lines (HO-8910PM, HO-8910), and normal ovarian tissues (C), to identify metastasis-associated signal transduction genes and determine their chromosomal localizations. A total of 37 signal transduction genes showed more than twofold differences in expression levels between the H and L metastatic ovarian cancer cell lines; of these, 21 genes were up-regulated [signal log ratio (SLR)≥1], and 16 genes were down-regulated (SLR≤-1). Most genes were located on chromosome 1 (7 genes, 18.9%), followed by chromosome 8 (5 genes, 13.5%), then chromosomes 6, 11, and 17 (3 genes each, 8.1%). A total of 21 of the differentially expressed genes (56.7%) were localized on the short arm of the chromosome (q). The disruption of signal transduction gene expression may be an important factor associated with ovarian cancer metastasis. The affected signal transduction genes were localized to chromosomes 1, 8, 6, 11, and 17.

Topics: Cell Line, Tumor; Chromosome Mapping; ErbB Receptors; Female; Humans; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Receptors, CXCR4; Signal Transduction; Transforming Growth Factor beta

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Disease Progression; Extracellular Matrix; Losartan; Mice; Mice, Knockout; Neoplasm Invasiveness; Neoplasm Metastasis; Osteonectin; Pancreatic Neoplasms; Survival Rate; Transforming Growth Factor beta; Treatment Outcome

Response gene to complement-32 (RGC-32) is comprehensively expressed in many kinds of tissues and has been reported to be expressed abnormally in different kinds of human tumors. However, the role of RGC-32 in cancer remains controversial and no reports have described the effect of RGC-32 in pancreatic cancer. The present study investigated the expression of RGC-32 in pancreatic cancer tissues and explored the role of RGC-32 in transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human pancreatic cancer cell line BxPC-3.. Immunohistochemical staining of RGC-32 and E-cadherin was performed on specimens from 42 patients with pancreatic cancer, 12 with chronic pancreatitis and 8 with normal pancreas. To evaluate the role of RGC-32 in TGF-β-induced EMT in pancreatic cancer cells, BxPC-3 cells were treated with TGF-β1, and RGC-32 siRNA silencing and gene overexpression were performed as well. The mRNA expression and protein expression of RGC-32 and EMT markers such E-cadherin and vimentin were determined by quantitative reverse transcription-PCR (qRT-PCR) and western blot respectively. Finally, migration ability of BxPC-3 cells treated with TGF-β and RGC-32 siRNA transfection was examined by transwell cell migration assay.. We found stronger expression of RGC-32 and higher abnormal expression rate of E-cadherin in pancreatic cancer tissues than those in chronic pancreatitis tissues and normal pancreatic tissues. Immunohistochemical analysis revealed that both RGC-32 positive expression and E-cadherin abnormal expression in pancreatic cancer were correlated with lymph node metastasis and TNM staging. In addition, a significant and positive correlation was found between positive expression of RGC-32 and abnormal expression of E-cadherin. Furthermore, in vitro, we found sustained TGF-β stimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of siRNA silencing and gene overexpression, we further demonstrated that RGC-32 mediated TGF-β-induced EMT and migration in BxPC-3 cells.. The results above indicated that RGC-32 might be a novel metastasis promoting gene in pancreatic cancer and it enhances metastatic phenotype by mediating TGF-β-induced EMT in human pancreatic cancer cell line BxPC-3.

Topics: Cadherins; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Muscle Proteins; Neoplasm Metastasis; Nerve Tissue Proteins; Pancreatic Neoplasms; Phenotype; RNA Interference; 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

The Krüppel-like zinc finger protein ZNF217 is a candidate oncogene in breast cancer. In this study, we showed that high levels of expression of ZNF217 mRNA are associated with poor prognosis and the development of metastases in breast cancer. Overexpression of ZNF217 in breast cancer cells stimulated migration and invasion in vitro and promoted the development of spontaneous lung or node metastases in mice in vivo. ZNF217 also promoted epithelial-mesenchymal transition (EMT) in human mammary epithelial cells, and the TGF-β-activated Smad signaling pathway was identified as a major driver of ZNF217-induced EMT. In addition, a TGF-β autocrine loop sustained activation of the TGF-β pathway in ZNF217-overexpressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2 or TGFB3. Inhibition of the TGF-β pathway led to the reversal of ZNF217-mediated EMT. Together, our findings indicate that ZNF217 mRNA expression may represent a novel prognostic biomarker in breast cancer. Therapeutic targeting of ZNF217 of the TGF-β signaling pathway may benefit the subset of patients whose tumors express high levels of ZNF217.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Lymphatic Metastasis; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; RNA, Messenger; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Transplantation, Heterologous

To investigate the inhibitory effects of heparin on PC-3M cells proliferation in vitro and B16-F10-luc-G5 cells metastasis in Balb/c nude mice and identify the protein expression patterns to elucidate the action mechanism of heparin.. Human prostate cancer PC-3M cells were incubated with heparin 0.5 to 125 μg/mL for 24 h. The proliferation of PC-3M cells was assessed by MTS assay. BrdU incoporation and Ki67 expression were detected using a high content screening (HCS) assay. The cell cycle and apoptosis of PC-3M cells were tested by flow cytometry. B16-F10-luc-G5 cardinoma cells were injected into the lateral tail vein of 6-week old male Balb/c nude mice and heparin 30 mg/kg was administered iv 30 min before and 24 h after injection. The metasis of B16-F10-luc-G5 cells was detected by bioluminescence assay. Activated partial thromboplastin time (APTT) and hemorheological parameters were measured on d 14 after injection of B16-F10-luc-G5 carcinoma cells in Balb/c mice. The global protein changes in PC-3M cells and frozen lung tissues from mice burdened with B16-F10-luc-G5 cells were determined by 2-dimensional gel electrophoresis and image analysis. The protein expression of vimentin and 14-3-3 zeta/delta was measured by Western blot. The mRNA transcription of vimentin, transforming growth factor (TGF)-β, E-cadherin, and α(v)-integrin was measured by RT-PCR.. Heparin 25 and 125 μg/mL significantly inhibited the proliferation, arrested the cells in G(1) phase, and suppressed BrdU incorporation and Ki67 expression in PC-3M cells compared with the model group. But it had no significant effect on apoptosis of PC-3M cells. Heparin 30 mg/kg markedly inhibits the metastasis of B16-F10-luc-G5 cells on day 8. Additionally, heparin administration maintained relatively normal red blood hematocrit but had no influence on APTT in nude mice burdened with B16-F10-luc-G5 cells. Thirty of down-regulated protein spots were identified after heparin treatment, many of which are related to tumor development, extracellular signaling, energy metabolism, and cellular proliferation. Vimentin and 14-3-3 zeta/delta were identified in common in PC-3M cells and the lungs of mice bearing B16-F10-luc-G5 carcinoma cells. Heparin 25 and 125 μg/mL decreased the protein expression of vimentin and 14-3-3 zeta/delta and the mRNA expression of α(v)-integrin. Heparin 125 μg/mL decreased vimentin and E-cadherin mRNA transcription while increased TGF-β mRNA transcription in the PC-3M cells, but the differences were not significant. Transfection of vimentin-targeted siRNA for 48 h significantly decreased the BrdU incoporation and Ki67 expression in PC-3M cells.. Heparin inhibited PC-3M cell proliferation in vitro and B16-F10-luc-G5 cells metastasis in nude mice by inhibition of vimentin, 14-3-3 zeta/delta, and α(v)-integrin expression.

Topics: 14-3-3 Proteins; Animals; Anticoagulants; Antineoplastic Agents; Apoptosis; Cadherins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Heparin; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasms; Prostate; Prostatic Neoplasms; Transforming Growth Factor beta; Vimentin

TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling.. To test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging.. Abrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma.. The observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; HEK293 Cells; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Microscopy, Video; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Experimental; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transplantation, Heterologous; X-Linked Inhibitor of Apoptosis Protein

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

Recent studies have shown the contribution of miRNAs to cancer pathogenesis. Prostate cancer is the most commonly diagnosed cancer in men. Unlike other major types of cancer, no single gene has been identified as being mutated in the majority of prostate tumors. This implies that the expression profiling of genes, including the non-coding miRNAs, may substantially vary across individual cases of this cancer. The within-class variability makes it possible to reconstruct or infer disease-specific miRNA-mRNA correlation and regulatory modular networks using high-dimensional microarray data of prostate tumor samples. Furthermore, since miRNAs and tumor suppressor genes are usually tissue specific, miRNA-mRNA modules could potentially differ between primary prostate cancer (PPC) and metastatic prostate cancer (MPC). We herein performed an in silico analysis to explore the miRNA-mRNA correlation network modules in the two tumor subtypes. Our analysis identified 5 miRNA-mRNA module pairs (MPs) for PPC and MPC, respectively. Each MP includes one positive-connection (correlation) module and one negative-connection (correlation) module. The number of miRNAs or mRNAs (genes) in each module varies from 2 to 8 or from 6 to 622. The modules discovered for PPC are more informative than those for MPC in terms of the implicated biological insights. In particular, one negative-connection module in PPC fits well with the popularly recognized miRNA-mediated post-transcriptional regulation theory. That is, the 3'UTR sequences of the involved mRNAs (∼620) are enriched with the target site motifs of the 7 modular miRNAs, has-miR-106b, -191, -19b, -92a, -92b, -93, and -141. About 330 GO terms and KEGG pathways, including TGF-beta signaling pathway that maintains tissue homeostasis and plays a crucial role in the suppression of the proliferation of cancer cells, are over-represented (adj.p<0.05) in the modular gene list. These computationally identified modules provide remarkable biological evidence for the interference of miRNAs in the development of prostate cancers and warrant additional follow-up in independent laboratory studies.

Topics: Algorithms; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Male; MicroRNAs; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Messenger; Signal Transduction; Transcription, Genetic; 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

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1(+/-)ErbB2/Neu(+) tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1(+/+)Neu(+) mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1(-/-)Neu(+) or Hsf1(+/+)Neu(+) cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.

Topics: Animals; Cadherins; Cell Transformation, Neoplastic; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Heat Shock Transcription Factors; HSP90 Heat-Shock Proteins; Mammary Neoplasms, Animal; MAP Kinase Signaling System; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 3; Neoplasm Metastasis; Proto-Oncogene Proteins c-raf; Receptor, ErbB-2; Transcription Factors; Transforming Growth Factor beta

Aberrant activation of Cyclin D-Cdk4/6 signaling pathway is commonly found in pancreatic ductal adenocarcinoma (PDAC). Here, we show that PD-0332991, a highly specific inhibitor for Cdk4 and Cdk6, exerted growth inhibitory effects on three human PDAC cell lines. Microarray analysis revealed that PD-0332991 downregulated cell-cycle-related genes, but upregulated genes implicated in extracellular matrix (ECM) remodeling and pancreatic cancer cell invasion and metastasis. Moreover, PD-0332991 enhanced invasion in TGF-β-responsive PDAC cell lines that harbor a wild-type SMAD4 gene (COLO-357, PANC-1), but not in TGF-β-resistant AsPC-1 cells that harbor a mutated SMAD4. PD-0332991 also induced epithelial-mesenchymal transition (EMT) in COLO-357 and PANC-1, but not in AsPC-1 cells. Inhibition of CDK4/6 using shRNA mimicked the effects of PD-0332991 on EMT induction. Furthermore, PD-0332991 increased Smad transcriptional activity in luciferase readout assays and activated TGF-β signaling. SB-505124, an inhibitor of the type-I TGF-β receptor (TβRI) kinase, completely blocked EMT induction by PD-0332991. When combined with PD-0332991, SB-505124 inhibited the growth of COLO-357 and PANC-1 cells. Taken together, these data suggest that anti-Cdk4/6 therapy could induce EMT and enhance pancreatic cancer cell invasion by activating Smad-dependent TGF-β signaling, and that combining PD-0332991 and SB-505124 may represent a novel therapeutic strategy in PDAC.

Topics: Benzodioxoles; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Screening Assays, Antitumor; Drug Synergism; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Imidazoles; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Piperazines; Pyridines; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Stem Cell Assay

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

TGF-β promotes epithelial-mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative advantage to cancer cells. However, the molecular mechanisms by which TGF-β regulates CLU expression and CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates TGF-β-induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1 regulated basal and TGF-β-induced CLU transcription. In addition, CLU reduction reduced TGF-β induction of the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our findings indicate that CLU is an important mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression.

Topics: Animals; Base Sequence; Blotting, Western; Chromatin Immunoprecipitation; Clusterin; DNA Primers; Epithelial-Mesenchymal Transition; Humans; Male; Mice; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Twist-Related Protein 1

In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.

Topics: Aged; Carcinoma, Hepatocellular; Cytokines; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Liver Neoplasms; Male; Mesenchymal Stem Cells; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Phenotype; Proportional Hazards Models; Transforming Growth Factor beta; Treatment Outcome; Wound Healing

Portal vein tumor thrombus (PVTT) is strongly correlated to a poor prognosis for patients with hepatocellular carcinoma (HCC). In this study, we uncovered a causative link between hepatitis B virus (HBV) infection and development of PVTT. Mechanistically, elevated TGF-β activity, associated with the persistent presence of HBV in the liver tissue, suppresses the expression of microRNA-34a, leading to enhanced production of chemokine CCL22, which recruits regulatory T (Treg) cells to facilitate immune escape. These findings strongly suggest that HBV infection and activity of the TGF-β-miR-34a-CCL22 axis serve as potent etiological factors to predispose HCC patients for the development of PVTT, possibly through the creation of an immune-subversive microenvironment to favor colonization of disseminated HCC cells in the portal venous system.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chemokine CCL22; Female; Hepatitis B; Hepatitis B virus; Humans; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Metastasis; Portal Vein; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Adipocytes represent a major cell type in the mammary tumor microenvironment and are important for tumor growth. Collagen VI (COL6) is highly expressed in adipose tissue, upregulated in the obese state, and enriched in breast cancer lesions and is a stimulator of mammary tumor growth. Here, we have described a cleavage product of the COL6α3 chain, endotrophin (ETP), which serves as the major mediator of the COL6-mediated tumor effects. ETP augmented fibrosis, angiogenesis, and inflammation through recruitment of macrophages and endothelial cells. Moreover, ETP expression was associated with aggressive mammary tumor growth and high metastatic growth. These effects were partially mediated through enhanced TGF-β signaling, which contributes to tissue fibrosis and epithelial-mesenchymal transition (EMT) of tumor cells. Our results highlight the crucial role of ETP as an obesity-associated factor that promotes tumor growth in the context of adipocyte interactions with tumor and stromal cells.

Topics: Adipocytes; Animals; Cell Communication; Cell Line, Tumor; Collagen Type VI; Endothelial Cells; Epithelial-Mesenchymal Transition; Female; Fibrosis; Gene Expression Regulation, Neoplastic; Macrophages; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; Peptide Fragments; Proteolysis; Stromal Cells; Transforming Growth Factor beta

To evaluate CD4(+)CD25(+)FOXP3(+) T regulatory cells (T(REG)) and associated immune-regulatory pathways in peripheral blood lymphocytes (PBL) of metastatic renal cell carcinoma (mRCC) patients and healthy volunteers. We subsequently investigated the effects of immunotherapy on circulating T(REG) combining an extensive phenotype examination, DNA methylation analysis and global transcriptome analysis.. Eighteen patients with mRCC and twelve volunteers (controls) were available for analysis. T(REG) phenotype was examined using flow cytometry (FCM). T(REG) were also quantified by analyzing the epigenetic status of the FOXP3 locus using methylation specific PCR. As a third approach, RNA of the PBL was hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays and the gene signatures were explored using pathway analysis.. We observed higher numbers of T(REG) in pre-treatment PBL of mRCC patients compared to controls. A significant increase in T(REG) was detected in all mRCC patients after the two cycles of immunotherapy. The expansion of T(REG) was significantly higher in non-responders than in responding patients. Methylation specific PCR confirmed the FCM data and circumvented the variability and subjectivity of the FCM method. Gene Set Enrichment Analysis (GSEA) of the microarray data showed significant enrichment of FOXP3 target genes, CTLA-4 and TGF-ß associated pathways in the patient cohort.. Immune monitoring of the peripheral blood and tumor tissue is important for a wide range of diseases and treatment strategies. Adoption of methodology for quantifying T(REG) with the least variability and subjectivity will enhance the ability to compare and interpret findings across studies.

Topics: Carcinoma, Renal Cell; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; CTLA-4 Antigen; DNA Methylation; Female; Flow Cytometry; Forkhead Transcription Factors; Gene Expression Profiling; Humans; Immunotherapy; Interleukin-2; Male; Metabolic Networks and Pathways; Neoplasm Metastasis; Neoplastic Cells, Circulating; Oligonucleotide Array Sequence Analysis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vaccination

A large proportion of colorectal cancers (CRCs) display mutational inactivation of the TGF-β pathway, yet, paradoxically, they are characterized by elevated TGF-β production. Here, we unveil a prometastatic program induced by TGF-β in the microenvironment that associates with a high risk of CRC relapse upon treatment. The activity of TGF-β on stromal cells increases the efficiency of organ colonization by CRC cells, whereas mice treated with a pharmacological inhibitor of TGFBR1 are resilient to metastasis formation. Secretion of IL11 by TGF-β-stimulated cancer-associated fibroblasts (CAFs) triggers GP130/STAT3 signaling in tumor cells. This crosstalk confers a survival advantage to metastatic cells. The dependency on the TGF-β stromal program for metastasis initiation could be exploited to improve the diagnosis and treatment of CRC.

Topics: Animals; Colorectal Neoplasms; Cytokine Receptor gp130; HT29 Cells; Humans; Interleukin-11; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recurrence; Signal Transduction; STAT3 Transcription Factor; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Topics: Colorectal Neoplasms; Humans; Models, Biological; Neoplasm Metastasis; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer-peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR-5 and SKOV-3) and peritoneal (LP-9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI-TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor-beta-induced protein (TGFBIp, also known as βig-H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR-5 and SKOV-3 cells and significantly increased ovarian cancer cell (OVCAR-5, OVCAR-3 and SKOV-3) adhesion to LP-9 cells. TGFBIp was found to be processed at both the N- and C-terminus in the secretome of the ovarian cancer-peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR-5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Coculture Techniques; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Neoplasm Metastasis; Ovarian Neoplasms; Ovary; Peritoneal Neoplasms; Transforming Growth Factor beta

Endoglin is a transmembrane receptor that suppresses human prostate cancer (PCa) cell invasion. Small molecule therapeutics now being tested in humans can activate endoglin signaling. It is not known whether endoglin can regulate metastatic behavior, PCa tumor growth, nor what signaling pathways are linked to these processes. This study sought to investigate the effect of endoglin on these parameters. We used a murine orthotopic model of human PCa metastasis, designed by us to measure effects at early steps in the metastatic cascade, and implanted PCa cells stably engineered to express differing levels of endoglin. We now extend this model to measure cancer cells circulating in the blood. Progressive endoglin loss led to progressive increases in the number of circulating PCa cells as well as to the formation of soft tissue metastases. Endoglin was known to suppress invasion by activating the Smad1 transcription factor. We now show that it selectively activates specific Smad1-responsive genes, including JUNB, STAT1, and SOX4. Increased tumor growth and increased Ki67 expression in tissue was seen only with complete endoglin loss. By showing that endoglin increased TGFβ-mediated suppression of cell growth in vitro and TGFβ-mediated signaling in tumor tissue, loss of this growth-suppressive pathway appears to be implicated at least in part for the increased size of endoglin-deficient tumors. Endoglin is shown for the first time to suppress cell movement out of primary tumor as well as the formation of distant metastasis. It is also shown to co-regulate tumor growth and metastatic behavior in human PCa.

Topics: Animals; Antigens, CD; Cell Proliferation; Disease Models, Animal; Endoglin; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, Cell Surface; Signal Transduction; Smad1 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

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

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial-mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFβ, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell-cell junction disassembly and cell migration. Further, gradients of TGFβ induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFβ. Interestingly, VEGF suppresses TGFβ-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFβ might modulate HGF signaling to influence route selection during cancer progression.

Topics: Animals; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Hepatocyte Growth Factor; Intercellular Signaling Peptides and Proteins; Neoplasm Metastasis; PC12 Cells; Rats; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.. In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.. Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Proteasome Inhibitors; Proto-Oncogene Proteins; RNA Interference; Skin Neoplasms; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of function of Smad4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major proangiogenic factor. Pharmacologic inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK (mitogen-activated protein kinase) auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in matrix metalloprotease 9 enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis, and resistance to 5'-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with hypoxia-inducible factor (HIF) 1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, and p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention.

Topics: Cell Hypoxia; Cell Movement; Cell Transformation, Neoplastic; Colonic Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; Gene Silencing; HCT116 Cells; Humans; Matrix Metalloproteinase 9; Neoplasm Metastasis; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Smad4 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Topics: Animals; Bone Morphogenetic Proteins; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Transgenic; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Osteopontin; Penetrance; Prognosis; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; PTEN Phosphohydrolase; Smad4 Protein; Transforming Growth Factor beta

Altered natural killer (NK) cell function is a component of the global immune dysregulation that occurs in advanced malignancies. Another condition associated with altered NK homeostasis is normal pregnancy, where robust infiltration with CD16- CD9+ NK cells can be identified in decidual tissues, along with a concomitant expansion of CD16- NK cells in the maternal peripheral blood. In metastatic melanoma, we identified a similar expansion of peripheral blood CD16- NK cells (median 7.4% in 41 patients with melanoma compared with 3.0% in 29 controls, P < .001). A subset of NK cells in melanoma patients also expresses CD9, which is characteristically expressed only on NK cells within the female reproductive tract. Expansion of CD16- NK cells was associated with elevated plasma transforming growth factor-beta (TGF-β levels (median 20  ng/ml, Spearman's ρ = 0.81, P = .015)). These findings suggest the possibility of exploring anti-TGF-β therapy to restore NK function in melanoma.

Topics: Antigens, CD; Case-Control Studies; Female; Humans; Killer Cells, Natural; Male; Melanoma; Membrane Glycoproteins; Neoplasm Metastasis; Pregnancy; Receptors, IgG; Skin Neoplasms; Tetraspanin 29; Transforming Growth Factor beta

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) is a secreted glycoprotein that is overexpressed in lung cancer tumor tissues and is correlated with the invasive ability in a lung cancer cell line model. These observations suggest that SCUBE3 may have a role in lung cancer progression. By exogenous SCUBE3 treatment or knockdown of SCUBE3 expression, we found that SCUBE3 could promote lung cancer cell mobility and invasiveness. Knockdown of SCUBE3 expression also suppressed tumorigenesis and cancer metastasis in vivo. The secreted SCUBE3 proteins were cleaved by gelatinases (matrix metalloprotease-2 (MMP-2) and MMP-9) in media to release two major fragments: the N-terminal epidermal growth factor-like repeats and the C-terminal complement proteins C1r/C1s, Uegf and Bmp1 (CUB) domain. Both the purified SCUBE3 protein and the C-terminal CUB domain fragment, bound to transforming growth factor-β (TGF-β) type II receptor through the C-terminal CUB domain, activated TGF-β signaling and triggered the epithelial-mesenchymal transition (EMT). This process includes the induction of Smad2/3 phosphorylation, the increase of Smad2/3 transcriptional activity and the upregulation of the expression of target genes involved in EMT and cancer progression (such as TGF-β1, MMP-2, MMP-9, plasminogen activator inhibitor type-1, vascular endothelial growth factor, Snail and Slug), thus promoting cancer cell mobility and invasion. In conclusion, in lung cancer cells, SCUBE3 could serve as an endogenous autocrine and paracrine ligand of TGF-β type II receptor, which could regulate TGF-β receptor signaling and modulate EMT and cancer progression.

Topics: Calcium-Binding Proteins; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Ligands; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Microsatellite instability (MSI), which occurs in 15% of colorectal cancer, has been shown to have a lower incidence of metastasis and better patient survival rates compared with microsatellite stable colorectal cancer. However, a mechanistic understanding of the basis for this difference is very limited. Here, we show that restoration of TGFβ signaling by re-expression of TGFβ receptor II in MSI colon cancer cells increased PI3K/AKT activation, conferred resistance to growth factor deprivation stress-induced apoptosis, and promoted cell motility in vitro. Treatment with a potent PI3K inhibitor (LY294002) blocked the prosurvival and promotility effects of TGFβ, indicating that TGFβ-mediated promotion of cell survival and motility is dependent upon activation of the PI3K/AKT pathway. Analysis of apoptotic effectors that are affected by TGFβ signaling indicated that Bim is an effector of TGFβ-mediated survival. In addition, TGFβ-induced down-regulation of E-cadherin contributed to the prosurvival effect of TGFβ, and restoration of TGFβ signaling in MSI colon cancer cells increased liver metastasis in an orthotopic model in vivo. Taken together, our results demonstrate that restoration of TGFβ signaling promotes cell survival, motility, and metastatic progression in MSI colon cancer cells and indicate that TGFβ receptor II mutations contribute to the favorable outcomes in colon cancer patients with MSI.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Survival; Chromones; Colonic Neoplasms; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microsatellite Repeats; Morpholines; Neoplasm Metastasis; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

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

Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.. Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.. This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.

Topics: Animals; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Signal Transduction; 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

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

Recently, small molecule inhibitors of transforming growth factorβ (TGF-β) type I receptor kinase ⁄ activin receptor-like kinase-5 (ALK5) have been developed to target TGF-β signalling as a therapeutic strategy for combating cancer. In the present study, the authors examined a novel small molecule inhibitor of ALK5, 3-((5- ([1,2,4]triazolo[1,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2-ylamino)methyl)benzonitrile (EW-7203) in breast cancer cells to determine if it has potential for cancer treatment. The inhibitory effects of EW-7203 on TGF-β-induced Smad signalling and epithelial- to-mesenchymal transition (EMT) were investigated in mammary epithelial cells using luciferase reporter assays, immunoblotting, confocal microscopy and wound healing assays. In addition, the suppressive effects of EW-7203 on mammary cancer metastasis to the lung were examined using a Balb ⁄ c xenograft model system. The novel ALK5 inhibitor, EW-7203, inhibited the TGF-β1-stimulated transcriptional activation of p3TP-Lux and pCA-GA₁₂- Luc. In addition, EW-7203 decreased phosphorylated Smad2 levels and the nuclear translocation of Smad2 was increased by TGF-β1. In addition, EW-7203 inhibited TGF-β1-induced EMT and wound healing of NMuMG cells. Furthermore, in xenografted Balb ⁄ c mice, EW-7203 inhibited metastasis to the lung from breast tumors. The novel ALK5 inhibitor, EW-7203, efficiently inhibited TGF-β1-induced Smad signalling, EMT and breast tumor metastasis to the lung in vivo, demonstrating that EW-7203 has therapeutic potential for breast cancer metastasis to the lung.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Mammary Glands, Animal; Mammary Glands, Human; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Pyridines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Thiazoles; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The microRNA-200 (miR-200) family restricts epithelial-mesenchymal transition (EMT) and metastasis in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma. To determine the mechanisms responsible for EMT and metastasis regulated by this microRNA, we conducted a global liquid chromatography/tandem mass spectrometry analysis to compare metastatic and nonmetastatic murine lung adenocarcinoma cells which had undergone EMT because of loss of miR-200. An analysis of syngeneic tumors generated by these cells identified multiple novel proteins linked to metastasis. In particular, the analysis of conditioned media, cell surface proteins, and whole-cell lysates from metastatic and nonmetastatic cells revealed large-scale modifications in the tumor microenvironment. Specific increases were documented in extracellular matrix (ECM) proteins, peptidases, and changes in distribution of cell adhesion proteins in the metastatic cell lines. Integrating proteomic data from three subproteomes, we defined constituents of a multilayer protein network that both regulated and mediated the effects of TGFβ. Lastly, we identified ECM proteins and peptidases that were directly regulated by miR-200. Taken together, our results reveal how expression of miR-200 alters the tumor microenvironment to inhibit the processes of EMT and metastasis.

Topics: Adenocarcinoma; Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromatography, Liquid; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasms; Oligonucleotide Array Sequence Analysis; Proteomics; Signal Transduction; Tandem Mass Spectrometry; Transforming Growth Factor beta; Tumor Microenvironment

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

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

Androgens are functionally required for the normal growth of the prostate gland and in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development and in cancer cell metastasis induced by factors within the microenvironment, such as transforming growth factor-beta (TGF-beta). This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells. The EMT pattern was evaluated on the basis of expression of the epithelial markers E-cadherin/beta-catenin, and the mesenchymal markers N-cadherin, as well as cytoskeleton reorganization in response to 5alpha-dihydrotestosterone (DHT; 1 nM) and/or TGF-beta (5 ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and lead to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. These findings indicate the ability of androgens to induce EMT by potentially bypassing the functional involvement of TGF-beta, thus contributing to metastatic behavior of prostate cancer cells.-Zhum, M.-L., Kyprianou, N. Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells.

Topics: Androgen Receptor Antagonists; Blotting, Western; Cell Dedifferentiation; Cell Line, Tumor; Cell Movement; Dihydrotestosterone; Epithelial Cells; Flow Cytometry; Humans; Male; Mesoderm; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Topics: Animals; Biocatalysis; Cell Line, Tumor; Cell Movement; Cell Nucleus; Gene Knockdown Techniques; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Smad2 Protein; Smad3 Protein; Substrate Specificity; Threonine; Transforming Growth Factor beta

Loss of expression of the type III transforming growth factor-beta receptor (TbetaRIII or betaglycan), a transforming growth factor-beta (TGF-beta) superfamily co-receptor, is common in human breast cancers. TbetaRIII suppresses cancer progression in vivo by reducing cancer cell migration and invasion by largely unknown mechanisms. Here, we demonstrate that the cytoplasmic domain of TbetaRIII is essential for TbetaRIII-mediated downregulation of migration and invasion in vitro and TbetaRIII-mediated inhibition of breast cancer progression in vivo. Functionally, the cytoplasmic domain of TbetaRIII is required to attenuate TGF-beta signaling, whereas TbetaRIII-mediated attenuation of TGF-beta signaling is required for TbetaRIII-mediated inhibition of migration and invasion. Mechanistically, both TbetaRIII-mediated inhibition of TGF-beta signaling and TbetaRIII-mediated inhibition of invasion occur through the interaction of the cytoplasmic domain of TbetaRIII with the scaffolding protein GAIP-interacting protein C-terminus (GIPC). Taken together, these studies support a functional role for the TbetaRIII cytoplasmic domain interacting with GIPC to suppress breast cancer progression.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunoenzyme Techniques; Mammary Neoplasms, Animal; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RGS Proteins; RNA, Messenger; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

The elucidation of biological concepts enriched with differentially expressed genes has become an integral part of the analysis and interpretation of genomic data. Of additional importance is the ability to explore networks of relationships among previously defined biological concepts from diverse information sources, and to explore results visually from multiple perspectives. Accomplishing these tasks requires a unified framework for agglomeration of data from various genomic resources, novel visualizations, and user functionality.. We have developed ConceptGen, a web-based gene set enrichment and gene set relation mapping tool that is streamlined and simple to use. ConceptGen offers over 20,000 concepts comprising 14 different types of biological knowledge, including data not currently available in any other gene set enrichment or gene set relation mapping tool. We demonstrate the functionalities of ConceptGen using gene expression data modeling TGF-beta-induced epithelial-mesenchymal transition and metabolomics data comparing metastatic versus localized prostate cancers.

Topics: Animals; Computational Biology; Databases, Genetic; Gene Expression Profiling; Gene Regulatory Networks; Humans; Male; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Pattern Recognition, Automated; Software; 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

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

Transforming growth factor beta (TGF-beta) acts as a tumor promoter by inducing epithelial-mesenchymal transition (EMT), which leads to a motile phenotype, enabling invasion and metastasis of cancer cells. Cancer-related inflammation, mediated by prostaglandins, has been proposed as a critical mechanism in conversion of benign cells to malignant.. Induction of cyclooxygenase 2 (COX-2), producer of prostaglandins, is thought to be a prerequisite for TGF-beta-induced EMT in benign cells. We used HaCaT derivatives, representative of skin cancer progression, to investigate TGF-beta1 mediated EMT response, and the role of COX-2 in it.. Effect of TGF-beta1 was investigated by analyzing cell proliferation, morphology and protein expression. Chemotaxis and scratch-wound assays were used to study migration.. TGF-beta1 caused proliferation arrest of benign and malignant HaCaT cells, and changed the epithelial morphology of benign and low-grade malignant cells, but not metastatic cells, to mesenchymal spindle-shape. Epithelial junction proteins ZO-1 and E-cadherin were downregulated in all cell lines in response to TGF-beta1, but mesenchymal markers were not induced, suggesting a partial EMT response. COX-2 and migration were induced only in benign HaCaT derivatives. Malignant derivatives did not induce COX-2 in response to TGF-beta 1 treatment, thus emphasizing the role of inflammation in EMT response of benign cells.. TGF-beta1 operates via distinct mechanisms in inducing EMT and metastasis, and supporting this we show that TGF-beta1 induces COX-2 and promotes the migration of benign cells, but does not further augment the migration of malignant cells, indicating their resistance to TGF-beta1 in the context of motility.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemotaxis; Epithelium; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Keratinocytes; Mesoderm; Models, Biological; Neoplasm Metastasis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

The immune modulating molecules cyclooxygenase-2 (COX-2), transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) have regulatory roles in cancer progression. There are conflicting data regarding the roles of these molecules in prostate cancer. To elucidate the prognostic impact of these proteins and provide information on prognosis and treatment, we compared the expression of COX-2, TGF-beta, and IL-10 in prostate cancer specimens with or without metastases. Ki67 was included as a measure of growth fraction of tumor cells.. Digital video analysis images from tumor cell areas and tumor stromal areas were analyzed on formalin fixed, paraffin-embedded and immunohistochemical stained cancer specimens from 59 patients: 32 patients with metastases and 27 patients without clinical, biochemical, or radiological evidence of metastases within 10 years after diagnosis. The expression of COX-2 was scored as negative, weak, moderate, or strong. The expressions of TGF-beta and IL-10 were assessed as proportions of moderately or strongly stained cells. Ki67 was detected as strong nuclear staining in proliferating cells.. In primary cancers in the metastatic group, COX-2, TGF-beta and Ki67 were stronger expressed in epithelial tumor cell and tumor stromal areas compared with non-metastatic cancers (for all markers, p<0.0001). High intensity of COX-2 staining in tumor areas was strongly associated with death from prostate cancer in univariate analyses (hazard ratio [HR] 95% CI, 4.0 (1.1-14.5)). In multivariate analyses, the risk estimate was strengthened but did not reach significance. No associations to death were found for the other markers.. High expression of COX-2, TGF-beta and Ki67 were in metastatic primary prostate carcinoma compared to non-metastatic cancers. High expression of COX-2 was associated to death from prostate carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Cyclooxygenase 2; Humans; Interleukin-10; Ki-67 Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Metastasis; Prostatic Neoplasms; Rectal Neoplasms; Transforming Growth Factor beta; Urinary Bladder Neoplasms

Bone invasion is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). We established an orthotropic implantation model using the murine OSCC cell line, SCCVII, showing direct invasion of the mandible by OSCC. Using this model, we examined the molecular mechanisms of bone invasion and the role of parathyroid-related protein (PTHrP). We established PTHrP, stable, knock-down SCCVII cells. Knock-down of PTHrP caused decreased osteoclast formation in vitro relative to expression levels of PTHrP. In vivo models showed dramatic suppression of bone invasion in PTHrP knock-down cells, and the degree of suppression was more pronounced than the level of PTHrP knock-down. We looked at an additive role of transforming growth factor-beta (TGF-beta) in PTHrP-mediated bone invasion. TGF-beta induced mRNA expression of PTHrP, showed no inhibitory effect on SCCVII cell proliferation, and caused epithelial mesenchymal trans-differentiation such as changes in the cells. Sections of resected mandibles from patients with invasive OSCC showed a great number of osteoclasts at bone invasion sites, strong expression of PTHrP, and decreased expression of E-cadherin in the tumour cells. Cancer-derived PTHrP appears to play a critical role in bone invasion by OSCC, mediated by osteoclasts. Moreover, TGF-beta appears to act synergistically to accelerate mandibular bone invasion.

Topics: Animals; Bone Neoplasms; Carcinoma, Squamous Cell; Disease Models, Animal; Gene Knockdown Techniques; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Metastasis; Osteoclasts; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; Tomography, X-Ray Computed; Transforming Growth Factor beta

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

Whereas the accumulation of fibroblasts and macrophages in breast cancer is a well-documented phenomenon and correlates with metastatic disease, the functional contributions of these stromal cells on breast cancer progression still remain largely unclear. Previous studies have uncovered a potentially important role for CCL2 inflammatory chemokine signaling in regulating metastatic disease through a macrophage-dependent mechanism. In these studies, we demonstrate a significant regulatory mechanism for CCL2 expression in fibroblasts in mediating mammary tumor progression and characterize multiple functions for CCL2 in regulating stromal-epithelial interactions. Targeted ablation of the transforming growth factor-beta (TGF-beta) type 2 receptor in fibroblasts (Tgfbr2(FspKO)) results in a high level of secretion of CCL2, and cografts of Tgfbr2(FspKO) fibroblasts with 4T1 mammary carcinoma cells enhanced tumor progression associated with recruitment of tumor-associated macrophages (TAMs). Antibody neutralization of CCL2 in tumor-bearing mice inhibits primary tumor growth and liver metastases as evidenced by reduced cell proliferation, survival, and TAM recruitment. Both high and low stable expressions of small interfering RNA to CCL2 in Tgfbr2(FspKO) fibroblasts significantly reduce liver metastases without significantly affecting primary tumor growth, cell proliferation, or TAM recruitment. High but not low knockdown of CCL2 enhances tumor cell apoptosis. These data indicate that CCL2 enhances primary tumor growth, survival, and metastases in a dose-dependent manner, through TAM-dependent and -independent mechanisms, with important implications on the potential effects of targeting CCL2 chemokine signaling in the metastatic disease.

Topics: Animals; Cell Separation; Chemokine CCL2; Coculture Techniques; Disease Progression; Female; Fibroblasts; Flow Cytometry; Gene Knockout Techniques; Immunohistochemistry; Macrophages; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

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

Previous studies have found that matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta (TGF-beta) can be considered as biomarkers and indices of disease progression in several human cancers. In this study, we investigated the plasma levels of MMP-2 and TGF-beta and their correlation in 49 primary cutaneous melanoma and 10 metastatic melanoma. Plasma MMP-2 and TGF-beta levels in patients with primary melanoma were significantly higher than those of healthy controls. These protein levels were significantly higher in patients with metastatic melanoma. A positive correlation between plasma levels of MMP-2 and TGF-beta in melanoma patients supports the hypothesis that TGF-beta triggers the release of MMP-2. The immunohistochemistry analysis shows that MMP-2 and TGF-beta were highly expressed in tumor tissues as well as in matched plasma samples. This finding suggests that these proteins are released from tumor cells. Overall, our data indicate that MMP-2 and TGF-beta may represent novel diagnostic markers and therapeutic targets in melanoma and the determination of their concentration could be a useful diagnostic and prognostic indicator. TGF-beta, leading the tissue invasion mediated by MMP-2, is a strong promoter of tumor progression. Therefore, reducing or blocking the activity of TGF-beta may represent a promising target in therapeutic strategies for limiting the growth of melanoma.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; Cell Transformation, Neoplastic; Disease Progression; Female; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Melanoma; Middle Aged; Neoplasm Metastasis; Skin Neoplasms; Transforming Growth Factor beta

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Cell Line, Transformed; Cell Transdifferentiation; Disease Progression; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta

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

It is now generally accepted that TGF-β acts as a pro-metastatic factor in advanced human breast cancer. However, it is well documented, that TGF-β is context dependent, and whether the TGF-β pathway switches to promote metastasis during the progression of squamous cell carcinoma (SCC) is unknown. This study examined the role of TGF-β signalling in SCC using a series of genetically related keratinocyte cell lines representing later stages of the disease, stably transduced with a dominant negative TβRII cDNA (dnTβRII). We demonstrated that clones expressing dnTβRII lost their growth inhibitory response to TGF-βin vitro, while ligand expression remained unchanged. Following transplantation of transduced cells to athymic mice in vivo, we showed that attenuation of the TGF-β signal resulted in a loss of differentiation and increased metastasis. In human tissue samples loss of TGF-β signal transduction as measured by pSmad2 activity also correlated with a loss of differentiation. Id1, previously shown to be down regulated by TGF-β, an inhibitor of differentiation and associated with metastasis, was weakly expressed in focal areas of a small number of human tumours but expression did not correlate with low levels of pSmad2. Our data demonstrate that TGF-β does not switch to promote metastasis in late stage human SCC of the skin and that inhibition of TGF-β signalling results in a loss of differentiation and increased metastasis in the later stages of this disease.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Humans; Immunohistochemistry; Inhibitor of Differentiation Protein 1; Mice; Mice, Nude; Neoplasm Metastasis; Signal Transduction; Skin Neoplasms; Transfection; Transforming Growth Factor beta; Up-Regulation

Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. The influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant down-regulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PC3 and DU145. Interestingly, TGF-β down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and α5β1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after cross-talk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with "activated" stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM.

Topics: Animals; Antibodies, Neutralizing; Cell Communication; Cell Differentiation; Cell Movement; Coculture Techniques; Cytoskeleton; Down-Regulation; Extracellular Matrix; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Mice; Neoplasm Metastasis; Prostatic Neoplasms; Protein Transport; Proteoglycans; Receptors, Transforming Growth Factor beta; Receptors, Vitronectin; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

As a tumor marker for colorectal cancers, carcinoembryonic antigen (CEA) enhances the metastatic potential of cancer cells. CEA functions as an intercellular adhesion molecule and is upregulated in a wide variety of human cancers. However, the molecular mechanisms by which CEA mediates metastasis remain to be understood. Transforming growth factor-β (TGF-β) signaling regulates both tumor suppression and metastasis, and also contributes to the stimulation of CEA transcription and secretion in colorectal cancer cells. However, it remains unknown whether CEA, in turn, influences TGF-β functions and if a regulatory cross-talk exists between CEA and the TGF-β signaling pathway. Here, we report that CEA directly interacts with TGF-β receptor and inhibits TGF-β signaling. Targeting CEA with either CEA-specific antibody or siRNA rescues TGF-β response in colorectal cancer cell lines with elevated CEA, thereby restoring the inhibitory effects of TGF-β signaling on proliferation. CEA also enhances the survival of colorectal cancer cells in both local colonization and liver metastasis in animal study. Our study provides novel insights into the interaction between CEA and TGF-β signaling pathway and establishes a negative feedback loop in amplifying the progression of colon cancer cells to more invasive phenotypes. These findings offer new therapeutic opportunities to inhibit colorectal cancer cell proliferation by cotargeting CEA in promoting tumor-inhibitory action of the TGF-β pathway.

Topics: Carcinoembryonic Antigen; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; DNA Primers; Genes, myc; Humans; Microsatellite Repeats; Microscopy, Confocal; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

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

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

Epithelial-mesenchymal transition (EMT) was shown to confer tumor cells with abilities essential for metastasis, including migratory phenotype, invasiveness, resistance to apoptosis, evading immune surveillance, and tumor stem cell traits. Therefore, inhibition of EMT can be an important therapeutic strategy to inhibit tumor metastasis. Here, we show that activation of peroxisome proliferator-activated receptor γ (PPAR-γ) inhibits transforming growth factor β (TGF-β)-induced EMT in lung cancer cells and prevents metastasis by antagonizing Smad3 function. Activation of PPAR-γ by synthetic ligands (troglitazone and rosiglitazone) or by a constitutively active form of PPAR-γ prevents TGF-β-induced loss of E-cadherin expression and inhibits the induction of mesenchymal markers (vimentin, N-cadherin, fibronectin) and matrix metalloproteases. Consistently, activation of PPAR-γ also inhibited EMT-induced migration and invasion of lung cancer cells. Furthermore, effects of PPAR-γ ligands were attenuated by siRNA-mediated knockdown of PPAR-γ, indicating that the ligand-induced responses are PPAR-γ dependent. Selective knockdown of Smad2 and Smad3 by siRNA showed that TGF-β-induced EMT is Smad3 dependent in lung cancer cells. Activation of PPAR-γ inhibits TGF-β-induced Smad transcriptional activity but had no effect on the phosphorylation or nuclear translocation of Smads. Consistently, PPAR-γ activation prevented TGF-β-induced transcriptional repression of E-cadherin promoter and inhibited transcriptional activation of N-cadherin promoter. Finally, treatment of mice with troglitazone or knockdown of Smad3 in tumor cells significantly inhibited TGF-β-induced experimental metastasis in SCID-Beige mice. Together, with the low toxicity profile of PPAR-γ ligands, our data show that these ligands may serve as potential therapeutic agents to inhibit metastasis.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Cell Nucleus; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Ligands; Lung Neoplasms; Mice; Neoplasm Metastasis; Phenotype; Phosphorylation; PPAR gamma; Promoter Regions, Genetic; Protein Transport; Smad2 Protein; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

TGFbeta is an important cell growth regulator which may have a role in metastasis formation. Microsatellite unstable (MSI-H) colon cancer serves as a unique model to demonstrate this as most MSI-H colon cancers have a mutation in the transforming growth factor beta receptor II (TGFbetaRII) gene and a low metastatic rate.. To demonstrate an increase in invasion and metastasis in a MSI-H colorectal cancer cell line with a known mutation in TGFbetaRII.. By restoring the wild-type TGFbetaRII gene in the KM12C MSI-H colorectal carcinoma cell line with a known mutation in TGFbetaRII, we have demonstrated that both invasion and metastasis in this cell line was significantly increased. A mouse metastatic model have shown that liver metastases were increased in mice inoculated with cells containing a wild-type TGFbetaRII gene (42% for the transfected group compared with 15% for the control group; p = 0.0379), despite a reduction in the size of primary tumours.. This study highlights an important mechanism which may contribute to the low metastatic rate of MSI-H colon cancers and demonstrates the importance of TGFbeta signalling in metastasis formation. Previous studies involving breast cancer cell lines have shown that blocking TGFbeta signalling results in a reduction in metastasis formation. This study is the first study to use a cell line with a low metastatic rate and TGFbetaRII mutations to demonstrate that restoring TGFbeta signalling increases the metastatic rate.

Topics: Animals; Cell Line, Tumor; Collagen; Colorectal Neoplasms; Drug Combinations; Humans; Laminin; Mice; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta

Immunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC.. MHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression.. In vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia.. The immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC.

Topics: Actins; Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; DNA Primers; Fibroblast Growth Factor 2; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Messenger; Sirolimus; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Neutrophil infiltrates into tumors have been reported in certain circumstances to reduce tumor growth and in other circumstances to augment tumor growth, particularly by facilitating metastasis. Neutrophil chemotaxis can be facilitated by both interleukin-8 (IL-8) and transforming growth factor-beta (TGF-beta). However, the combined effects of these two cytokines on neutrophil tumor infiltrates is unknown, and we considered the possibility that studying the combined effects might resolve apparent contradictions with regard to neutrophil effects on tumor development. First, we determined that a simultaneous IL-8 and TGF-beta blockade is far more efficient at eliminating the neutrophil infiltrate from an A549 derived tumor than is blockade of either cytokine alone. Blockade of IL-8 alone, led to smaller tumors, consistent with the known inhibitory role of TGF-beta on A549 cell proliferation. Blockade of TGF-beta alone rescued the tumor growth but led to reduced metastasis volume. Surprisingly, blockade of both cytokines rescued both tumor volume and metastasis, underscoring the difficulty of understanding the effects of complete tumor cytokine elaboration profiles by isolating the effects of only one cytokine.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Humans; Injections, Intraperitoneal; Interleukin-8; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Neutrophil Infiltration; Transforming Growth Factor beta; Tumor Burden

Topics: Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromatography, Liquid; Epithelium; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mass Spectrometry; Mesoderm; Neoplasm Metastasis; Proteomics; Transforming Growth Factor beta

To observe the expression of Smad4, the core of TGF-beta/Smads signal transduction pathway in different prostate cancer cell lines, and explore their molecular mechanism of bone metastatic potential.. The Millicell polycarbonate filter coated with matrigel was used to confirm the invasive potency of LNCaP and ARCaP cell lines (IF11 and IA8). The expressions of the Smad4 protein and mRNA in these prostate cancer cells with different metastatic potentials were detected by Western blotting and RT-PCR, respectively.. ARCaP cell lines (IF11 and IA8) exhibited a stronger potency of invasion than LNCaP (P < 0.01). The Smad4 protein and mRNA highly expressed in the LNCaP cell line that was well-known with a low metastatic potential, but not in the ARCaP (IF11 or IA8) cells with high metastatic potentials (P < 0.01).. Smad4 expresses differently in LNCaP and ARCaP cell lines with different metastatic potentials and, as a tumor suppressive gene, its deficient expression may play an important role in the invasion and metastasis of advanced prostate cancer.

Topics: Cell Line, Tumor; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Messenger; Smad4 Protein; Transforming Growth Factor beta

Although much is known about the genes that promote metastasis, few suppressors of metastasis have been found. Adorno et al. (2009) now identify p63 as a potent suppressor of metastasis and uncover an intricate mechanism for the inactivation of metastasis in cancer cells in response to transforming growth factor beta.

Topics: Animals; Humans; Neoplasm Metastasis; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

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

Evidence suggests that multiple tumors, including pancreatic adenocarcinoma, display heterogeneity in parameters that are critical for tumor formation, progression and metastasis. Understanding heterogeneity in solid tumors is increasingly providing a plethora of new diagnostic and therapeutic approaches. In this study, a particular focus was put on identifying a subpopulation of stem cell-like, slow cycling tumor cells in a pancreas adenocarcinoma cell lines. Using a label retention technique a subpopulation of slow cycling cells (DiI+/SCC) was identified and further evaluated in the BxPC-3 and Panc03.27 cell lines. These slowly cycling cells managed to retain the lipophilic labeling dye DiI, while the bulk of the cells (>94%) did not. The DiI+/SCC population, showed only a partial overlap with the CSC markers CD24(+)/CD44(+), CD133(+) and ALDH but they survived chemotherapeutic treatment, and were able to recreate the initial heterogeneous tumor cell population. DiI+/SCCs exhibited an increased invasive potential as compared with their non-label retaining, faster cycling cells (DiI-/FCC). They also had increased tumorigenic potential and morphological changes resembling cells that have undergone an epithelial to mesenchymal transition (EMT). Analysis of DiI+/SCC cells by real time PCR revealed a selective up-regulation of tell tale components of the Hedgehog/TGFbeta pathways, as well as a down-regulation of EGFR, combined with a shift in crucial components implied in EMT. The presented findings offer an expanded mechanistic understanding that associates tumor initiating potential with cycling speed and EMT in pancreatic cancer cell lines.

Topics: Adenocarcinoma; Animals; Antigens, CD; Antimetabolites, Antineoplastic; Cell Line, Tumor; ErbB Receptors; Female; Flow Cytometry; Fluorouracil; Hedgehog Proteins; Humans; Mice; Mice, SCID; Neoplasm Metastasis; Neoplastic Stem Cells; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

Colorectal cancer is the second leading cause of cancer related mortality, with a majority of deaths resulting from metastases. Few in vivo models allow for the study of the complex process of metastasis. The purpose of this study was to determine the effects of epidermal growth factor receptor activation and TGFbeta pathway attenuation in FET, a weakly tumorigenic human colon cancer cell line, in an orthotopic model.. Using FET, FETalpha, FETalphaDNRII, and FETDNRII cells were constructed. Tumors were orthotopically implanted onto the colons of BALB/c nude mice. After 7 wk, the mice were euthanized and organs extracted for examination. All cell lines demonstrated primary invasion. FETalpha was weakly metastatic compared with FETalphaDNRII and FETDNRII, which demonstrated metastases to the lung and liver, respectively.. Epidermal growth factor receptor (EGFR) activation transforms a nontumorigenic cell line into a tumorigenic but not metastatic one. The tumorigenic line becomes metastatic with the attenuation of TGFbeta signaling. Loss of EGFR activation in the TGFbeta inhibited line results in a decreased metastatic burden, but importantly, changes the organotropic homing from lung to liver. Thus, these in vivo studies demonstrate that EGFR activation and TGFbeta signaling pathways play a role in tumorigenicity and in pattern of metastases.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; ErbB Receptors; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta

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

Transforming growth factor-beta (TGF-beta) is involved in actin cytoskeleton reorganization and tumor progression. Fascin1, an actin-binding protein, increases cell invasiveness and motility in various transformed cells. To determine whether fascin1 is an important mediator of the tumor response to TGF-beta, we applied the small interfering RNA (siRNA) technique to silence fascin1 in gastric cancer (GC) cells MKN45. Results showed that the effects of TGF-beta1 on GC cells invasion and metastasis were mediated by tumor production of fascin1; furthermore, it was found that TGF-beta1- induced fascin1 expression was suppressed by the specific inhibitors of JNK and ERK pathways, SP6001125 and PD98059, respectively, but not by transient transfection of Smad2 and Smad4 siRNA. Our data for the first time demonstrated that fascin1 is an important mediator of TGF-beta1-induced invasion and metastasis of GC cells, which involves JNK and ERK signaling pathways.

Topics: Animals; Carrier Proteins; Cell Line, Tumor; Chemotaxis; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; Microfilament Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; RNA Interference; Smad2 Protein; Smad4 Protein; Stomach Neoplasms; Transforming Growth Factor beta

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

Metastatic disease is a primary cause of cancer-related death, and factors governing tumor cell metastasis have not been fully elucidated. Here, we address this question by using tumor cell lines derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53. Despite having widespread somatic genetic alterations, the metastasis-prone tumor cells retained a marked plasticity. They transited reversibly between epithelial and mesenchymal states, forming highly polarized epithelial spheres in three-dimensional culture that underwent epithelial-to-mesenchymal transition (EMT) following treatment with transforming growth factor-beta or injection into syngeneic mice. This transition was entirely dependent on the microRNA (miR)-200 family, which decreased during EMT. Forced expression of miR-200 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize, and conferred transcriptional features of metastasis-incompetent tumor cells. We conclude that tumor cell metastasis is regulated by miR-200 expression, which changes in response to contextual extracellular cues.

Topics: Adenocarcinoma; Animals; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Disease Models, Animal; Epithelial Cells; Extracellular Space; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice; MicroRNAs; Neoplasm Metastasis; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous beta-catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of beta-catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E-cadherin transcription.. Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.

Topics: Adult; Aged; Animals; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Epithelial Cells; Female; gamma Catenin; Humans; Liver Neoplasms; Male; Mesoderm; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Nuclear Proteins; Retrospective Studies; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Twist-Related Protein 1; Vimentin

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

Although Akt is known to play a role in human cancer, the relative contribution of its three isoforms to oncogenesis remains to be determined. We expressed each isoform individually in an Akt1(-/-)/Akt2(-/-)/Akt3(-/-) cell line. MicroRNA profiling of growth factor-stimulated cells revealed unique microRNA signatures for cells with each isoform. Among the differentially regulated microRNAs, the abundance of the miR-200 family was decreased in cells bearing Akt2. Knockdown of Akt1 in transforming growth factor-beta (TGFbeta)-treated MCF10A cells also decreased the abundance of miR-200; however, knockdown of Akt2, or of both Akt1 and Akt2, did not. Furthermore, Akt1 knockdown in MCF10A cells promoted TGFbeta-induced epithelial-mesenchymal transition (EMT) and a stem cell-like phenotype. Carcinomas developing in MMTV-cErbB2/Akt1(-/-) mice showed increased invasiveness because of miR-200 down-regulation. Finally, the ratio of Akt1 to Akt2 and the abundance of miR-200 and of the messenger RNA encoding E-cadherin in a set of primary and metastatic human breast cancers were consistent with the hypothesis that in many cases breast cancer metastasis may be under the control of the Akt-miR-200-E-cadherin axis. We conclude that induction of EMT is controlled by microRNAs whose abundance depends on the balance between Akt1 and Akt2 rather than on the overall activity of Akt.

Topics: Animals; Cadherins; Carcinoma; Cell Line, Tumor; Epithelium; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Mice; MicroRNAs; Neoplasm Metastasis; Protein Isoforms; Proto-Oncogene Proteins c-akt; Stem Cells; 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

Topics: Animals; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Neoplasms; Signal Transduction; 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

The role of Smad4 in transforming growth factor beta (TGFbeta)-mediated epithelial-mesenchymal transition (EMT), invasion, and metastasis was investigated using isogenically matched pancreatic cancer cell lines that differed only in expression of Smad4. Cells expressing Smad4 showed an enhanced TGFbeta-mediated EMT as determined by increased expression of vimentin and decreased expression of beta-catenin and E-cadherin. TGFbeta-mediated invasion was suppressed in Smad4-intact cells as determined by in vitro assays, and these cells showed a reduced metastasis in an orthotopic model of pancreatic cancer. Interestingly, TGFbeta inhibited STAT3(Tyr705) phosphorylation in Smad4-intact cells. The decrease in STAT3(Tyr705) phosphorylation was linked to a TGFbeta/Smad4-dependent and enhanced activation of extracellular signal-regulated kinases, which caused an increase in serine phosphorylation of STAT3(Ser727). Down-regulating signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA in Smad4-deficient cells prevented TGFbeta-induced invasion. Conversely, expressing a constitutively activated form of STAT3 (STAT3-C) in Smad4-intact cells enhanced invasion. This study indicates the requirement of STAT3 activity for TGFbeta-induced invasion in pancreatic cancer cells and implicates Smad4-dependent signaling in regulating STAT3 activity. These findings further suggest that loss of Smad4, leading to aberrant activation of STAT3, contributes to the switch of TGFbeta from a tumor-suppressive to a tumor-promoting pathway in pancreatic cancer.

Topics: Blotting, Western; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Phosphorylation; Smad4 Protein; STAT3 Transcription Factor; Transforming Growth Factor beta; Tyrosine

Transforming growth factor-beta (TGF-beta) stimulates phosphorylation of TGF-beta type I receptor. This receptor is now shown to be sumoylated, leading to enhanced activation and modulation of the downstream Smad signalling pathway.

Topics: Animals; Gene Expression Regulation; Humans; Models, Biological; Mutation; Neoplasm Metastasis; Neoplasms; Phosphorylation; Signal Transduction; SUMO-1 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is important during embryonic cell layer movement and tumor cell invasiveness. EMT converts adherent epithelial cells to motile mesenchymal cells, favoring metastasis in the context of cancer progression. Transforming growth factor-beta (TGF-beta) triggers EMT via intracellular Smad transducers and other signaling proteins. We previously reported that the high mobility group A2 (HMGA2) gene is required for TGF-beta to elicit EMT in mammary epithelial cells. In the present study we investigated the molecular mechanisms by which HMGA2 induces EMT. We found that HMGA2 regulates expression of many important repressors of E-cadherin. Among these, we analyzed in detail the zinc-finger transcription factor SNAIL1, which plays key roles in tumor progression and EMT. We demonstrate that HMGA2 directly binds to the SNAIL1 promoter and acts as a transcriptional regulator of SNAIL1 expression. Furthermore, we observed that HMGA2 cooperates with the TGF-beta/Smad pathway in regulating SNAIL1 gene expression. The mechanism behind this cooperation involves physical interaction between these factors, leading to an increased binding of Smads to the SNAIL1 promoter. SNAIL1 seems to play the role of a master effector downstream of HMGA2 for induction of EMT, as SNAIL1 knock-down partially reverts HMGA2-induced loss of epithelial differentiation. The data propose that HMGA2 acts in a gene-specific manner to orchestrate the transcriptional network necessary for the EMT program.

Topics: Animals; Cell Differentiation; Cell Movement; Chlorocebus aethiops; COS Cells; Epithelium; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; HMGA2 Protein; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mesoderm; Mice; Neoplasm Metastasis; Neoplasms; Promoter Regions, Genetic; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; 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

Epithelial-mesenchymal transition (EMT) has been considered essential for metastasis, a multistep process including local invasion, intravasation, extravasation, and proliferation at distant sites. However, controversy remains as to whether EMT truly happens and how important it is to metastasis. We studied the involvement of EMT in individual steps of metastasis and found that p12(CDK2-AP1), a down-stream effector of transforming growth factor beta, induced EMT of hamster cheek pouch carcinoma-1 cells by promoting the expression of Twist2. EMT cells have an increased invasive but decreased metastatic phenotype. When s.c. inoculated, both EMT and non-EMT cells established primary tumors, but only EMT cells invaded into the adjacent tissues and blood vessels; however, neither cells formed lung metastases. When i.v. inoculated, only non-EMT cells established lung metastases. Moreover, s.c. inoculation of a mixture of the two cell types resulted in intravasation of both cell types and formation of lung metastasis from non-EMT cells. Our results allowed us to propose a novel model for the role of EMT in cancer metastasis. We showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. We thus hypothesize that EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites.

Topics: Animals; Cadherins; Cricetinae; Epithelial Cells; Humans; Keratinocytes; Lung Neoplasms; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Kinases; Transfection; Transforming Growth Factor beta; Tumor Suppressor Proteins

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

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

It is known that the activation of hedgehog (Hh) signaling is involved in the progression and invasion of various tumors, including gastric carcinoma. In this study, we investigated the impact of transforming growth factor (TGF)-beta signaling on the sonic hedgehog (Shh)-mediated invasion of gastric cancer cells. We found that higher concentrations of N-Shh enhanced cell motility and invasiveness in gastric cancer cells, whereas no increase was observed in cells that were treated with KAAD-cyclopamine (a Shh signaling inhibitor) or anti-Shh blocking antibodies. In addition, the N-Shh-induced migration and invasiveness of gastric cancer cells were reduced by treatment with anti-TGF-beta blocking antibody or TGF-beta1 small interfering RNA (siRNA) in presence of N-Shh when compared with control groups. Furthermore, TGF-beta1 secretion, TGF-beta-mediated transcriptional response, expression of activin receptor-like kinase (ALK) 5 protein and phosphorylation of Smad 3 were also enhanced by treatment with N-Shh, but not KAAD-cyclopamine, anti-Shh or TGF-beta1 blocking antibodies. Blockade of the ALK5 kinase in the presence of N-Shh significantly inhibited phosphorylation of Smad 3, activity of matrix metalloproteinases and Shh-induced cell motility/invasiveness. Importantly, transient expression of ALK5 siRNA or Smad 3 siRNA reduced the ability of N-Shh to stimulate migration and invasion of those cells compared with the cells treated with non-specific control siRNA. In summary, these results indicate that Shh promotes motility and invasiveness of gastric cancer cells through TGF-beta-mediated activation of the ALK5-Smad 3 pathway. Additionally, our findings are the first to suggest a role and mechanism for Shh signaling as it relates to the metastatic potential of gastric cancer, thereby indicating potential therapeutic molecular targets to decrease metastasis.

Topics: Base Sequence; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA Primers; Hedgehog Proteins; Humans; Immunohistochemistry; Lymphatic Metastasis; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad3 Protein; Stomach Neoplasms; Transforming Growth Factor beta

We demonstrated previously that growth factor receptor-bound protein 2 (Grb2) associates with the transforming growth factor-beta (TGF-beta) type II receptor [TbetaR-II] upon its phosphorylation on Tyr284 by Src. Although this phosphotransferase reaction is critical in mediating TGF-beta stimulation of epithelial-mesenchymal transition (EMT) and invasion in mammary epithelial cells (MECs), the necessity of Grb2 in promoting these TGF-beta-dependent events remain purely correlative. Herein, we further evaluated the role of Grb2 in mediating the oncogenic activities of TGF-beta and show that the binding of Grb2 to TbetaR-II paralleled the induction of EMT in MECs stimulated by TGF-beta. Introducing siRNAs against Grb2 or expression of a TbetaR-II mutant that cannot bind Grb2 (i.e. Y284F-TbetaR-II) had no effect on the ability of TGF-beta to activate Smad3, but significantly impaired its stimulation of p38 mitogen-activated protein kinase (MAPK) in MECs. Importantly, these same cellular conditions also prevented the ability of MECs to undergo EMT in response to TGF-beta, and to invade synthetic basement membranes when stimulated by beta3 integrin and TGF-beta. Finally, we show that the ability of TGF-beta to stimulate breast cancer growth and pulmonary metastasis in mice required TbetaR-II to be phosphorylated on Tyr284, which activated p38 MAPK in developing and progressing mammary tumors. Collectively, our findings have established the necessity of Grb2 in mediating TGF-beta stimulation of EMT and invasion in MECs, as well as demonstrated the essential function of the alphavbeta3 integrin:Src:phospho-Y284-TbetaR-II:Grb2:p38 MAPK signaling axis to promote breast cancer growth and metastasis in vivo.

Topics: Animals; Cell Proliferation; Female; GRB2 Adaptor Protein; Humans; Mammary Neoplasms, Animal; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Phosphotransferases; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; src-Family Kinases; Transforming Growth Factor beta; Tyrosine

Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFbeta signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases.. Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (beta-catenin), Notch (HES1) and TGFbeta (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed.. When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear beta-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear beta-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear beta-catenin.. Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFbeta signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFbeta pathways is reduced in secondary colorectal tumors.

Topics: Adult; Aged; Brain Neoplasms; Cadherins; Colorectal Neoplasms; Female; Fibronectins; Humans; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Metastasis; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, Notch; Signal Transduction; Tissue Array Analysis; Transforming Growth Factor beta; Vimentin; Wnt Proteins

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-beta (TGF-beta). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-beta signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-beta. In 3D spheroid confrontation assays between breast tumor cells and TGF-beta-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-beta antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

Topics: Animals; Antigens, CD; Cell Line, Tumor; Cell Movement; Endoglin; Gene Expression Regulation, Neoplastic; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Matrix Metalloproteinase 1; Matrix Metalloproteinases, Secreted; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Receptors, Cell Surface; 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

Existing prostate cancer cell lines have limitations.. Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors.. We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p.. The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Humans; Immunohistochemistry; Karyotyping; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, Androgen; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Topics: Animals; Cell Communication; Extracellular Matrix; Fibroblasts; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Macrophages; Matrix Metalloproteinases; Mesenchymal Stem Cells; Myeloid Cells; Neoplasm Metastasis; Neoplasms; 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

MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-beta-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.

Topics: Animals; Cadherins; Cell Movement; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genome, Human; HeLa Cells; Homeodomain Proteins; Humans; Kruppel-Like Transcription Factors; Mice; MicroRNAs; Multigene Family; Neoplasm Metastasis; Neoplasms; Repressor Proteins; Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.

Topics: Animals; DNA-Binding Proteins; Female; Genes, Tumor Suppressor; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Nuclear Proteins; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Smad Proteins; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Post-translational sumoylation, the covalent attachment of a small ubiquitin-like modifier (SUMO), regulates the functions of proteins engaged in diverse processes. Often associated with nuclear and perinuclear proteins, such as transcription factors, it is not known whether SUMO can conjugate to cell-surface receptors for growth factors to regulate their functions. Here we show that the type I transforming growth factor-beta (TGF-beta) receptor, T beta RI, is sumoylated in response to TGF-beta and that its sumoylation requires the kinase activities of both T beta RI and the type II TGF-beta receptor, T beta RII. Sumoylation of T beta RI enhances receptor function by facilitating the recruitment and phosphorylation of Smad3, consequently regulating TGF-beta-induced transcription and growth inhibition. T beta RI sumoylation modulates the dissemination of transformed cells in a mouse model of T beta RI-stimulated metastasis. T beta RI sumoylation therefore controls responsiveness to TGF-beta, with implications for tumour progression. Sumoylation of cell-surface receptors may regulate other growth factor responses.

Topics: Animals; Chlorocebus aethiops; COS Cells; Cytoplasm; Gene Expression Regulation; Humans; Lysine; Mice; Models, Biological; Neoplasm Metastasis; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; SUMO-1 Protein; Transforming Growth Factor beta

Overexpression of transforming growth factor beta (TGF-beta) is frequently associated with metastasis and poor prognosis, and TGF-beta antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity. Here, we have used the transplantable 4T1 model of metastatic breast cancer to address underlying mechanisms. We showed that efficacy of the anti-TGF-beta antibody 1D11 in suppressing metastasis was dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment. The main outcome was a highly significant enhancement of the CD8+ T-cell-mediated antitumor immune response, but effects on the innate immune response and on angiogenesis also contributed to efficacy. Treatment with 1D11 increased infiltration of natural killer cells and T cells at the metastatic site, and enhanced expression of coactivators (NKG2D) and cytotoxic effectors (perforin and granzyme B) on CD8+ T cells. On the tumor cells, increased expression of an NKG2D ligand (Rae1gamma) and of a death receptor (TNFRSF1A) contributed to enhanced immune cell-mediated recognition and lysis. The data suggest that elevated TGF-beta expression in the tumor microenvironment modulates a complex web of intercellular interactions that aggregately promote metastasis and progression. TGF-beta antibodies reverse this effect, and the absence of a major effect of TGF-beta antagonism on any one cell compartment may be critical for a good therapeutic window and the avoidance of autoimmune complications.

Topics: Animals; Apoptosis; CD8-Positive T-Lymphocytes; Culture Media, Conditioned; Immunotherapy; Killer Cells, Natural; Ligands; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Prognosis; Transfection; 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

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. 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 pancreatic tumor cells.

Topics: Activin Receptors, Type I; Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Line; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression; Humans; Immunoblotting; Mice; Mice, SCID; Mutation; Neoplasm Metastasis; Neoplasms, Experimental; Pancreatic Neoplasms; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; Transfection; Transforming Growth Factor beta; Tumor Burden

HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.

Topics: Antibodies; Basigin; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Line, Tumor; Down-Regulation; Extracellular Matrix Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Up-Regulation

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-beta (TGF-beta). Because TGF-beta promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-beta in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-beta/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-beta target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-beta and that Smad7 and the TbetaRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-beta promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-beta signaling may be an effective treatment for melanoma metastasis to bone.

Topics: Animals; Bone Neoplasms; Female; Humans; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Smad7 Protein; Survival Analysis; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

A dynamic interplay between prostate cancer cells and reactive bone stroma modulates growth of metastases within bone. We used microarray analysis to screen for changes in gene expression in bone marrow stromal cells cocultured with prostate cancer cells and found reduced expression of endoglin, a transmembrane glycoprotein that functions as an auxiliary coreceptor for members of the transforming growth factor beta (TGF-beta) family of cytokines. The downstream TGF-beta/bone morphogenetic protein signaling pathway including Smad1 and Smad2/3 also was attenuated, as was Smad-dependent gene transcription. Smad1/5/8-dependent inhibitor of DNA binding 1 expression and Smad2/3-dependent plasminogen activator inhibitor I expression both were decreased and were accompanied by decreased cell proliferation. Small interfering RNA-mediated knockdown of endoglin in HS-5 cells verified that the effects on signaling were a direct result of the attenuation of endoglin. These data illustrate that endoglin acts as a positive regulator of both activin receptor-like kinase 1-induced Smad1/5/8 activation and activin receptor-like kinase 5-induced Smad2/3 activation in bone marrow stromal cells. In addition, the data illustrate that one early event of metastasis upon the arrival of prostate cancer cells into the bone stroma is attenuated endoglin expression in the stromal cells, which subsequently alters Smad signaling and cell proliferation. We hypothesize that coculture of bone marrow stromal cells with prostate cancer cells alters TGF-beta signaling in the stromal cells, ultimately facilitating growth of the cancer cells in the bone compartment. Collectively, these studies suggest that prostate cancer cells modulate TGF-beta responsiveness of bone marrow stroma as one means of facilitating their own growth in bone.

Topics: Antigens, CD; Apoptosis; Bone and Bones; Bone Marrow Cells; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Endoglin; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, Cell Surface; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Tumor cell invasion through basement membranes and into stromal tissue are key steps for promoting growth and metastasis. Tumor cells express various extracellular-matrix-degrading enzymes such as matrix metalloproteinases (MMPs) to degrade extracellular matrix components to facilitate tumor migration and invasion. Histological and clinical studies suggest a role for MMP-1 (collagenase-1) in malignant melanoma invasion. In this study, we evaluated MMP-1 in regulating malignant phenotypes of human melanoma cells by generating human melanoma cells stably transfected with pro-MMP-1 cDNA. The transfectants expressed the active form of MMP-1 associated with cells and showed enhanced invasive and growth abilities in type I collagen gel. Furthermore, MMP-1 expression promoted anchorage-independent growth, which was inhibited in the presence of type II transforming growth factor (TGF)-beta receptor:Fc fusion protein that scavenges TGF-beta receptors. Finally, we demonstrated that MMP-1 directly generated active TGF-beta from its latent form. Thus, these results suggest that MMP-1 produced from melanoma cells would play a role in tumor progression by both degrading matrix proteins and generating active growth factors such as TGF-beta in vivo.

Topics: Animals; Cell Line, Tumor; Collagen Type I; Epithelial Cells; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung; Matrix Metalloproteinase 1; Melanoma; Mink; Models, Biological; Neoplasm Metastasis; Transfection; Transforming Growth Factor beta

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

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Bone Neoplasms; Epithelial Cells; Humans; Male; Neoplasm Metastasis; Prostate; Prostatic Neoplasms; Transforming Growth Factor beta

The 1,029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val(12)ras. These cell lines respond to TGFbeta by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFbeta. In vitro, vimentin expression is induced in all the cell lines by TGFbeta treatment, whereas cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFbeta is mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFbeta treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half-life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFbeta, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.

Topics: Animals; Cell Line, Transformed; Epithelial Cells; Gene Expression Regulation, Developmental; Gene Silencing; Humans; Keratin-18; Mesoderm; Mice; Neoplasm Metastasis; Promoter Regions, Genetic; ras Proteins; Transforming Growth Factor beta; Vimentin

During development, stem and progenitor cells gradually commit to differentiation pathways. Cell fate decisions are regulated by differentiation factors, which activate transcription programmes that specify lineage and differentiation status. Among these factors, the transforming growth factor (TGF)-beta family is important in both lineage selection and progression of differentiation of most, if not all, cell and tissue types. There is now increasing evidence that TGF-beta family proteins have the ability to redirect the differentiation of cells that either have fully differentiated or have engaged in differentiation along a particular lineage, and can thereby elicit 'transdifferentiation'. This capacity for cellular plasticity is critical for normal embryonic development, but when recapitulated in the adult it can give rise to, or contribute to, a variety of diseases. This is illustrated by the ability of TGF-beta family members to redirect epithelial cells into mesenchymal differentiation and to cause switching of mesenchymal cells from one lineage to another. Hence, various pathologies in adults may be considered diseases of abnormal development and differentiation.

Topics: Animals; Cell Differentiation; Cell Lineage; Epithelium; Fibrosis; Humans; Mesoderm; Morphogenesis; Neoplasm Metastasis; Stem Cells; Transforming Growth Factor beta

For a better understanding of the factors contributing to tumor progression in metastatic renal cell carcinoma and to identify possible targets for immunotherapeutic approaches, we characterized several primary cultures from renal cell carcinoma.. Cell cultures were tested for activity of telomerase, secretion of immunosuppressive cytokines and others. The induction of cytotoxic activity against the autologous tumor was tested in a cytotoxicity assay after coculture of immunological effector cells with antigen-pulsed dendritic cells. The data were tested for influence on survival.. We were able to establish primary cell cultures from 58 patients with renal cell carcinoma and their metastasis. 48/58 were positive for telomerase activity and all secreted IL-6, TGF-beta, VEGF and IL-8. High TGF-beta secretion, the activity of telomerase and the induction of a telomerase-specific immune response against telomerase peptides in telomerase-positive tumors had a significant impact on survival.. TGF-beta secretion, activity of telomerase in telomerase-positive tumors and the ability to generate a telomerase-specific immune response might serve as a prognostic marker for RCC. New approaches might focus on attacking the TGF-beta pathway and on induction of telomerase-specific immune cells.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Culture Techniques; Cytokines; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy; Interleukin-6; Interleukin-8; Kaplan-Meier Estimate; Kidney Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Telomerase; Transforming Growth Factor beta; Treatment Outcome; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Although a majority of metastatic prostate cancer lesions are osteoblastic in nature, some are mixed or lytic; and, osteoblastic lesions require osteolytic activity in order to progress. The role of BMPs in the formation of prostate cancer metastases to bone remains unknown. We hypothesized that BMPs influence the development and progression of osteolytic prostate cancer lesions.. RT-PCR and Western blot analysis were used to determine BMP receptor expression on the osteolytic prostate cancer cell line PC-3. Migration, invasion, and cellular proliferation assays were performed on PC-3 cells to quantify the effects of BMP-2, -4, and -7. In vivo, PC-3 cells were injected alone, with an empty retroviral vector, or with a retroviral vector overexpressing noggin, into the tibias of SCID mice. The animals were followed for 8 weeks, and histologic and radiographic analysis were performed at 2, 4, 6, and 8 weeks.. BMP receptors are expressed on PC-3 cells, suggesting that they would be responsive to host BMP secretion. BMP-2, and to a lesser extent, BMP-4, stimulated PC-3 cell migration and invasion in a dose-dependent fashion. Noggin inhibited cellular migration and invasion of BMP-2 and -4 stimulated PC-3 cells. BMP-2 alone stimulated PC-3 cell proliferation, but BMP-4 had no effect. BMP-7 had no effect on proliferation, migration, or invasion. PC-3 cells implanted into SCID mouse tibias formed osteolytic lesions as early as 2 weeks and completely destroyed the proximal tibia by 8 weeks. Overexpression of noggin in PC-3 cells inhibited the expansion of the lesion in vivo.. BMPs influence the formation of the osteolytic prostate cancer metastases, and treatment modalities that inhibit BMP activity may limit the progression of the lytic component of prostate cancer metastases.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Bone Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Gene Expression; Humans; Male; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Osteoclasts; Osteolysis; Prostatic Neoplasms; Rats; Transfection; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.

Topics: Animals; Cadherins; Cell Line, Tumor; Female; Lymph Nodes; Matrix Metalloproteinases; Mice; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Metastasis; Nitric Oxide Synthase Type II; Platelet Endothelial Cell Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinases; Tongue Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vaccination; Vaccinia virus; Vascular Endothelial Growth Factor Receptor-2; Viral Vaccines

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, exerts contradictory roles in different kinds of cells. A number of studies have revealed its involvement in the progression of many types of tumors. To investigate the effect of TGF-beta on gastric carcinoma, SGC7901, BGC823 and MKN28 (a TGF-beta-resistant cell line) adenocarcinoma clones were used. After pretreatment in serum-free medium with or without 10 ng/ml TGF-beta1, their experimental metastatic potential, chemotaxis, and invasive and adhesive ability were measured. Furthermore, zymography for gelatinase was processed. Liver colonies were also measured 4 weeks after inoculation of SGC7901, BGC823 and MKN28 in Balb/c nude mice, and an increase in the number of surface liver metastases was seen in SGC7901 (from 11.0+/-3.0 to 53.3+/-3.3) and BGC823 (from 9.3+/-2.5 to 60.0+/-2.8) groups, whereas there was no difference between MKN28 groups (from 35.2+/-3.8 to 38.5+/-2.7). In vitro experiments showed that TGF-beta1 increased the adhesion capacity of SGC7901 and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted their penetration through reconstituted basement membrane barriers. Zymography demonstrated that enhanced invasive potential was partly due to the increased type IV collagenolytic (gelatinolytic) activity, but there was no difference in type IV collagenolytic activity and other biological behaviors between MKN28 groups. These results suggested that TGF-beta1 might modulate the metastatic potential of gastric cancer cells by promoting their ability to break down and penetrate basement membrane barriers and their adhesive and motile activities. We speculated that TGF-beta1 might act as a progression-enhancing factor in gastric cancer. Therefore blockage of TGF-beta or TGF-beta signaling might prevent gastric cancer cells from invading and metastasizing.

Topics: Animals; Basement Membrane; Cell Adhesion; Collagenases; Fibronectins; Gelatinases; Liver Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Stomach Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

To determine the expression of bone morphogenetic protein 2 and 4 (BMP-2 and BMP-4) in prostatic carcinoma (PCa) and investigate their relationship with clinical stage and Gleason score of tumor.. Forty-eight PCa cases and 5 normal prostatic tissue were analysed for the expressions of BMP-2 and BMP-4 by Western bolt assay.. The optical densities of BMP-2 expressions in the tumor with Gleason score < or =5, 6-8, and > or = 9 were 7547.1 +/- 1964.12, 9657.4 +/- 2010.54, 12467.7 +/- 2496.75 and of BMP-4 expressions were 5174.4 +/- 1400.54, 5940.3 +/- 1587.42, 6332.1 +/- 1647.83, respectively. The optical densities of BMP-2 expressions in the tumor in T1 - T2 and T3 - T4 stages were 8003.37 +/- 1889.23, 12385.55 +/- 2506.72 and of BMP4 expressions were 5267.41 +/- 1 464.19, 6543.75 +/- 1668.46, respectively. There were significant differences between tissues with Gleason score < or =5 and > or =9 (P <0.01), and tissues in T1 - T2 and T3 - T4 stages, in expressions of BMP-2 protein. The expression of BMP-2 protein was significantly high in the PCa with bone metastasis compared with that without bone metastasis.. The expressions of BMP-2 and BMP-4 increase with the progression of clinical stage and Gleason score compared with normal prostatic tissue. The expression of BMP-2 protein is significantly upregulated in bone metastasis of PCa, which indicates a poor prognosis.

Topics: Aged; Aged, 80 and over; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Prostate; 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

Since modern treatment of liver metastases includes serial hepatectomies and portal vein obstruction, we investigated the effects of portal vein ligation (PVL) and partial hepatectomy (PH) on tumor growth.. The effects of 70% PH and PVL on liver and lung metastases were evaluated in mice. Tumor growth and liver regeneration were assessed by morphometry and immunohistochemistry for PCNA and BrdU. The effect of growth factors of liver regeneration on CT-26 cells was tested in vitro, and TGF-beta secretion of CT-26 cells was measured by ELISA. RNA synthesis of TGF-beta and activin A was measured by RT-PCR.. Liver regeneration after PH and PVL was similar in tumor-free mice. Intrahepatic tumor growth was lower after PH than after PVL (p=0.016). Extrahepatic tumor growth was not different. In contrast to PVL, liver regeneration was delayed after PH in metastatic livers (p=0.001). Tested growth factors of liver regeneration stimulated CT-26 cells in vitro, and CT-26 secreted significant amounts of TGF-beta in vitro and in vivo.. Although similar in tumor-free mice, liver regeneration significantly differed between PVL and PH in metastatic livers. In addition, PH and PVL differently affected intrahepatic tumor growth.

Topics: Animals; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Hepatectomy; Liver Neoplasms; Liver Regeneration; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Portal Vein; Transforming Growth Factor beta

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

Immunosuppressed individuals undergoing organ transplantation are at increased risk of recurrences of initial cancers, although how immunosuppressive therapy increases early cancer metastasis remains unclear.. The metastatic fate of luciferase-expressing rat metastatic colon cancer cells (luc-RCN-H4) injected intravenously into the liver of syngeneic and allogeneic rats was examined in the presence of the immunosuppressant cyclosporin A (CsA) by in vivo luminescent technique. With respect to potential tumor-progressing factors, contribution of chemokine receptors and transforming growth factor (TGF)-beta1 to early metastasis was evaluated using their specific signaling inhibitors.. F344 rats injected in the liver with luc-RCN-H4 cells did not always exhibit the formation of tumors and showed a dormant state as long as 60 days after inoculation without CsA. However, CsA released early luc-RCN-H4 cells from dormancy within 2 weeks at nearly 100% in liver and preferentially promoted metastasis to the lymph nodes (approximately 40%). A similar dissemination occurred even in minor histocompatibility complex-disparate hosts. As a tumor-progressing factor, RCN-H4 cells aberrantly expressed chemokine receptors CXCR4 and CCR7. The chemokine receptor (CXC) R4-specific antagonist AMD3100 decreased early metastasis of luc-RCN-H4 cells in rats with ischemic liver conditions (P<0.05), but CsA treatment did not enhance early adhesion. Use of CsA was able to facilitate TGF-beta1 expression and the subsequent TGF-beta-mediated random migration was blocked by the use of the specific signaling inhibitor SB431542 in vitro.. Whereas the chemokine receptor expression by cancer cells is implicated with early organotropic dissemination even under CsA-mediated immune suppression, rather, CsA enhances the late-phase progression after tumor adhesion through TGF-beta1 expression.

Topics: Adenocarcinoma; Animals; Benzamides; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclosporine; Dioxoles; Disease Progression; Gene Expression Regulation, Neoplastic; Image Processing, Computer-Assisted; Killer Cells, Natural; Liver Neoplasms; Luminescence; Lymphatic Metastasis; Male; Neoplasm Metastasis; Rats; Rats, Inbred F344; Receptors, Chemokine; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Topics: Animals; Antibodies, Monoclonal; Collagen; Disease Models, Animal; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Integrin-Binding Sialoprotein; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Sialoglycoproteins; Transforming Growth Factor beta

To determine the expression of Smad4 and TGF-beta1 in bladder transitional cell carcinoma (BTCC), and to understand the mechanism of invasion, angiogenesis, and metastasis of BTCC.. The expressions of Smad4 and TGF-beta1 in samples of 42 human bladder carcinoma and 12 normal bladder mucosa tissues were determined with standard immunohistochemical analysis. We also analyzed the relationship among the expressions of Smad4 and TGF-beta1 and invasion, angiogenesis, and metastasis of BTCC, and the correlation between Smad4 and TGF-beta1.. The positive rate of Smad4 in BTCC was significantly lower than those in normal bladder mucosa tissues (33.3% vs 83.3%, P < 0.01). The expressions of Smad4 in poorly differentiated, invasive, recurrent, or with lymph node metastasis of BTCCs were lower than those in well differentiated, superficial, nonrecurrent, or without lymph node metastasis ones (P <0.05). The positive rate of TGF-beta1 in BTCC was significantly lower than that in normal bladder mucosa tissues (64.3% vs 100%, P <0.01), which was positively correlated to that of Smad4 (P = 0.000).. The expressions of Smad4 and TGF-beta1 in BTCC decrease with the increase in clinical stage, poor pathological grade, and the recurrence and metastasis of BTCC.

Topics: Adult; Aged; Carcinoma, Transitional Cell; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Smad4 Protein; Transforming Growth Factor beta; Urinary Bladder Neoplasms

In this paper we present a two-compartment model for tumor dormancy based on an idea of Zetter [1998, Ann. Rev. Med. 49, 407-422] to wit: The vascularization of a secondary (daughter) tumor can be suppressed by an inhibitor originating from a larger primary (mother) tumor. We apply this idea at the avascular level to develop a model for the remote suppression of secondary avascular tumors via the secretion of primary avascular tumor inhibitors. The model gives good agreement with the observations of [De Giorgi et al., 2003, Derm. Surgery 29, 664-667]. These authors reported on the emergence of a polypoid melanoma at a site remote from a primary polypoid melanoma after excision of the latter. The authors observed no recurrence of the melanoma at the primary site, but did observe secondary tumors at secondary sites 5-7 cm from the primary site within a period of 1 month after the excision of the primary site. We attempt to provide a reasonable biochemical/cell biological model for this phenomenon. We show that when the tumors are sufficiently remote, the primary tumor will not influence the secondary tumor while, if they are too close together, the primary tumor can effectively prevent the growth of the secondary tumor, even after it is removed. It should be possible to use the model as the basis for a testable hypothesis.

Topics: Algorithms; Cell Movement; Cell Proliferation; Computer Simulation; Enzymes; Fibrinolysin; Fibroblast Growth Factor 2; Humans; Kinetics; Melanoma; Models, Biological; Neoplasm Metastasis; Neoplasms; Plasminogen; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

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

Erk/MAPK and TGFbeta signaling cause epithelial to mesenchymal transition (EMT) and metastasis in mouse mammary epithelial cells (EpH4) transformed with oncogenic Ras (EpRas). In trials to unravel underlying mechanisms, expression profiling for EMT-specific genes identified a secreted interleukin-related protein (ILEI), upregulated exclusively at the translational level. Stable overexpression of ILEI in EpH4 and EpRas cells caused EMT, tumor growth, and metastasis, independent of TGFbeta-R signaling and enhanced by Bcl2. RNAi-mediated knockdown of ILEI in EpRas cells before and after EMT (EpRasXT) prevented and reverted TGFbeta-dependent EMT, also abrogating metastasis formation. ILEI is overexpressed and/or altered in intracellular localization in multiple human tumors, an event strongly correlated to invasion/EMT, metastasis formation, and survival in human colon and breast cancer.

Topics: Animals; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cytokines; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Neoplasms; Prognosis; Protein Biosynthesis; RNA Interference; RNA, Messenger; Signal Transduction; Survival Rate; Time Factors; 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

We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin beta3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin beta3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin beta3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin alphav subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin beta3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin beta3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin beta3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin alphav beta3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Homeodomain Proteins; Humans; Integrin alphaVbeta3; Lung Neoplasms; Mice; Neoplasm Metastasis; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Wound Healing

Cell-matrix adhesive interaction has an important role in the invasive process of tumor cells, and integrins are the major receptors mediating cell-matrix adhesion. The current study is to investigate the modulation of basement membrane (BM) proteins, especially collagen IV (C IV), laminin (LN), and fibronectin (FN) in the invasive processes of human hepatocellular carcinoma (HCC) cells in vitro, and to reveal the roles of beta1 integrins and RGD-containing oligopeptide in the cell-matrix interaction.. Static adhesion assay was performed to study the rates of adhesion of MHCC97-H cells, treated or untreated with anti-beta1 (2 microg ml(-1)) and GRGDS, to C IV (50 microg ml(-1)), LN (50 microg ml(-1)) or FN (50 microg ml(-1)). Gelatin zymography was used to detect the secretion of MMPs in the conditioned medium of MHCC97-H cells incubated 24 h by C IV, LN or FN, and interactions with anti-beta1 and GRGDS. Transwell chamber assay was used to investigate the influence of C IV, LN or FN, interacting with anti-beta1 and GRGDS, on the cellular mobility of MHCC97-H cells.. Compared with blank control group, MHCC97-H cells showed significantly higher rates of adhesion to C IV, LN, and FN. Pretreatment with anti-beta1 could suppress adhesion to C IV, LN or FN, but GRGDS inhibited adhesion to FN (P<0.05) only. LN and FN could stimulate the secretion of MMPs by MHCC97-H cells cultured in vitro, especially MMP-9 and its activated type. Treatment with anti-beta1 could partly counteract the effects of LN and FN. GRGDS could prominently induce the secretion of MMPs, but the effect could be inhibited by pretreatment of anti-beta1. The results of Transwell chamber assay showed that LN, FN, and GRGDS could increase the number of tumor cells penetrating the microporous membrane, but the data of C IV did not reach significance. The effects were partly counteracted by anti-beta1.. BM proteins play an active role in the invasive process of human hepatocellular carcinoma cells. Integrin beta1 is an important molecule which mediates the cell-matrix adhesive interaction of tumor cells. RGD-containing peptides competitively combine with the binding site of integrin beta1, and the effects of FN are RGD sequence-dependent.

Topics: Basement Membrane; Carcinoma, Hepatocellular; Cell Adhesion; Collagen Type IV; Extracellular Matrix Proteins; Fibronectins; Humans; Integrin beta Chains; Laminin; Liver Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

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

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

We investigated the role of transforming growth factor-beta (TGF-beta) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-beta receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-beta-induced cell growth inhibition and TGF-beta1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen-positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-beta signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.

Topics: Androgens; Animals; Blotting, Northern; Cell Line, Tumor; Cell Proliferation; Disease Progression; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Immunohistochemistry; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasm Metastasis; Platelet Endothelial Cell Adhesion Molecule-1; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-beta (TGF-beta) in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-beta receptor kinase inhibitors (TRKI) are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-beta, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-beta-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-beta, including TGF-beta-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-beta, whereas it reduces colony formation by cells that are growth-promoted by TGF-beta. However, SB-431542 has no effect on a cell line that failed to respond to TGF-beta. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-beta-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-beta.

Topics: Activin Receptors, Type I; Animals; Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Movement; Dioxoles; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Mice; Mink; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Time Factors; Transcription, Genetic; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

We previously identified constitutive Smad signaling in human melanoma cells despite resistance to transforming growth factor-beta (TGF-beta) control of cell proliferation. This led us to investigate the effect of inhibitory Smad7 overexpression on melanoma cell behavior. Using the highly metastatic cell line, 1205-Lu, we thus generated melanoma cell clones constitutively expressing Smad7, and their mock-transfected counterparts. Stable expression of Smad7 resulted in an inhibition of constitutive Smad2/3 phosphorylation, and in a reduced TGF-beta response of Smad3/Smad4-driven gene transactivation, as measured using transfected Smad3/4-specific reporter gene constructs. Smad7 overexpression, however, did not alter their proliferative capacity and resistance to TGF-beta-driven growth inhibition. On the other hand, expression of Smad7 efficiently reduced the capacity of human melanoma cells to invade Matrigel in Boyden migration chambers, while not affecting their motility and adhesion to collagen and laminin. Gelatin zymography identified reduced MMP-2 and MMP-9 secretion by Smad7-expressing melanoma cells as compared with their control counterparts. Smad7-expressing melanoma cells exhibited a dramatically reduced capacity to form colonies under anchorage-independent culture conditions, and, when injected subcutaneously into nude mice, were largely delayed in their ability to form tumors. These results suggest that TGF-beta production by melanoma cells not only affects the tumor environment but also directly contributes to tumor cell aggressiveness through autocrine activation of Smad signaling.

Topics: Animals; Cell Adhesion; Cell Movement; Gene Expression Profiling; Humans; Matrix Metalloproteinase 9; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Skin Neoplasms; Smad7 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Angiogenesis and tumor-associated immunosuppression are two of the hallmarks of carcinogenesis. In previous studies we demonstrated in vitro that HNSCC tumor cells attract monocytes via monocyte chemotactic protein-1 (MCP-1) and activate them via transforming growth factor-beta 1(TGF-beta1) to secrete interleukin (IL)-1alpha, which in turn stimulates tumor cells to secrete increased levels of the angiogenic and immunosuppressive vascular endothelial growth factor (VEGF). These findings suggest that interaction between the immune system and VEGF-mediated angiogenesis is important for progression of HNSCC. Recent studies in vitro show that retinoic acid (RA) downregulates the release of MCP-1 and TGF-beta1 by tumor cells. Therefore, we investigated the ability of RA to modulate the ability of tumor cells to recruit and activate monocytes for participation in VEGF-mediated angiogenesis and immunosuppression in vivo.. Mice (ten/group) were injected daily with RA (160 microg/kg) for 3 weeks. After that time mice were sacrificed, and paraffin sections of tumors were obtained and stained for VEGF-A, CD68, and PECAM (CD31) by immunohistochemistry. The lungs, liver, and myocardium were analyzed for macro- and micrometastases. The plasma protein levels of VEGF-A and MCP-1 were determined by ELISA.. In RA-treated mice tumors regressed completely and RA prevented metastases (p=0.00) and macrophage infiltration (p=0.007). Treated mice downregulated VEGF-A (0 pg/ml) and MCP-1 (12 pg/ml) in peripheral blood (p=0.001).. Our findings suggest a new therapeutic possibility: the development of treatment protocols that can block each of the ways in which tumors induce new blood vessel growth and immunosuppression of the host.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokine CCL2; Disease Progression; Down-Regulation; Humans; Immune Tolerance; Interleukin-1; Macrophage Activation; Male; Mice; Mice, Inbred A; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Transplantation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Vascular Endothelial Growth Factor A

Tumor-stroma interactions play a decisive role in the growth and metastasis of solid tumors, and involve signalling either by soluble mediators or direct cell-cell interaction. Here, we report the isolation and characterisation of a novel cDNA (MEL4B3), which is induced in cultured dermal fibroblasts exposed to supernatants of melanoma cell lines. MEL4B3 shares high homology with two predicted cDNA sequences for which no activity has so far been described. In situ hybridisation revealed the expression of MEL4B3 in malignant melanoma increasing with tumor depth; in basal cell carcinoma and in squamous cell carcinoma. MEL4B3 was barely detectable in normal skin or non-malignant melanocytic naevi. Furthermore, MEL4B3 was expressed at high level in the epidermis of psoriatic skin. In vitro, the expression of MEL4B3 was found to be induced by the exposure of human dermal fibroblasts to melanoma cell culture supernatants or to transforming growth factor-beta, interleukin-1 and tumor necrosis factor-alpha. The expression MEL4B3 therefore reflects closely cell activation occurring during tumor growth, metastasis and inflammation.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cells, Cultured; Culture Media; Culture Media, Conditioned; Cytokines; DNA, Complementary; Fibroblasts; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Inflammation; Keratinocytes; Microcirculation; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Complementary; RNA, Messenger; Skin; Skin Neoplasms; Transforming Growth Factor beta

Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on the Rath multitargeted approach to cancer using natural substances to control ECM stability and enhancing its strength, we developed a novel formulation (NM) of lysine, proline, ascorbic acid and green tea extract that has shown significant anti-cancer activity against a number of cancer cell lines. The aim of the present study was to determine whether NM exhibits anti-angiogenic and anti-metastatic effects using in vitro and in vivo experimental models. Angiogenesis was measured using a chorioallantoic membrane (CAM) assay in chick embryos and bFGF-induced vessel growth in C57BL/6J female mice. To determine the in vivo effect of NM on the tumor xenograft growth, male nude mice were inoculated with 3 x 10(6) MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed a mouse chow diet supplemented with 0.5% NM for 4 weeks. In vitro studies on cell proliferation (MTT assay), MMP expression (zymography) and Matrigel invasion were conducted on human osteosarcoma U2OS, maintained in McCoy medium, supplemented with 10% FBS, penicillin and streptomycin in 24-well tissue culture plates and tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose. NM at 250 microg/ml caused a significant (p<0.05) reduction in bFGF-induced angiogenesis in CAM. NM inhibited tumor growth of osteosarcoma MNNG-HOS cell xenografts in nude mice by 53%; furthermore, tumors in NM-treated mice were less vascular and expressed lower levels of VEGF and MMP-9 immunohistochemically than tumors in the control group. In addition, NM exhibited a dose-dependent inhibition of osteosarcoma U2OS cell proliferation (up to 60% at 1000 microg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 microg/ml NM), and invasion through Matrigel (with total inhibition at 100 microg/ml NM). Moreover, NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGFbeta-1. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation, which inhibits growth, invasion, metastasis, and angiogenesis of tumor cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Extracellular Matrix; Fibroblast Growth Factor 2; Formaldehyde; Humans; Immunohistochemistry; In Vitro Techniques; Laminin; Lysine; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Osteosarcoma; Plant Extracts; Platelet-Derived Growth Factor; Proline; Proteoglycans; Tea; Tetrazolium Salts; Thiazoles; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

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

Topics: Animals; Cell Adhesion; Cell Movement; Fibronectins; Hematopoietic Stem Cells; Humans; Integrins; Mice; Neoplasm Metastasis; Neoplasms; Organ Specificity; Transforming Growth Factor beta; Vascular Endothelial Growth Factor Receptor-1

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.

Topics: Blotting, Northern; Blotting, Western; Cell Division; Cell Line, Tumor; Densitometry; DNA, Complementary; Down-Regulation; Enzyme Inhibitors; Female; Humans; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Oligonucleotides, Antisense; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Time Factors; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Kunitz Soybean; Up-Regulation; Urokinase-Type Plasminogen Activator

Runx transcription factors are important regulators of lineage specific gene expression, cell proliferation, and differentiation. Runx3 expression is lost in a high proportion of gastric cancers, suggesting a tumour suppressive role in this malignancy. This study investigates the expression and localisation of Runx3 in pancreatic tissues.. Quantitative polymerase chain reaction was used to measure Runx3 mRNA. Immunohistochemistry was carried out to localise Runx3 in normal pancreatic tissues, and in primary and metastatic pancreatic ductal adenocarcinoma (PDAC). Basal and transforming growth factor beta1 (TGFbeta1) induced Runx3 expression was analysed in cultured pancreatic cancer cell lines.. Runx3 expression was low to absent in normal pancreatic tissues, but increased in a third of cancer tissues. Runx3 was present only in islets in normal pancreas, whereas in pancreatic cancers, Runx3 was detected in the cancer cells of seven of 24 samples analysed. In addition, it was expressed by lymphocytes in six of the 16 cases with lymphocyte infiltration. In pancreatic cancer cell lines, Runx3 mRNA was present in Colo-357 and T3M4 cells, but was low to absent in the other cell lines tested. TGFbeta1 repressed Runx3 mRNA expressed in Colo-357 cells, and had no effect on Runx3 expression in the other pancreatic cancer cell lines.. Runx3 expression is restricted to islets in the normal pancreas. In contrast, a considerable proportion of pancreatic tumours express Runx3, and its expression is localised in the tumour cells and in the infiltrating lymphocytes. Thus, Runx3 might play a role in the pathogenesis of PDAC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Blotting, Western; Core Binding Factor Alpha 3 Subunit; DNA-Binding Proteins; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Islets of Langerhans; Lymphocytes; Male; Middle Aged; Neoplasm Metastasis; Pancreas; Pancreatic Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

We reported that orthotopic xenograft of human gallbladder cancer (Mz-ChA-2) produced a greater amount of endogenous angiogenic inhibitory factors, however, only TGFbeta1 suppressed angiogenesis and tumor growth at the distant site (intracranium). The aim of this study was to confirm the validity of our previous findings that the site of the primary tumor would influence the angiogenesis in the distant site in a different xenograft of human gallbladder cancer (Mz-ChA-1). The growth rates, histology of the ectopic (flank) and orthotopic (gallbladder) xenografts, the plasma level of TGFbeta1, micro-circulation and angiogenesis in the distant site (intracranium) were estimated by size-measurement, hematoxylin and eosin staining, ELISA, intravital fluorescence microscopic observation and cranial window gel assay for angiogenesis. All experiments were performed in severe combined immunodeficient (SCID) mice. Orthotopic tumors grew faster and were less necrotic than ectopic tumors. Angiogenesis, vessel diameters, vessel density and leukocyte-rolling count in the distant site were significantly decreased in orthotopic tumor-bearing mice compared to those in either ectopic or no tumor-bearing mice. The plasma level of TGFbeta1 was significantly elevated in mice bearing orthotopic tumor as compared with ectopic and no tumor-bearing mice. Angiogenesis at the distant site was inhibited by the orthotopic xenograft of Mz-ChA-1 by the greatest amount of TGFbeta1 production. The results of the present study together with our previous study imply that the primary tumor microenvironment is conducive to the angiogenesis at a distant site by the production of the endogenous angiogenesis inhibitor TGFbeta1.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Gallbladder Neoplasms; Humans; Leukocyte Rolling; Mice; Mice, SCID; Neoplasm Metastasis; Neovascularization, Pathologic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

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

To understand alterations to the urokinase system that may occur in progressively metastatic prostate cancer cells, we assessed urokinase plasminogen activator receptor (uPAR) expression, in vitro motility towards vitronectin, urokinase plasminogen activator (uPA)-induced growth and growth factor regulation of uPAR expression in three cell lines--PC-3 and two derivatives from secondary metastases, PC-3M and PC-3MM2. DU-145 and Tsu-Pr1 cells were included for comparative purposes. uPAR expression increases with metastatic passage in these cell lines and accompanies increased growth and motility responses in the presence of uPA. Growth factors TGFbeta1 and IGF-1 induce uPAR in all three prostate cancer lines; however, PC-3M and PC-3MM2 cells also respond to bFGF. Of the cell lines tested, PC-3MM2 most uniformly respond to added TGFbeta1, IGF-1 and bFGF. These results show that in two progressive derivatives from repeated metastasis of PC-3 cells, constitutive and growth factor-induced uPAR expression is enhanced. This increased uPAR facilitates the properties of growth and motility.

Topics: Animals; Cell Division; Cell Line, Tumor; Cell Movement; Fibroblast Growth Factor 2; Insulin-Like Growth Factor I; Male; Mice; Mice, Nude; Neoplasm Metastasis; Plasminogen Activators; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator

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

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

Transforming growth factor (TGF) beta mediates a tumor suppressor pathway in human colon epithelial cells. We were interested in identifying and characterizing novel genes regulated by the TGF beta pathway in the colon. We employed expression microarrays to identify transcripts induced by TGF beta in Vaco 330, a colon adenoma cell line. We then used expression microarrays to determine which of these TGF beta induced transcripts are down-regulated in metastatic colon cancer. Northern analysis and real-time reverse transcription PCR confirmed and quantified our findings from the microarrays. These analyses highlighted C8orf4 as induced by TGF beta in colon cells. Moreover, C8orf4 is expressed in most normal colon mucosa samples, and is not expressed in most colon cancer metastases or colon cancer cell lines. Colon cancer primary tumors showed reduced expression of C8orf4 relative to normal mucosa, possibly reflecting contributions of C8orf4 expression in stromal cells. C8orf4 is a gene regulated by TGF beta signaling and loss in advanced colon cancer suggests C8orf4 may play a role in colon cell differentiation or growth regulation.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Gene Expression Profiling; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stromal Cells; Transcription, Genetic; Transforming Growth Factor beta

Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors.. We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines.. N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change.. This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Topics: Aged; Blotting, Western; Cadherins; Cell Adhesion; Cell Proliferation; Epithelium; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Mesoderm; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreas; Pancreatic Neoplasms; Time Factors; Transforming Growth Factor beta; Up-Regulation; Vimentin

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

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.

Topics: Angiogenesis Inducing Agents; Angiogenic Proteins; Gene Expression Regulation, Neoplastic; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Prostatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Cells, Cultured

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

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.

Topics: Actinin; Actins; Animals; Calcium-Binding Proteins; Calponins; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Humans; MAP Kinase Signaling System; Mice; Microfilament Proteins; Molecular Sequence Data; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; ras Proteins; RNA, Small Interfering; Smad Proteins; Stress Fibers; Trans-Activators; Transforming Growth Factor beta; Tropomyosin

We have shown recently that the hyaluronan receptor, CD44, and matrix metalloproteinase 9 (MMP-9) form a complex on the surface of TA/St mouse mammary carcinoma cells that activates latent transforming growth factor-beta (TGF-beta) and is required for tumor invasion. Disruption of the CD44/MMP-9 complex by expression of soluble CD44 results in the loss of tumor invasiveness and abrogates tumor cell survival in host lung parenchyma following intravenous injection into syngeneic mice. To explore the molecular nature of the survival signals derived from the CD44/MMP-9 complex during the development of tumor metastasis, we investigated the possibility that activation of latent TGF-beta by the CD44/MMP-9 complex is responsible for tumor cell survival in host lung parenchyma. TA3 cells overexpressing dominant negative soluble CD44 (TA3sCD44), which compromises native CD44 function and the ability of TA3 cells to develop metastases, were transfected with constitutively active or latent TGF-beta2 and tested for their ability to form tumors in syngeneic mice. Our results demonstrate that expression of the constitutively active, but not the latent, form of TGF-beta2 rescues TA3sCD44 cells from apoptosis during lung colonization. These observations provide evidence that activation of latent TGF-beta constitutes an event downstream of CD44-dependent signals that is required for tumor cell survival and metastatic colony formation. The functional axis composed of CD44, MMP-9 and TGF-beta may therefore play an important role in the metastatic proclivity of selected tumor types.

Topics: Apoptosis; Base Sequence; Cell Survival; DNA Primers; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Transforming Growth Factor beta

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

Topics: Animals; Cell Movement; DNA-Binding Proteins; DNA, Antisense; Female; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neoplasm Metastasis; Oncogenes; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Antibodies; Bone Neoplasms; Cell Line, Tumor; Disease Models, Animal; Lung Neoplasms; Mice; Mice, Inbred Strains; Neoplasm Metastasis; Species Specificity; Transforming Growth Factor beta; Treatment Outcome

A pilot study was conducted to assess whether plasma levels of transforming growth factor-beta1 (TGF-beta1) might facilitate biological subgrouping of postmenopausal metastatic breast cancer patients, and, accordingly, its applicability in clinical oncology. This study included 29 postmenopausal metastatic breast cancer patients. Plasma TGF-beta1 levels were detected by enzyme-linked immunosorbent assay (ELISA). Estrogen and progesterone receptors were assayed by radioligand binding, in accordance with the recommendation of the EORTC. Concentrations of 17-beta estradiol were determined by using ELISA-microwell method (DIALAB). Overall survival was followed for 24 months for each individual patient. Stratification of the patients by ER/PR status showed that 14 patients with estrogen receptor-negative, progesterone receptor-negative carcinomas displayed a statistically significant increase in plasma TGF-beta1 levels when compared to plasma TGF-beta1 levels of 6 patients with ER-positive, PR-positive carcinomas (P=0.04). In this study, 7 out of 14 patients with negative receptors' status had no plasma TGF-beta1 values overlapping with patients having positive receptors' status. The TGF-beta1 cut-off value was defined as the highest plasma TGF-beta1 level of ER-positive, PR-positive patients: 3.28 ng/ml. This plasma TGF-beta1 cut-off value defined low-risk subgroup of 19 patients (< or = 3.28 ng/ml) and high-risk subgroup of 10 patients (> 3.28 ng/ml) (P=0.047). Plasma TGF-beta1-related survival was independent of the classical prognostic factors of metastatic breast cancer. Accordingly, a clinical significance of elevated plasma TGF-beta1 levels may be suggested.

Topics: Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Estradiol; Female; Humans; Neoplasm Metastasis; Postmenopause; Radioligand Assay; Receptors, Estrogen; Receptors, Progesterone; Survival Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta1

TGFbeta signaling controls a plethora of cellular responses in human development and disease. Recent cellular, biochemical, and structural studies have revealed significant insight into the mechanisms of the activation of TGFbeta receptors, the receptor-mediated activation of Smad transcription factors, the Smad-mediated regulation of target gene expression, and the negative control of the cell cycle by these signals. Loss of TGFbeta cytostatic responsiveness and gain of metastastic activity are common alterations of this pathway in cancer. The analysis of these normal and altered states is providing new insights into physiology, pathology and therapy.

Topics: Animals; Cell Cycle; Humans; Neoplasm Metastasis; Transforming Growth Factor beta

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Adhesion; Cell Movement; Chemokines; Embryo, Nonmammalian; Gene Expression Profiling; Genes, Tumor Suppressor; Humans; Neoplasm Metastasis; Neoplasm Seeding; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Receptors, Chemokine; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the tumor growth factor beta (TGF-beta) superfamily. Several observations suggest that it plays a role in colorectal carcinoma (CRC). In particular, MIC-1 is markedly up-regulated in colorectal cancers as well as in premalignant adenomas. This study examines the relationship of serum MIC-1 levels and genotypes to clinical and pathologic features of colonic neoplasia.. We confirmed the presence of MIC-1 in CRC tissue and the cell line CaCo-2. The normal range for serum MIC-1 levels was defined in 260 healthy blood donors, and the differences between normal subjects and 193 patients having adenomatous polyps or CRC were then determined. In a separate cohort of 224 patients, we evaluated the relationship of MIC-1 serum level and genotype to standard tumor parameters and outcome measures.. MIC-1 was expressed in CRC tissue and the cancer cell line CaCo-2. There was a progressive increase in serum MIC-1 levels between normal individuals [mean (M) = 495 pg/ml, SD = 210), those with adenomatous polyps (M = 681 pg/ml, SD = 410), and those with CRC (M = 783 pg/ml, SD = 491)]. Serum MIC-1 level was correlated with the extent of disease so that the levels were higher in patients with higher Tumor-Node-Metastasis stage. There were significant differences in time to relapse and overall survival between subjects with different MIC-1 levels and genotypes.. This study identifies a strong association between MIC-1 serum levels and neoplastic progression within the large bowel. We suggest that the measurement of serum MIC-1 levels and determination of MIC-1 genotype may have clinical use in the management of patients with CRC.

Topics: Adenoma; Adenomatous Polyps; Alleles; Carcinoma; Cell Line, Tumor; Cohort Studies; Colorectal Neoplasms; Cytokines; Disease-Free Survival; Female; Genotype; Growth Differentiation Factor 15; Humans; Immunohistochemistry; Logistic Models; Lymphocytes; Male; Neoplasm Metastasis; Prognosis; Time Factors; Transforming Growth Factor beta; Treatment Outcome; Up-Regulation

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and beta-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-beta increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

Topics: Animals; beta Catenin; Blotting, Northern; Blotting, Western; Cadherins; Cell Line, Tumor; Cytoskeletal Proteins; Disease Models, Animal; Female; Glypicans; Heparan Sulfate Proteoglycans; Immunohistochemistry; Insulin-Like Growth Factor II; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Rats; Trans-Activators; Transfection; Transforming Growth Factor beta

Immunosuppressive therapy is a risk factor for the increased incidence and metastatic progression of malignancies in organ graft recipients. Transforming growth factor (TGF)-beta(1) has been associated with tumor invasion and metastasis, and we have implicated cyclosporine-associated TGF-beta(1) hyperexpression in tumor progression in mice.. BALB/c mice or severe combined immunodeficient-beige mice were treated with 2 or 4 mg/kg of tacrolimus, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. We also determined whether tacrolimus induces TGF-beta(1) expression. Spleens from tacrolimus-treated mice were analyzed for level of expression of TGF-beta(1) mRNA with the use of competitive-quantitative polymerase chain reaction assay, and circulating levels of TGF-beta(1) protein were measured with the use of an enzyme-linked immunosorbent assay.. Treatment with tacrolimus resulted in a dose-dependent increase in the number of pulmonary metastases in the BALB/c mice (197+/-16 in untreated mice, 281+/-26 in mice treated with 2 mg/kg of tacrolimus, and 339+/-25 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, Bonferroni's P<0.001) and in the severe combined immunodeficient-beige mice (117+/-18 in untreated mice, 137+/-19 in mice treated with 2 mg/kg of tacrolimus, and 216+/-29 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, P<0.05). Treatment with 4 mg/kg but not 2 mg/kg of tacrolimus resulted in a significant increase in the levels of expression of TGF-beta(1) mRNA and circulating levels of TGF-beta(1) protein.. Tacrolimus has a dose-dependent effect on tumor progression and TGF-beta(1) expression, and tacrolimus-induced TGF-beta(1) overexpression may be a pathogenetic mechanism in tumor progression.

Topics: Animals; Carcinoma, Renal Cell; Disease Progression; Dose-Response Relationship, Drug; Immunosuppressive Agents; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Metastasis; Neoplasms; Spleen; Tacrolimus; Transforming Growth Factor beta; Transforming Growth Factor beta1

We studied the expression of TGF-beta/T(beta)R system and its biological role in tumor development, in M3 and MM3 murine mammary adenocarcinomas with different metastasizing capability and in LM3 and LMM3 derived cell lines. All the studied cells secreted TGF-beta(s) and expressed T(beta)Rs. While the proliferation of the poorly metastatic M3 cells was significantly inhibited by 4 ng/ml TGF-beta(s), the highly metastatic MM3 cells were only slightly inhibited in response to the highest dose used. LM3 and LMM3 cells, highly invasive and metastatic, were totally refractory to TGF-beta antiproliferative effect. The role of TGF-beta in modulating key proteolytic cascades in tumor progression was also studied. TGF-beta(s) enhanced metalloproteinases production in all the studied cells while induced a stimulatory net effect on plasmin system activity only in the more metastatic cells. Our results in this murine mammary tumor lineage support the concept that dissociation of TGF-beta regulated growth control versus proteolytic enzyme pathways promotes tumor dissemination.

Topics: Adenocarcinoma; Animals; Cell Differentiation; Cell Division; Disease Progression; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Metalloproteases; Mice; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

The TGF-beta signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-beta can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-beta receptor into a series of genetically related human breast-derived cell lines representing different stages in the progression process. We show that decreased TGF-beta responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-beta responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-beta responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-beta switches from tumor suppressor to prometastatic factor.

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Disease Progression; Female; Humans; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.

Topics: Apoptosis; Cell Cycle Proteins; Cell Division; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Humans; Neoplasm Metastasis; NF-kappa B; Phenotype; Thyroid Gland; Thyroid Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Proteins

The degradation of basement membranes by tumor cells involves secretion and activation of proteinases, such as matrix metalloproteinases (MMPs) and the plasminogen activation system (uPA, tPA, PAI-1), and results from an imbalance between their inhibitors and activators, controlled by various growth factors or cytokines. Among them, the TGF-beta family is one of the most intriguing because it has been reported either to decrease or promote cancer progression. In the present paper, we studied the effect of TGF-beta1 in a mouse melanoma model. In vivo, TGF-beta1 inhibited tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. In vitro, TGF-beta1 did not alter B16F1 cell proliferation, but strongly decreased their migration through Matrigel-coated membranes. The protease production was analyzed by zymography, Western blot, or RT-PCR. MMP-2 and TIMP-2 expression were not altered by TGF-beta1. In contrast, TGF-beta1 triggered a large decrease of uPA and tPA, as well as a decrease of uPA and uPAR mRNAs. By Western blot and RT-PCR analyses, TGF-beta1 was shown to induce a strong increase of PAI-1 synthesis. Collectively, these results suggest that TGF-beta1 may inhibit melanoma tumor growth by specifically decreasing plasmin activity of tumor cells and play a protective role during the earliest stages of tumor progression.

Topics: Animals; Cell Division; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Down-Regulation; Female; Fibrinolysin; Matrix Metalloproteinases; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Plasminogen Activator Inhibitor 1; Plasminogen Activators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Skin Neoplasms; Tissue Plasminogen Activator; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator

Cancers of the breast, prostate, and lung commonly metastasize to the bone resulting in osteolysis, pathologic fracture, pain and significant clinical morbidity. To date, the reason for such selectivity in the site of metastasis remains largely unknown. The bone is a rich source of many chemokines and growth factors, including: insulin-like growth factor (IGF) I and II, transforming growth factor-beta (TGF-beta), interleukins, and tumour necrosis factor-alpha (TNF-alpha). We propose that exposure of breast cancer cells to the bone microenvironment results in alterations in gene expression that favour the growth and proliferation of tumour cells in the bone. To investigate this hypothesis, MDA-MB-231 breast carcinoma cells were exposed to bone-derived conditioned media (BDCM) generated by culturing fetal rat calvaria for 24 h under serum free conditions. Using cDNA microarray technology, we have identified the insulin-like growth factor family of binding proteins (IGFBPs) as genes whose expression profiles are consistently and significantly altered with exposure to this simulated bone environment in vitro, when compared to untreated controls. Our data suggests that the upregulation of IGFBP-3 seen with exposure to the bone microenvironment is directly linked to an increase in TGF-beta mediated cell proliferation. Furthermore, this process appears to be functioning through an IGF-independent mechanism.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Division; Culture Media, Conditioned; Culture Media, Serum-Free; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor II; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone metastasis from primary tumors is a clinically important complication of neoplastic progression. The role of parathyroid hormone-related protein (PTHrP) and transforming growth factor (TGF)-beta1 in this process has been clearly established. The current study describes an in vivo model of 13762 rat mammary carcinoma tumor cell-induced osteolysis in which PTHrP and TGF-beta1 expression is observed. Exposure of in vitro-cultured 13762 cells to doxorubicin, cis-platinum, carboplatin, methotrexate, 5-fluorouracil, paclitaxel, alendronate, risedronate, or pamidronate for 72 h resulted in varying effects on cell proliferation (IC(50) values of 0.005, 0.4, 1.9, >40, 17.9, 0.003, >40, >40, and 33.6 micro M, respectively). Tumor cells were implanted into the intramedullary space of the proximal tibia of rats, and the time course of tumor progression was evaluated using radiographic and microcomputed tomography scanning techniques. Trabecular bone mineral density, cortical bone mineral density, and whole bone mineral density were measured (in mg/cm(3)). In untreated animals, radiographic evidence of osteolysis was evident 7 days after implantation. Trabecular bone mineral density and whole bone mineral density were significantly decreased by 21 days after implantation (48% and 26%, respectively). Bisphosphonates showed broad protective activity against tumor-driven osteolysis, Immunohistochemical evaluation of s.c. and intratibially implanted cells demonstrated the expression of PTHrP and TGF-beta1. The results of this study demonstrate the ability of 13762 rat mammary carcinoma cells to elicit a measurable osteolysis and that bisphosphonates inhibit the tumor-induced bone resorption in this model.

Topics: Animals; Antineoplastic Agents; Bone Resorption; Cell Division; Cell Line, Tumor; Cell Survival; Diphosphonates; Disease Progression; Female; Humans; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Osteolysis; Parathyroid Hormone-Related Protein; Rats; Rats, Inbred F344; Transforming Growth Factor beta

This study was to measure the expression of TGF-beta 1 in larynx squamous carcinoma, to investigate the relationship between TGF-beta 1 and pathological grading of tumours, the clinical staging and, through which the effect of TGF-beta 1 on the pathogenesis of the larynx squamous carcinoma was to be known.. RNA was extracted from the larynx squamous carcinoma tissues of 21 patients and from normal laryngeal tissues of 15 cases. Reverse transcriptional polymerase chain reaction (RT-PCR) was applied to detect the expression of the TGF-beta 1 mRNA.. Ten of the 21 specimens of the larynx squamous carcinoma showed positive expression of TGF-beta 1 mRNA, then the expression ratio was 47.62%. The expression of TGF-beta 1 mRNA had relationship to the pathological grading and the clinical staging of the larynx carcinoma.. TGF-beta 1 can be used as a marker to evaluate the progression of laryngeal squamous cell carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Female; Humans; Laryngeal Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The role of transforming growth factor beta in breast cancer is controversial with tumor suppressor and pro-oncogenic activities having been demonstrated. To address whether the same or different signal transduction pathways mediate these opposing activities, we manipulated the Smad2/3 signaling pathway in cells of common origin but differing degrees of malignancy derived from MCF10A human breast cells. We show that interference with endogenous Smad2/3 signaling enhances the malignancy of xenografted tumors of premalignant and well-differentiated tumor cells but strongly suppresses lung metastases of more aggressive carcinoma cells after tail vein injection. Overexpression of Smad3 in the same cells has opposite effects. The data demonstrate that the Smad2/3 signaling pathway mediates tumor suppressor and prometastatic signals, depending on the cellular context.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Female; Humans; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous

Topics: Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neovascularization, Pathologic; Prostatic Neoplasms; Protein Kinase Inhibitors; Receptors, Growth Factor; Transforming Growth Factor beta

Topics: Animals; Fibroblast Growth Factors; Growth Substances; Humans; Models, Biological; Neoplasm Metastasis; Proto-Oncogene Proteins c-myc; Transforming Growth Factor beta; Wound Healing

Several cytokines including members of the transforming growth factor-beta (TGF-beta) and tumor necrosis factor (TNF) families have been implicated in the homing mechanism of breast cancer metastasis. We hypothesize that primary breast tumor tissues differentially express modulators of bone cell function and that this expression pattern contributes to their aggressive and metastatic potential and to their capacity to establish and grow in bone. We, therefore, examined the gene expression pattern of the TGF-beta family members (inhibin/activin betaA subunit (activin betaA), inhibin alpha subunit, and bone morphogenetic protein-2 (BMP-2)), the TNF family members (receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG)), and osteopontin (OPN) in normal, non-invasive, invasive, and metastatic human breast cancer specimens. The mRNA transcript levels of these genes were quantified by reverse transcription (RT) and fluorescent-based kinetic PCR in 18 normal breast tissues, five ductal carcinoma in situ (DCIS). 24 primary breast tumor tissue, and five distant metastases. The mRNA transcript level of each gene was normalized to the amount of beta-actin present in the samples. We observed differential gene expression of the selected TGF-beta family members as well as OPN in breast cancer progression. The average gene expression of the putative tumor suppressor, inhibin alpha, did not significantly change in any of the tumor tissues examined compared to normal breast tissue. The mRNA level of BMP-2, a protein with anti-proliferative effects in breast cancer cell lines and involved in bone formation, significantly decreased in non-invasive, invasive, and liver metastatic breast tumor tissue compared to normal breast tissue. The gene expression of activin betaA, a protein involved in cell proliferation and osteoclast induction, increased in invasive and bone metastatic tumor tissue compared to normal breast tissue. The mRNA level of OPN, a bone matrix protein associated with enhanced malignancy, increased in non-invasive, invasive, and liver and bone metastatic breast tumor tissue compared to normal breast tissue. In contrast, the average gene expressions of the TNF family members, RANKL and OPG, proteins involved in the regulation of osteoclastogenesis, were only slightly if at all changed in the different stage breast tumor tissues. These results suggest that differential gene expression of bone-related proteins, especially OPN, activin betaA, and BMP-

Topics: Adult; Aged; Aged, 80 and over; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Inhibin-beta Subunits; Liver Neoplasms; Middle Aged; Neoplasm Metastasis; Osteopontin; Ovarian Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta

This study was to determine the role of tumor-infiltrating macrophages in IFN-beta-induced host defense against prostate cancer.. Efficacy of adenovirus-mediated IFN-beta gene therapy against orthotopic xenografts of human prostate cancer was tested in macrophage-compromised nude mice. Immunohistochemistry and Northern blotting were used to elucidate mechanisms responsible for the IFN-beta gene therapy.. PC-3MM2 human prostate cancer cells were inoculated into the prostates of nude mice. Intralesional injection of an adenoviral vector-encoding murine IFN-beta (AdmIFN-beta) but not control vector AdE/1 suppressed growth of PC-3MM2 tumors in a dose-dependent manner, with a maximal reduction of tumor weight by approximately 85% at 2 x 10(9) plaque-forming units. The therapy prevented metastasis, eradicated established metastases in some mice, and prolonged the survival of tumor-bearing mice. The efficacy of AdmIFN-beta therapy was reduced significantly in mice treated with macrophage-selective anti-Mac-1 and anti-Mac-2 antibodies. Moreover, the i.p. injection of the antibodies restored the tumorigenicity of PC-3MM2 cells stably engineered with murine IFN-beta gene. Tumor-infiltrating macrophages, significantly increased in AdmIFN-beta-injected lesions, were depleted by the antibodies. The therapy stimulated expression of the inducible nitric oxide synthase, down-regulated transforming growth factor-beta1 and interleukin-8, reduced microvessel density, and resulted in apoptosis of endothelial cells in the lesions. These effects of AdmIFN-beta were partially diminished in mice treated with the antibodies.. These data suggest that macrophages play an important role in IFN-beta gene therapy and that intralesional delivery of the IFN-beta gene could be an effective therapy for clinically localized human prostate cancer.

Topics: Adenoviridae; Animals; Cell Division; Gene Expression Regulation, Neoplastic; Genetic Therapy; Genetic Vectors; Humans; Immunologic Factors; Injections, Intralesional; Interferon-beta; Interleukin-8; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostatic Neoplasms; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Reduced retinyl ester synthesis has been associated with several forms of cancer; we therefore proposed studying melanoma development from the perspective of this biochemical pathway. Cultures of human melanoma cells with fibroblastoid morphology showed negligible retinyl ester synthesis; in sharp contrast, those with epithelioid morphology were capable of retinol esterification. Further, isolated proliferating epidermal melanocytes (HFSC/2) esterified retinol, whereas proliferating normal skin fibroblasts (F:CCD-1121.Sk) did not. A primary site cutaneous melanoma and its metastatic match (both of epithelioid morphology) were capable of retinol esterification, while a matched fibroblastoid tumor pair did not synthesize retinyl esters; nevertheless, LRAT (lecithin:retinol acyltransferase) protein was found in microsomal fractions from all four tumors. A mutation screen in the LRAT coding region and adjacent intronic sequences revealed several novel mutations in these melanomas as well as in HFSC/2 and F:CCD-1121.Sk cells: a single nucleotide polymorphism in exon 1(37A-->G), a silent mutation in exon 2a (188 A-->G/186 G-->A), and an insertion in the 5'UTR (9-10insC). CRBP-1 basal expression was present in the HFSC/2, and in both sets of matched tumor pairs; however, steady-state levels in the fibroblastoid melanoma pair were one-third that found in the epithelioid matched tumor pair. Co-culture of human primary site epithelioid melanoma with proliferating normal human skin fibroblasts abrogated retinol esterification within 96 h and increased the expression of the active form of TGFbeta-1 by 2.4-fold. A concomitant 3.2-fold downregulation of CRBP-1 expression took place. This is the first study to (1) demonstrate an association between retinyl ester synthesis and cutaneous melanoma morphological phenotypes; (2) suggest the existence of a soluble, diffusible inhibitor of the retinol esterification pathway; (3) report the ability of the isolated, proliferating human epidermal melanocyte to esterify retinol; and (4) provide evidence of DNA variants in the coding region of LRAT.

Topics: Acyltransferases; Biological Factors; Cells, Cultured; Diffusion; DNA Mutational Analysis; Epithelial Cells; Esterification; Esters; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes; Humans; Introns; Melanoma; Microsomes; Mutagenesis, Insertional; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Stem Cells; Polymorphism, Single Nucleotide; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Skin Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Vitamin A

We investigated the effects of transforming growth factor-beta (TGF-beta) on biological behavior of a weakly malignant rat mammary carcinoma ER-1 cell line. TGF-beta enhanced the tumorigenic and metastatic capacity of ER-1 cells and their in vitro invasiveness to rat mesothelial and endothelial cell. Further cell biological analysis indicated that the increased invasive and metastatic capacity of ER-1 cells by TGF-beta was due to the increase in cell motility and adhesion to the mesothelial and endothelial cell monolayers. Thus, it is suggested that TGF-beta acts on ER-1 cells as a progression-enhancing factor which stimulates their adhesive and motile activities.

Topics: Animals; Antibodies; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Chemotaxis; DNA-Binding Proteins; Dose-Response Relationship, Drug; Female; Hyaluronan Receptors; Hyaluronic Acid; Hyaluronoglucosaminidase; Immediate-Early Proteins; Kinetics; Mammary Neoplasms, Experimental; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Nuclear Proteins; Trans-Activators; Transforming Growth Factor beta; Xenopus Proteins

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Epithelial Cells; Hepatocyte Growth Factor; Mammary Glands, Animal; MAP Kinase Signaling System; Mesoderm; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Molecular Weight; Mutation; Neoplasm Metastasis; Oncogene Protein p21(ras); Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.

Topics: Bone Neoplasms; Breast Neoplasms; DNA-Binding Proteins; Female; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Parathyroid Hormone-Related Protein; Protease Inhibitors; Proteins; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

Topics: Animals; Antigens, Polyomavirus Transforming; Apoptosis; Autocrine Communication; Breast; Cell Movement; Cell Survival; Female; Genetic Vectors; Immunoglobulin Fc Fragments; Immunoglobulin G; Lung Neoplasms; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred BALB C; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Solubility; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

TGF-betas play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-beta antagonist of the soluble type II TGF-beta receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-beta null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-beta associated with metastasis, while sparing the regulatory roles of TGF-betas in normal tissues. Thus this soluble TGF-beta antagonist has potential for long-term clinical use in the prevention of metastasis.

Topics: Animals; Female; Genetic Vectors; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Liver Neoplasms; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Melanoma, Experimental; Mice; Mice, Transgenic; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Solubility; Transforming Growth Factor beta; Tumor Cells, Cultured

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cell Differentiation; Cell Division; Cells, Cultured; Centrosome; Glioma; Guanosine Triphosphate; Immunohistochemistry; Isoenzymes; Microtubules; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Precipitin Tests; Rats; Recombinant Proteins; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tubulin

The mechanism of metastasis of osteosarcoma cells to other bones has not yet fully been clarified. The purpose of the present study was to examine whether various factors involve the formation of osteosarcoma metastatic foci in other bones. Immunohistochemically, CD31 expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 10 and 75% of cases, respectively. Met/hepatocyte growth factor (HGF) receptor expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 90 and 25% of cases, respectively. Bone morphogenetic protein (BMP) expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 20 and 75% of cases, respectively. Metastasis of osteosarcoma cells to other bones was significantly correlated with expression of BMP and CD31 and with no expression of Met/HGF receptor protein in osteosarcoma cells. In contrast, expression of insulin-like growth factor receptor in osteosarcoma cells did not correlate significantly with bone metastasis. These results suggest that formation of metastatic foci of osteosarcoma cells in other bones is regulated by CD31, which is associated with migration between endothelial cells, by BMP, which can induce and activate various mesenchymal cells affecting bone formation, and by escape of effect by HGF, which promotes differentiation of osteosarcoma cells.

Topics: Adolescent; Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Bone Neoplasms; Child; Hepatocyte Growth Factor; Humans; Immunoenzyme Techniques; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Osteosarcoma; Platelet Endothelial Cell Adhesion Molecule-1; Receptor, IGF Type 1; Transforming Growth Factor beta

Bone matrix serves as a reservoir of growth factors important in growth and tissue remodeling, and transforming growth factor-beta (TGF-beta) is abundant in bone matrix. Normal processes, such as remodeling, and pathological processes, such as osteolytic metastasis, cause the release of growth factors from the matrix, allowing them to influence the behavior of cells within their microenvironment. Breast cancer metastases frequently establish themselves in the bone compartment, often causing localized osteolysis. Stromelysin-3 is a matrix metalloproteinase associated with tumor metastases. Its expression in host tissues favors the homing and survival of malignant epithelial cells in early tumorigenesis by releasing and/or activating growth factors sequestered in the extracellular matrix. Osteoblasts express stromelysin-3, and Northern and Western blot analysis show that its messenger RNA and protein levels are increased by TGF-beta. Nuclear run-off assays demonstrate activation of gene transcription, and experiments using transcription inhibitors demonstrate stabilization of stromelysin-3 messenger RNA by TGF-beta. Importantly, TGFbeta induces stromelysin-3 in fibroblasts by similar mechanisms, indicating that it is likely to stimulate stromelysin-3 expression in breast stroma. Stimulation of stromelysin-3 expression by TGF-beta in fibroblasts and osteoblasts could play a role in the metastasis of breast cancer cells and their homing and survival in bone.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Line; Cell Nucleus; Cycloheximide; Fibroblasts; Matrix Metalloproteinase 11; Metalloendopeptidases; Mice; Neoplasm Metastasis; Osteoblasts; Protein Synthesis Inhibitors; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

To assess the expression of the homeogene Pax-2 in adult renal cell carcinomas, we did a retrospective immunohistochemical analysis of 56 frozen tumor samples representing all major histologic subtypes of renal tumors. There were 33 conventional renal cell carcinomas (58.9%), 12 papillary renal cell carcinomas (21.4%), 4 chromophobe cell renal carcinomas, 4 urothelial cell renal carcinomas, and 3 oncocytomas. Forty-five tumors (62.5%) were localized, and 21 tumors had extrarenal involvement. Eight patients (14%) had metastatic disease at the end of the follow-up. We searched for relationships between Pax-2 expression and nuclear grading, TNM staging, Ki-67 proliferation index, expression of transforming growth factor-beta1 (TGF-beta 1), an in vitro down-regulator of Pax-2 expression, and finally cytogenetic abnormalities. All histologic subtypes expressed Pax-2 protein, except urothelial renal carcinomas. The highest expression was in papillary renal cell carcinomas. In this subtype, all tumors and 83.3% +/- 12.3% of tumor cells were immunoreactive for Pax-2. All but 2 conventional renal cell carcinomas expressed Pax-2, but with 26.3% +/- 29.6% of immunoreactive cells (P <.001). Pax-2 expression was not correlated with nuclear grading (P =.6), tumor size (P =.3), and TGF-beta 1 expression (P =.1). Nevertheless, Pax-2 expression correlated with the Ki-67 proliferation index only for the conventional histologic subtype (P =.03). In this histologic subtype, Pax-2 expression was higher in patients with metastatic disease than in those without (P =.02). Pax-2 expression was not associated with specific cytogenetic abnormalities like trisomy 7 (P =.1), 3p deletion (P =.5), and hyperdiploidy (P =.2). TGF-beta 1 expression, positive in 33 tumors (59%), was not correlated with either Pax-2 expression (P =.1) or current prognostic factors such as nuclear grading (P =.2). Interestingly, we also observed an expression of TGF-beta RI and TGF-beta RII in the tumors with high nuclear grading (P =.005). We conclude that Pax-2 protein is expressed in all major histologic subtypes of renal cell carcinomas. The pattern of expression differs between these subtypes. Pax-2 expression in conventional renal cell carcinomas is correlated with the proliferation index and is significantly higher in patients with metastatic disease. HUM PATHOL 32:282-287.

Topics: Adenoma, Oxyphilic; Adult; Aged; Aged, 80 and over; Carcinoma, Papillary; Carcinoma, Renal Cell; Cell Division; Cell Nucleus; Chromosome Aberrations; Cryopreservation; Cytogenetic Analysis; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Kidney Neoplasms; Male; Middle Aged; Neoplasm Metastasis; PAX2 Transcription Factor; Retrospective Studies; Transcription Factors; Transforming Growth Factor beta

Plasma transforming growth factor beta1 (TGF-beta1) has been reported to be correlated with the extent of disease in colorectal cancer, but it is not known whether measuring this cytokine can help predict liver metastasis after curative resection. We prospectively studied whether plasma TGF-beta1 levels could predict liver metastasis in 117 patients with colorectal cancer before and after curative resection. Blood samples were drawn before and 2 weeks after surgery to determine the cytokine levels. Abdominal ultrasonography or computed tomography was done every 3 months after surgery. The primary end point for follow-up was recurrence. Seventy-seven of 117 cases (66%) had preoperative levels of the cytokine higher than the borderline limit of 7.5 ng/ml. Postoperative levels were >7.5 ng/ml in 29 of 117 patients (25%). The median follow-up period was 42 months (range, 5--66 months), with follow-up of all 117 patients. No recurrence was observed in 13 patients with Dukes' stage A lesions. Liver metastasis occurred in 18 of 104 patients (17%) with Dukes' stage B or C disease. Fourteen of 18 patients (78%) who developed liver metastasis had shown a postoperative plasma TGF-beta1 level of >7.5 ng/ml. Cox proportional hazards regression analysis showed that the postoperative level was a significant predictive factor for liver metastasis (P < 0.001). A single point measurement of plasma TGF-beta1 levels at 2 weeks after curative resection seems to be able to predict liver metastasis in colorectal cancer. This finding suggests the value of a prospective trial of liver-targeted adjuvant therapy for patients with elevated postoperative plasma TGF-beta1 levels.

Topics: Aged; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; Disease-Free Survival; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Postoperative Care; Preoperative Care; Prognosis; Recurrence; Transforming Growth Factor beta; Transforming Growth Factor beta1

Elevated local and circulating levels of transforming growth factor beta(1) (TGF-beta(1)) have been associated with prostate cancer invasion and metastasis. We tested the hypothesis that preoperative plasma TGF-beta(1) levels would independently predict cancer stage and prognosis in patients who undergo radical prostatectomy.. The study group consisted of 120 consecutive patients who underwent radical prostatectomy for clinically localized prostate cancer (median follow-up, 53.8 months). Preoperative plasma levels of TGF-beta(1) were measured and correlated with pathologic parameters and clinical outcomes. TGF-beta(1) levels also were measured in 44 healthy men without cancer, in 19 men with prostate cancer metastatic to regional lymph nodes, and in 10 men with prostate cancer metastatic to bone.. Plasma TGF-beta(1) levels in patients with lymph node metastases (14.2 +/- 2.6 ng/mL) and bone metastases (15.5 +/- 2.4 ng/mL) were higher than those in radical prostatectomy patients (5.2 +/- 1.3 ng/mL) and healthy subjects (4.5 +/- 1.2 ng/mL) (P <.001). In a preoperative analysis, preoperative plasma TGF-beta(1) level and biopsy Gleason sum both were predictors of organ-confined disease (P =.006 and P =.006, respectively) and PSA progression (P <.001 and P =.021, respectively). In a postoperative multivariate analysis, preoperative plasma TGF-beta(1) level, pathologic Gleason sum, and surgical margin status were predictors of PSA progression (P =.020,P =.020, and P =.022, respectively). In patients who progressed, preoperative plasma TGF-beta(1) levels were higher in those with presumed distant compared with local-only failure (P =.019).. Plasma TGF-beta(1) levels are markedly elevated in men with prostate cancer metastatic to regional lymph nodes and bone. In men without clinical or pathologic evidence of metastases, the preoperative plasma TGF-beta(1) level is a strong predictor of biochemical progression after surgery, presumably because of an association with occult metastatic disease present at the time of radical prostatectomy.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Disease Progression; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.

Topics: Animals; B7-1 Antigen; Cancer Vaccines; Carcinoma; Cell Division; Female; Genetic Therapy; Interferon-gamma; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligonucleotides, Antisense; RNA, Messenger; Survival Rate; T-Lymphocytes; Transforming Growth Factor beta; Transgenes

TGF-betas are multifunctional polypeptides that regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions. In this study, we investigated the effect of TGF-beta1 on liver metastasis and its mechanism by using human pancreatic cancer cell lines Panc-1, Capan-2, and SW1990. Capan-2 and SW1990 cells demonstrated enhanced liver metastatic potential by in vivo splenic injection with TGF-beta1. Consequently, we examined the role of TGF-beta1 on in vitro angiogenesis and received cytotoxicity by peripheral blood mononuclear leukocytes (PBMLs). While TGF-beta1 slightly decreased cell proliferation, it also upregulated VEGF production in all cancer cells examined. The binding of PBMLs to cancer cells and cancer cell cytotoxicity during co-culture with PBMLs were remarkably decreased by treatment with TGF-beta1. Panc-1 cells revealed no liver metastasis despite their high immunogenetic and angiogenetic abilities, which was attributed to a lack of expression of the cell surface carbohydrates that induce attachment to endothelial cells. We concluded that the presence of TGF-beta1 in the microenvironment of tumour site might play an important role in enhancing liver metastasis of pancreatic cancer by modulating the capacity of angiogenesis and immunogenicity.

Topics: Animals; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver Neoplasms; Lymphokines; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transforming growth factor beta1 (TGF-beta1) acts as a tumor suppressor at early stages of carcinogenesis, however, it has also been suggested to promote tumor progression at late stages. To determine at which stage and by what mechanisms this functional switch occurs, we have generated gene-switch-TGF-beta1 mice in which TGF-beta1 transgene expression can be induced in skin tumors at specific stages. These mice were exposed to a chemical carcinogenesis protocol, which allows tumorigenesis to develop in progressive stages from benign papillomas to malignant carcinomas. Remarkably, TGF-beta1 transgene induction in papillomas rapidly induced metastasis. This function is in sharp contrast to its tumor suppressive effect when TGF-beta1 transgene expression was induced early in the protocol. Transgenic papillomas exhibited down-regulation of TGF-beta receptors and their signal transducer, the Smads, and loss of the invasion suppressor E-cadherin/catenin complex in the cell membrane. These molecules were lost only in malignant carcinomas in control mice at a much later stage. Furthermore, transgenic papillomas exhibited elevated expression of matrix metalloproteinases and increased angiogenesis. Our study suggests that TGF-beta1 overexpression may directly induce tumor metastasis by initiating events necessary for invasion. Down-regulation of TGF-beta signaling components in tumor epithelia selectively abolishes growth inhibition, thus, switching the role of TGF-beta1 to a metastasis promoter.

Topics: Animals; beta Catenin; Cadherins; Cytoskeletal Proteins; Desmoplakins; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred ICR; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Papilloma; Signal Transduction; Skin Neoplasms; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Topics: Animals; Carcinoma, Squamous Cell; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; I-kappa B Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-1; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Transforming Growth Factor beta

Decorin, a member of the family of small leucin-rich proteoglycans, has originally been described as a secreted proteoglycan component of the connective tissues, and has been implicated in the negative regulation of cell proliferation directly or via interactions with TGF-beta. It was reported to be generally absent from tumor cells. Here we show that human melanoma cell lines express a decorin-like molecule. We detected decorin mRNA by RT-PCR in 7 out 7 human melanoma lines characterized by various metastatic potential. Using polyclonal antiserum against the core protein of decorin, the typical 80-120 kD glycanated form as well as a high molecular weight aberrant version (200-210 kD) of decorin were demonstrated by Western blot technique in the culture supernatants as well as in lysates of human melanoma cells. Finally, decorin epitope was also demonstrated immunohistochemically in human melanoma xenografts, as well as in tumor cells of surgically resected melanomas but not in melanocytes of nevi. The expression of this aberrant decorin did not inhibit the in vitroor in vivogrowth of human melanoma cells, and it was independent of their metastatic potential. Human melanoma cell lines expressing aberrant decorin retained sensitivity to the antiproliferative and gelatinase-stimulatory effects of exogenous TGF-beta.

Topics: Animals; Blotting, Southern; Cell Division; Cell Transformation, Neoplastic; Collagenases; Decorin; DNA Primers; Extracellular Matrix Proteins; Flow Cytometry; Immunoenzyme Techniques; Melanoma; Mice; Mice, SCID; Neoplasm Metastasis; Polymerase Chain Reaction; Proteoglycans; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) exerts an inhibitory effect on epithelial cell proliferation while insulin-like growth factor-1 (IGF-1) is a positive regulator of proliferation and together they may participate in driving neoplastic progression. The regulation of TGF-beta1 and IGF-1 gene expression was analyzed in an in vitro model of an estrogen receptor positive (ER+), non-metastatic (MCF-7) and an (ER-), metastatic (MDA-MB-435) breast cancer cell line, respectively. Our results indicate a loss of the regulation of TGF-beta1 and the gain of the expression and upregulation of IGF-1 pathways during malignant progression. These data demonstrate that two factors, convergent on cell growth, can have divergent roles in the regulation of the expression of TGF-beta1.

Topics: Blood; Breast Neoplasms; Cell Division; Disease Progression; Estradiol; Feedback; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Actins; Animals; Bone Marrow Cells; Cell Survival; Coculture Techniques; Colonic Neoplasms; Fibroblasts; Humans; Immunohistochemistry; Mesoderm; Mice; Neoplasm Metastasis; Transforming Growth Factor beta; Tumor Cells, Cultured

This study examined the effect of stable transfection of latent transforming growth factor-beta1 (TGF-beta1) cDNA into a predominantly polygonal, 4 nitroquinoline N-oxide (4NQO)-induced rat oral keratinocyte cell line. Seven polygonal and five spindle clonal populations were isolated that overexpressed TGF-beta1 protein by approximately two- to four-fold compared to vector-only transfected controls. Neutralisation experiments indicated that the majority of protein was in the latent form. There was no change in the proportion of polygonal and spindle cells in vitro after transfection with TGF-beta1 cDNA. Polygonal and spindle cells that overexpressed TGF-beta1 produced similar amounts of protein and grew more slowly in vitro than controls. The parent cell line and all transfected cells were growth inhibited (60-75%) by exogenous TGF-beta1. Orthotopic transplantation of the parent and the vector-only control cell lines resulted in primary tumours in the floor of the mouth in almost 100% (20/21) of athymic mice, with no evidence of bone resorption at the site of the primary tumour and pulmonary metastatic tumour deposits in some 40% (7/20) of these animals. The polygonal and spindle cells that overexpressed TGF-beta1 behaved similarly following orthotopic transplantation. A 96% (23/24) primary tumour take was evident following transplantation of cells that overexpressed TGF-beta1, with a significantly (P<0.02) higher number of animals showing bone resorption at the site of the primary tumour (35%; 8/23) compared to controls. By contrast, there was a significant (P<0.03) decrease in the number of animals with pulmonary metastases (4%; 1/23) following transplantation of TGF-beta1 overexpressing cells compared to controls. Overexpression of TGF-beta1 did not alter tumour cell differentiation in vivo. The results demonstrate that endogenous TGF-beta1 functions as a tumour suppressor in the rat-4NQO model of oral carcinogenesis without altering tumour cell morphology or differentiation but can also act to promote local bone resorption.

Topics: Animals; Bone Resorption; Cell Division; Cell Line, Transformed; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGFbeta) contributes to the promotion of invasion, metastasis, angiogenesis and even immunosuppression. Since overexpression of the delta isoform of protein kinase C (nPKCdelta) in BL6 murine melanoma cells (BL6T cells) increases their metastatic capacity, we investigated the possible involvement of TGFbeta1 in this process. Immunohistochemical analysis demonstrated lower levels of TGFbeta1 in BL6T lung metastases compared with BL6 lung metastases. On the other hand, higher levels of this cytokine, in particular in its active form, occur in the plasma of BL6T metastasized animals, suggesting a nPKCdelta-dependent TGFbeta1 release. Therefore, nPKCdelta-dependent TGFbeta1 release and activation may be involved in the greater angiogenic and metastatic capacity of murine melanoma BL6T cells.

Topics: Animals; Blotting, Western; Densitometry; Immunohistochemistry; Isoenzymes; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34(+) blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1beta (IL-1beta), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34(+) BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-beta1 (TGF-beta1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1beta + IL-3 + IL-6 + EPO, but was suppressed by TGF-beta1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 x 10(5)/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34(+) BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34(+) BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.

Topics: Antigens, CD34; Ascites; Breast Neoplasms; Cell Division; Cell Survival; Coculture Techniques; Culture Media; Culture Media, Serum-Free; Erythropoietin; Hematopoietic Stem Cells; Humans; Interleukin-1; Interleukin-3; Interleukin-6; Neoplasm Metastasis; Pleural Effusion; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Collagen; Gene Expression Regulation; Genes, ras; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Metastasis; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Thyroid Neoplasms; Time Factors; Transfection; Transforming Growth Factor beta

Malignancy is a common and dreaded complication following organ transplantation. The high incidence of neoplasm and its aggressive progression, which are associated with immunosuppressive therapy, are thought to be due to the resulting impairment of the organ recipient's immune-surveillance system. Here we report a mechanism for the heightened malignancy that is independent of host immunity. We show that cyclosporine (cyclosporin A), an immunosuppressant that has had a major impact on improving patient outcome following organ transplantation, induces phenotypic changes, including invasiveness of non-transformed cells, by a cell-autonomous mechanism. Our studies show that cyclosporine treatment of adenocarcinoma cells results in striking morphological alterations, including membrane ruffling and numerous pseudopodial protrusions, increased cell motility, and anchorage-independent (invasive) growth. These changes are prevented by treatment with monoclonal antibodies directed at transforming growth factor-beta (TGF-beta). In vivo, cyclosporine enhances tumour growth in immunodeficient SCID-beige mice; anti-TGF-beta monoclonal antibodies but not control antibodies prevent the cyclosporine-induced increase in the number of metastases. Our findings suggest that immunosuppressants like cyclosporine can promote cancer progression by a direct cellular effect that is independent of its effect on the host's immune cells, and that cyclosporine-induced TGF-beta production is involved in this.

Topics: Animals; Antibodies; Carcinogens; Carcinoma, Renal Cell; Cell Adhesion; Cell Division; Cell Movement; Cell Size; Cell Transformation, Neoplastic; Cyclosporine; Humans; Immunosuppressive Agents; Male; Mice; Mice, SCID; Microscopy, Electron, Scanning; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Phenotype; Pseudopodia; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies have suggested that transforming growth factor-beta 1 (TGF-beta1) acts as an autocrine growth inhibitor on normal human melanocytes, while melanoma cells may not respond to this stimulus. The role of other TGF-beta isoforms such as TGF-beta2 and TGF-beta3 remained less well characterized. In the present study, the mRNA and protein levels of all three isoforms of TGF-beta were analyzed in a panel of human melanoma cell lines and in cultures of normal human melanocytes in vitro. Northern analysis showed that the degree of TGF-beta1, -beta2, -beta3 mRNA expression varied considerably in melanoma cells, whereas TGF-beta expression was very low in melanocytes. In melanoma cells, secreted amounts of TGF-beta1 and TGF-beta3 were found increased in comparison to normal melanocytes: 615 pg/ml vs. 118 pg/ml and 193 pg/ml vs. 30 pg/ml (mean values). In addition, low levels of TGF-beta2 were detected (mean value: 28 pg/ml). Although TGF-beta secretion increased, the proliferation of melanoma cells was found to be only moderately inhibited by TGF-beta isoforms, in contrast to its strong antiproliferative effect on normal human melanocytes: - 15%, -11%, and -18% vs. -52%, -46%, and -50% average inhibition at 0.5 ng/ml TGF-beta1, -beta2, and -beta3, respectively. The different efficacy of TGF-beta on melanocyte and melanoma cells was highly significant (P<0.0001); in addition, TGF-beta-dependent growth inhibition of melanoma cells from primary tumors vs. cells from metastases showed a trend for further decreased response for the metastatic populations (P< or = 0.075). Measurements of DNA synthesis revealed even more pronounced differences between melanocytes (-86%, -78%, and -80% inhibition, respectively, for TGF-beta1, -beta2, and -beta3) and melanoma cells (no inhibition). Our data show loss of responsiveness of melanoma cells to the growth-inhibitory function of TGF-beta isoforms but not of melanocytes. Although melanoma cells are not growth-inhibited by all three TGF-beta isoforms, they secrete significantly higher levels of TGF-beta, as compared to melanocytes. The reduced response indicates their escape from TGF-beta surveillance with ongoing tumor progression.

Topics: Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; DNA, Neoplasm; Gene Expression; Growth Inhibitors; Humans; Melanocytes; Melanoma; Neoplasm Metastasis; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) is a major modulator of cellular proliferation and extracellular matrix formation. We determined the role of TGF-beta1 in invasion and metastasis in gastric cancer.. We detected TGF-beta1 expression in primary and lymph node metastatic lesions of gastric cancer, using an antibody and in situ hybridization. The plasma TGF-beta1 levels in the peripheral vein and in the tumor drainage vein were assayed.. In the cytoplasm of cancer cells, TGF- beta1 was immunostained in 35.9% (78 of 217) of primary gastric carcinomas, and this expression was confirmed by in situ hybridization. Of 59 gastric carcinomas with a TGF-beta1-negative primary tumor, metastatic lymph nodes were positive for TGF-beta1 staining in 32 cases (54.2%). Positive staining of TGF-beta1 in gastric cancer tissues was closely related to serosal invasion, infiltrative growth, and lymph node metastasis. Multivariate analysis showed that the expression of TGF-beta1 was an independent risk factor for serosal invasion and infiltrative growth of the tumor. The plasma level of TGF-beta1 did not differ between TGF-beta1-negative and -positive groups. There were also no differences in plasma TGF-beta1 levels among each tumor stage, between the peripheral and the tumor drainage veins, and between preoperative and postoperative testings.. Transforming growth factor-beta1 is closely related to the invasion and metastasis of gastric cancer, and production of TGF-beta1 in the tumor does not contribute to the total amount of TGF-beta1 in the blood circulation. We interpret our observations to mean that in a tumor microenvironment, TGF-beta1 alters the biologic behavior of the tumor.

Topics: Aged; Female; Humans; Immunohistochemistry; In Situ Hybridization; Lymph Nodes; Male; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Metastasis; Stomach Neoplasms; Survival Analysis; Transforming Growth Factor beta

Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis.

Topics: Animals; Colony-Forming Units Assay; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Gelatinases; Genetic Therapy; Humans; Immunohistochemistry; Interleukin-10; Male; Matrix Metalloproteinase 2; Metalloendopeptidases; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Prostatic Neoplasms; Survival Analysis; Tissue Inhibitor of Metalloproteinase-1; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation

Transforming growth factor-beta (TGF-beta) is known to inhibit primary epithelial ovarian carcinoma cells. The mechanism by which this inhibitory response is achieved is poorly understood. Furthermore, whether this response is consistent in cells from metastatic sites compared with the primary site cells is unknown. The authors wanted to determine whether TGF-beta differentially inhibited ovarian carcinoma cells from primary tumor sites compared with metastatic sites and to establish whether this response was associated with up-regulation of p21WAF1 or overexpression of p53.. Tumor cells were purified from primary and metastatic sites in five patients with advanced epithelial ovarian carcinoma. TGF-beta effect at concentrations of 10, 1, and 0.1 ng/mL was determined by tritiated thymidine incorporation assay. Expression of p21WAF1 was determined by Northern and slot blot analysis. p53 was detected by immunocytochemistry.. Metastatic tumor isolates were more responsive to the inhibitory effect of TGF-beta compared with their corresponding primary tumor isolates at 0.1 ng/mL. Increasing TGF-beta concentration conferred no additional inhibitory effect on the metastatic isolates; however, a dose-related phenomenon was observed in primary tumor isolates. p21WAF1 mRNA was up-regulated in only 2 of 10 primary and metastatic isolates. There was no correlation between TGF-beta responsiveness, p21WAF1 up-regulation, and p53 overexpression.. Differential inhibition was observed between primary and metastatic tumor isolates. p21WAF1 up-regulation and p53 overexpression were not major modulators in TGF-beta regulation of primary and metastatic tumor growth in early passaged ovarian carcinoma cells.

Topics: Carcinoma; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Topics: Adenocarcinoma; Animals; Cell Separation; Chemotaxis, Leukocyte; Flow Cytometry; Interleukin-10; Leukocyte Count; Lymphocytes, Tumor-Infiltrating; Male; Microscopy, Electron, Scanning; Neoplasm Metastasis; Neoplasm Transplantation; Phenotype; Prostatic Neoplasms; Prostheses and Implants; Rats; Surgical Sponges; Transforming Growth Factor beta

Topics: Apoptosis; Cell Division; Cellular Senescence; Japan; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Telomerase; Transforming Growth Factor beta

We have shown that metastasis is suppressed by low-dose total-body irradiation (TBI) in tumor-bearing rats. We have evaluated the immunological effects of low-dose TBI. Total-body irradiation with 0.2 Gy was given 14 days after the implantation of 5 x 10(5) allogenic hepatoma cells (KDH-8) which produce transforming growth factor beta (TGF-beta). On day 21, the splenocytes and tumor-tissue infiltrating lymphocytes were analyzed by FACScan and RT-PCR for the mRNA of the genes that encode tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), TGF-beta, interleukin (IL)-4, IL-10 and IL-6. The same procedure was conducted with untreated rats and with rats that underwent local irradiation with 0.2 Gy. The low-dose TBI significantly decreased the incidence of lung and lymph node metastasis (P < 0.01), whereas the same dose of local irradiation had no effect on the incidence of metastasis. The proportion of CD8+ cells in splenocytes increased in the low-dose TBI group (P < 0.01) compared to the locally irradiated and the untreated groups. The tumor-tissue infiltrating lymphocytes were also significantly increased after low-dose TBI (P < 0.01). The FACScan analysis revealed that 72% of the tumor-tissue infiltrating lymphocytes were CD8+. In both spleen and tumor tissue after low-dose TBI, mRNA expression of the genes that encode IFN-gamma and TNF-alpha increased, while that of the Tgfb gene decreased. There was no expression of the mRNAs of the Il4, Il6 and Il10 genes. CD8+ cells and the cytokine network may play an important role in the antitumor effect of low-dose TBI.

Topics: Animals; Interferon-gamma; Male; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Spleen; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Whole-Body Irradiation

Mutations in the transforming growth factor beta type II receptor (TGF-betaRII) have been identified in human cancers, which suggests a causal role for the loss of TGF-betaRII in cancer development. To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TGF-betaRII (delta betaRII) in the epidermis, using a truncated mouse loricrin promoter (ML). ML.delta betaRII transgenic mice exhibited a thickened skin due to epidermal hyperproliferation. When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they exhibited an increased sensitivity, with an earlier appearance and a 2-fold greater number of papillomas than control mice. In addition, papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; whereas ML.delta betaRII papillomas progressed to carcinomas. Furthermore, TPA promotion alone induced papilloma formation in ML.delta betaRII mice, which suggests an initiating role for delta betaRII in skin carcinogenesis. ML.delta betaRII tumors also exhibited increased neovascularization and progressed to metastases, although the primary tumors were still classified as carcinoma in situ or well-differentiated carcinomas. Increased expression of vascular endothelial growth factor, an angiogenesis factor, and decreased expression of thrombospondin-1, an angiogenesis inhibitor, were also observed in ML.delta betaRII tumors. The increased angiogenesis correlated with elevated endogenous TGF-beta1 in ML.delta betaRII tumors. These data provide in vivo evidence that inactivation of TGF-betaRII accelerates skin carcinogenesis at both earlier and later stages, and increased angiogenesis is one of the important mechanisms of accelerated tumor growth and metastasis.

Topics: Animals; Carcinogens; Epidermis; Genes, ras; Genetic Predisposition to Disease; Humans; Mice; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Papilloma; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Nasopharyngeal carcinomas (NPCs) of non-keratinizing type are strongly associated with Epstein-Barr virus (EBV). EBV and its gene products induce the synthesis and/or release of transforming growth factor beta1 (TGF-beta1) from human cells and platelets. TGF-beta1 is an immunosuppressive cytokine, and many tumors are known to secrete it, to counter the host immune response. To determine the potential role of this cytokine in the pathogenesis of NPC, 53 serum samples from patients with EBV-associated NPC and 20 from healthy donors were analyzed for total and active TGF-beta content using ELISA. Serum samples for TGF-beta content were also analyzed from NPC patients at different clinical stages of the tumors. Significantly higher (p < 0.01) levels of active and total TGF-beta were found in the sera of NPC patients than in control sera. The ratio of active:total TGF-beta was also significantly (p < 0.01) increased in the NPC sera. Levels of this cytokine were also significantly higher (p < 0.05) in the sera of patients with advanced stages of tumor compared to patients with earlier stages. Furthermore, higher levels were seen in patients with relapsing than with remitting tumors; even higher levels were observed in NPC patients who died of the tumor. Our data suggest a role of this cytokine in the pathogenesis of NPC; therefore, it may prove to be a valuable biomarker molecule for the diagnosis and prognosis of NPC. Int. J. Cancer (Pred. Oncol.) 84:396-399, 1999.

Topics: Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Herpesvirus 4, Human; Humans; Lymphatic Metastasis; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Neoplasm Staging; Predictive Value of Tests; Recurrence; Survival Analysis; Transforming Growth Factor beta

The transforming growth factor beta (TGF-beta) pathway is known to play an important role in both human and urine colon cancer. However, the staging, ligand specificity, and mechanism underlying the tumor suppressive activity of this pathway are unknown. We developed a mouse model for colon cancer that identifies an early role for TGF-beta1 in tumor suppression and implicates TGF-beta2 or TGF-beta3 in the prevention of metastasis. Analysis of the development of colon cancer in TGF-beta1 knockout mice pinpoints the defect to the hyperplasty/adenoma transition and reveals that the mechanism involves an inability to maintain epithelial tissue organization and not a loss of growth control, increased inflammatory activity, or increased genetic instability. These mice provide a unique opportunity to investigate the specific role of TGF-beta1 at this critical transition in the development of colon cancer.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli Protein; Animals; Apoptosis; beta Catenin; Biomarkers; Cecum; Cell Division; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Crosses, Genetic; Cytoskeletal Proteins; Disease Progression; DNA; DNA-Binding Proteins; DNA, Neoplasm; Genes, APC; Genetic Predisposition to Disease; Humans; Hyperplasia; Inflammation; Intestinal Mucosa; Mice; Mice, Knockout; Microsatellite Repeats; Neoplasm Metastasis; Nuclear Proteins; Specific Pathogen-Free Organisms; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) can promote late stage tumor progression in a number of model systems. In the present study, we have examined whether expression of a truncated soluble extracellular domain of TGF-beta type III receptor (sRIII) in human breast cancer MDA-MB-231 cells can antagonize the tumor-promoting activity of TGF-beta by sequestering active TGF-beta isoforms that are produced by the cancer cells. The secretion of sRIII reduced the amount of active TGF-beta1 and TGF-beta2 in the conditioned medium. This led to a significant reduction of the growth-inhibitory activity of the medium conditioned by sRIII-expressing cells on the growth of mink lung epithelial CCL64 cells in comparison with the medium conditioned by the control cells. The tumor incidence and growth rate of all of the three sRIII-expressing clones studied were significantly lower than those of the control cells in athymic nude mice. Four of five control cell-inoculated mice showed spontaneous metastasis in the lung, whereas none of the sRIII-expressing cell-inoculated mice had any lung metastasis. Thus, our results suggest that the sRIII may be used to antagonize the tumor-promoting activity of TGF-beta.

Topics: Animals; Breast Neoplasms; Cell Line; Culture Media, Conditioned; Female; Humans; Lung; Lung Neoplasms; Mice; Mice, Nude; Mink; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Urokinase (u-PA) and the u-PA receptor (u-PAR) influence tumor invasion and metastasis.. The purpose of this study is to determine whether u-PAR display in 4 human bladder cancer cell lines (RT4, 253J, EJ and T24) can be modulated with substances including phorbol 12-myristate 13-acetate, interferon gamma, epidermal growth factor, and transforming growth factor beta and to correlate changes with u-PAR expression with the ability of these cells to invade artificial basement membrane (Matrigel).. u-PAR display was determined using flow cytometry and immunohistochemical staining with an anti-u-PAR monoclonal antibody (Mab3936). Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines.. The T24, EJ and 253J cells expressed the u-PAR while the RT4 cells did not. The EJ cells (expressing the highest u-PA antigen levels and the u-PAR) invaded Matrigel. The T24 cells, which expressed the u-PAR but do not produce u-PA, invaded Matrigel only when pretreated with high molecular weight urokinase (HMW u-PA). HMW u-PA pretreatment of EJ and 253J cells also enhanced their invasive potential. Blocking u-PA/u-PAR attachment with an anti-u-PAR monoclonal antibody (Mab3936) inhibited invasion. Modulation of u-PAR expression on cell lines displaying the u-PAR directly affected in vitro invasiveness of the cell lines. The RT4 cells which lack the u-PAR were not invasive under conditions tested. Thus, bladder cancer cell lines require both u-PA and the u-PAR to invade Matrigel and modulation of u-PAR display directly affects their in vitro invasive capacity.. This study shows that bladder tumor cells produce u-PA and express the u-PAR and require both for in vitro invasion to occur. When bladder cancer cells express the u-PAR, invasiveness can be enhanced by exogenous u-PA and inhibited by anti-u-PAR antibodies. Modulation of u-PAR expression on the cell surface of bladder cancer cells can also affect their ability to invade Matrigel.. Histologically similar bladder tumors show differences in their propensity to invade locally or metastasize. Intrinsic differences in the tumor cells such as the production of u-PA antigen and u-PA receptor on the cell surface and extrinsic differences in the tumor cell environment such as substances influencing u-PAR display or antibodies blocking the u-PAR may affect the biological potential of human bladder cancers and offer one explanation for the aggressive or indolent tumor behavior observed in individual patients.

Topics: Basement Membrane; Collagen; Drug Combinations; Epidermal Growth Factor; Humans; Interferon-gamma; Laminin; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urokinase-Type Plasminogen Activator

Breast cancer remains one of the most common malignant diseases in women in North America and Western Europe, yet therapies for the more aggressive estrogen independent tumors are limited and few model systems are available for the study of this type of breast cancer. In these studies, we characterized a novel estrogen independent breast cancer cell line, SUM-159PT. SUM-159PT cells are epithelial in origin, demonstrated by expression of cytokeratin 18. SUM-159PT cells are estrogen independent, demonstrated by lack of estrogen receptor (ER) protein and ER ligand binding studies. Furthermore, SUM-159PT cells injected subcutaneously or orthotopically are tumorigenic in ovariectomized athymic nude mice in the absence of estradiol supplementation. SUM-159PT cells are capable of invading through an 8 microm Matrigel membrane and display a stellate morphology in Matrigel, indicative of a metastatic phenotype. Correlating with this phenotype, we have detected secondary tumors upon inoculation of SUM-159PT cells into the mammary fat pad. To further investigate the metastatic potential of the SUM-159PT cells, we examined the expression of two proteins, vimentin and E-cadherin, implicated in the transition of carcinoma cells to a metastatic phenotype. Western blot and immunohistochemical analysis demonstrated that both SUM-159PT cells and xenografts express vimentin. No expression of E-cadherin was detected in SUM-159PT cells. Our data indicate that despite estrogen independence, SUM-159PT cells are growth inhibited in vitro by compounds such as 1,25(OH)2D3, transforming growth factor beta (TGF-beta), and the phorbol ester TPA. These studies indicate that SUM-159PT cells represent a good model system for the study of late stage estrogen independent, invasive breast cancer.

Topics: Animals; Breast Neoplasms; Disease Models, Animal; Estradiol; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Estrogen; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

We demonstrate that targeted expression of SV40 large T antigen (TAg) to the urethral (periurethral) and bulbourethral gland epithelium leads to adenocarcinoma formation in these tissues after 7 months of age, which are extremely rare sites for spontaneous tumor formation in humans. The development of proliferative lesions in the urethral gland predictably follows a temporal course of progression with approximately one third of male animals developing urethral tumors by 1 year of age. Tumor progression in these organs correlates to the level of TAg and p53 expression. Immunoprecipitation confirmed that SV40 TAg protein was bound to p53 and Rb p110 in vivo. Expression of transforming growth factor beta (TGFbetas) was evaluated during tumor progression of urethral gland carcinomas. Elevations of intracellular and extracellular TGFbeta1 and extracellular TGFbeta3 were found in preneoplastic and neoplastic lesions, suggesting that increased TGFbetas may augment tumor growth. c-Met expression showed a tendency for increased expression in the urethral gland carcinomas. We speculate that the directed expression of SV40 TAg by the hormone responsive C3(1) gene and subsequent tumor formation in these organs is influenced by androgens, since these tissues and carcinomas express androgen receptor (AR) and arise only in male transgenic mice. Several cell lines established from the urethral carcinomas were also shown to express AR, but are not androgen dependent in culture. To our knowledge, this is the first transgenic animal model for urethral and bulbourethral carcinomas. This transgenic mouse model and the cell lines derived from it may provide a unique opportunity for dissecting molecular mechanisms involved in the tumorigenesis of these organs which otherwise rarely develop cancer.

Topics: Aging; Androgen-Binding Protein; Animals; Antigens, Polyomavirus Transforming; Bulbourethral Glands; Genital Neoplasms, Male; Liver Neoplasms; Male; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Binding; Recombinant Fusion Proteins; Retinoblastoma Protein; Simian virus 40; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Urethral Neoplasms

The interactions of the cells in the bone microenvironment play important roles in bone remodeling. Osteoblasts are involved in the bone remodeling through the production of soluble factors that regulate proliferation and differentiation of osteoclasts and through cell-cell interactions. Histological studies have suggested that endothelial cells are also associated with some osteolytic bone diseases. However, it is still unclear how endothelial cells contribute to bone resorption. We established bone-derived endothelial cells (BDECs) to study their roles in bone remodeling. The established BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting a soluble bone resorption-inducing factor(s) when stimulated by several inflammatory cytokines. This bone resorption-inducing factor was identified as interleukin-11 (IL-11). IL-11 is known to enhance bone resorption by promoting osteoclastogenesis and by suppressing the activity of osteoblasts. The production of IL-11 in BDECs was also promoted by conditioned medium of human melanoma A375M cells. Because A375M cells formed osteolytic bone metastasis in vivo, BDECs might be involved in pathological osteolysis by producing IL-11. These results suggest that endothelial cells in bone play important roles in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Cell Line, Transformed; Cells, Cultured; Culture Media, Conditioned; Endothelium; Femur; Humans; Interleukin-1; Interleukin-11; Knee Joint; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Osteolysis; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured

To identify genes associated with prostate cancer progression, we developed a strategy involving the use of differential display PCR and a panel of genetically matched primary tumor- and metastasis-derived mouse prostate cancer cell lines. We analyzed sequences that were differentially stimulated by transforming growth factor-beta1 in primary tumor-versus metastasis-derived cell lines, based on our previous studies indicating that acquisition of differential responses to this growth factor could result in phenotypic traits that facilitate tumor metastasis from specific cell clones within the primary tumor. Using this system, we isolated and sequenced a cDNA fragment that encoded mouse lysyl oxidase (LO) and was induced by transforming growth factor-beta1 in primary tumor but not in metastasis-derived cells. Northern blotting analysis revealed increased LO expression in a panel of primary tumor cell lines but significantly reduced or nondetectable expression in their matched metastatic counterparts. Further in situ hybridization analysis revealed LO expression in normal mouse prostate epithelium but, in most cases, progressive loss of expression in primary prostate cancer and associated metastatic lesions. Importantly, in situ hybridization studies of normal human prostate and prostate malignancies revealed a similar loss of expression during progression to metastasis. The progressive loss of LO expression during prostate cancer progression provides information that may increase our understanding of the mechanisms that underlie this disease. In addition, LO may provide a useful molecular marker and/or establish a novel therapeutic target for prostate cancer.

Topics: Animals; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Male; Mice; Neoplasm Metastasis; Prostatic Neoplasms; Protein-Lysine 6-Oxidase; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta

Even though adjuvant chemotherapy has improved the 5-year survival rate of osteosarcoma patients, a significant percentage of patients eventually die from lung metastasis. Since transforming growth faCtor-beta (TGF-beta) has been demonstrated to be related to the tumor progression, we investigated the clinical implications of the presence of TGF-beta isoforms in 16 human osteosarcoma tissue. There were 10 males and 6 females with a mean age of 20.8 years of age (range, 8 to 57 years). Biopsied specimen before chemotherapy was fixed in 10% formalin, demineralized and followed by paraffin embedding. The locations of tumor included femur (10), tibia (3), humerus (1), fibula (1), and ilium (1). Histologic subtypes included osteoblastic (11), chondroblastic (2), and fibroblastic (3). All patients were followed for a minimum of 1 year (range 12 to 44 months) or to the development of lung metastasis. Five patients (31.3%) developed subsequent lung metastasis during the follow up. We used immunohistochemistry technique to investigate the presence of the TGF-beta isoforms in osteosarcoma tissue and its relationship to the subsequent pulmonary metastasis. The results showed the presence of one or more TGF-beta isoforms in tumor cells in osteosarcoma tissues (13 of 16, 81.3%) in all of the subtypes. However, minimal presence of TGF-beta isoforms was shown in the tumor bone matrix. The expression of TGF-beta1 or TGF-beta3 isoforms was associated with a higher rate of subsequent lung metastasis (p < 0.05, chi-square test). Further research is warranted to determine the utility of routine TGF-beta analysis in the clinical practice.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Bone Neoplasms; Child; Female; Humans; Immunohistochemistry; Isomerism; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Osteosarcoma; Time Factors; Tissue Distribution; Transforming Growth Factor beta

The effect of a panel of cytokines on the proliferation and type IV collagenase production was studied in four melanoma cell lines of different origin, tumorigenicity and metastatic capacity. TGF-b, TNF-a and to a lesser extent, IL-1a exhibited antiproliferative effect on the cell lines, with some lines showing varying degree of resistance. The sensitivity did not correlate directly with the origin or the biological behavior of the tumor lines, suggesting that cytokine resistance of advanced stage melanoma cells may be relative. IL-2, IL-10 and IL-12 displayed little or no effect on proliferation. The effect of cytokines on metalloproteinase production showed a cell line dependent pattern. Interestingly, those cytokines that exhibited the most pronounced antiproliferative activity, also proved most effective in stimulating collagenase secretion, often simultaneously, in the same line. The results indicate that pleiotropic cytokines can have positive and negative effects simultaneously on various steps of tumor progression.

Topics: Animals; Cell Division; Cytokines; Gelatinases; Humans; Interleukin-1; Interleukin-10; Interleukin-12; Interleukin-2; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Recombinant Proteins; Skin Neoplasms; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Prostate tumors express high levels of transforming growth factor-beta1 (TGF-beta1) and seem to acquire resistance to its antiproliferative effects with tumor progression. Moreover, TGF-beta1 could be involved in tumor-promoting processes such as angiogenesis, cell migration, and immunosuppression.. Immunoreactivity for TGF-beta1 and its receptors type I and type II (TGFbeta-RI and TGFbeta-RII), tumor vascular count, and cell proliferation were studied in 73 cases of prostate cancer, diagnosed between 1975-1983 and followed with surveillance.. Patients with tumor overproduction of TGF-beta1 had shorter median cancer-specific survival than patients with normal TGF-beta1 immunoreactivity (5.0 vs. 10 years, P = 0.006). Furthermore, increased TGF-beta1 staining was associated with tumor grade, high vascular counts, and metastasis (P = 0.02, 0.02, and 0.01, respectively). Patients with loss of tumor TGFbeta-RII expression in combination with TGF-beta1 overproduction showed particularly short survival (2.6 vs. 10 years, P = 0.0000), when compared to patients with normal immunoreactivity.. Overproduction of TGF-beta1 and loss of TGFbeta-RII expression are associated with poor clinical outcome in prostate cancer, and TGF-beta1 may promote tumor progression by stimulating angiogenesis and metastasis.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Disease Progression; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Metastasis; Neovascularization, Pathologic; Prognosis; Prostatic Neoplasms; Receptors, Transforming Growth Factor beta; Survival Analysis; Transforming Growth Factor beta

Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor beta (TGFbeta) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. Many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFbeta, however, and even secrete TGFbeta. In line with this, TGFbeta is involved in angiogenesis, wound healing and epithelial-mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFbeta-induced EMT maintained via a TGFbeta autocrine loop. Thus, we have analyzed whether signal transduction by the TGFbeta receptor (TGFbetaR) is required for local tumor cell invasion and metastasis.. A dominant-negative type II TGFbetaR (TGFbetaRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFbetaRII-dn blocked TGFbetaR signaling and heavily delayed tumor formation. In EpRas cells, TGFbetaRII-dn prevented EMT. In the dedifferentiated mesenchymal CT26 cells, TGFbetaRII-dn caused mesenchymal-to-epithelial transition and inhibited their in vitro invasiveness in several assays. In addition, TGFbetaRII-dn completely abolished metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lost in vitro invasiveness when treated with neutralizing TGFbeta antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFbetaRII were non-invasive in vitro, a defect restored by re-expressing wild-type TGFbetaRII.. Cell-autonomous TGFbeta signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFbetaRII therefore represents a potential target for therapeutical intervention in human tumorigenesis.

Topics: Animals; Cell Cycle; Colonic Neoplasms; Epithelial Cells; Humans; Mesoderm; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

The ability of TGF-beta 1 (transforming growth factor-beta 1) to suppress growth factor induced proliferation of many cell types in vitro is well documented; however, TGF-beta 1 increases within a similar time frame as the hepatocyte mitogens HGF (hepatocyte growth factor), EGF (epidermal growth factor), and TGF-alpha (transforming growth factor-alpha) prior to hepatocyte proliferation during liver regeneration. This has raised the issue that TGF-beta 1 may have effects on hepatocytes additional to mito-inhibition and that these effects may be relevant to the regenerative process. To this end, we examined the effect of TGF-beta 1 on both the mitogenesis and the motility of growth factor stimulated primary rat hepatocytes and the hepatoblastoma cell line HepG2 in vitro. TGF-beta 1 significantly enhanced the chemotactic motility of EGF or TGF-alpha, and not HGF, stimulated hepatocytes on a collagen I substratum. TGF-beta 1 was not chemotactic when added alone and decreased the DNA synthesis of all hepatocyte cultures to near control levels. HepG2 cells were chemotactic toward HGF, EGF, and TGF-beta 1 alone and displayed an additive chemotactic response when TGF-beta 1 was added to either HGF or EGF. Additionally, HepG2 cells were refractory to the growth stimulatory effects of HGF or EGF and the growth inhibitory effects of TGF-beta 1. Hepatocytes plated onto other collagen-containing substrates (collagen IV, Matrigel, or ECL, an entactin-collagen IV-laminin matrix), but not on fibronectin or laminin alone, also displayed enhanced EGF stimulated motility by TGF-beta 1. The data indicate that an additional, novel role for TGF-beta 1 during liver tissue remodeling following PHx may include the synergistic enhancement EGF stimulated hepatocyte motility responses, and this enhancement is observed only on collagen-containing extracellular matrices.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Movement; Collagen; DNA; Drug Synergism; Epidermal Growth Factor; Extracellular Matrix; Hepatocyte Growth Factor; Liver Regeneration; Neoplasm Metastasis; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

A cell line designated CUMO-2 has been established from an undifferentiated ovarian carcinoma. The s.c. injection of cells into nude mice gave rise to fast-growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites. Histopathologically, the transplanted s.c. tumors closely resembled the original tumor, but tumors developed in the peritoneal cavity were highly anaplastic. The epithelial nature of the cells was confirmed by ultrastructural analysis. Sequential cytogenetic analyses on early and late passages revealed highly aneuploid tumor cells with consistent structural aberrations of chromosomes 1, 3, 8 and 11. CUMO-2 cells were found to produce CA 125 in vitro and in vivo. Cytosol estrogen receptor (ER) was found but progesterone receptor (PR) was not measured. HLA typing indicated the presence of DR8 and DQw4. A gonadotropin-releasing hormone (Gn-RH) analog inhibited cell growth and Gn-RH receptor mRNA was detected by reverse transcription/polymerase chain reaction in this cell line. Administration of transforming growth factor beta 1 inhibited both cell growth and c-myc mRNA expression. This cell line demonstrated a conformational band shift in exon 7 of the p53 gene. It was a frameshift mutation.

Topics: Animals; CA-125 Antigen; Carcinoma; Cell Differentiation; Cell Division; Cell Line; Chromosome Banding; Female; Gene Expression; Genes, myc; Growth Inhibitors; HLA Antigens; Humans; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Ovarian Neoplasms; Polymorphism, Single-Stranded Conformational; Receptors, Estrogen; Receptors, LHRH; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta

The occurrence of breast cancer metastases is preferential to certain organs. Astrocytes may play an important role in the development of brain metastases, as these cells have been shown to respond to extracellular stimuli by producing many cytokines and growth factors that can modulate tumor cell proliferation, growth, and/or metastases. To test this hypothesis, we analyzed the responses of the human breast cancer cell line MDA-MB-435 and its metastatic sublines to astrocyte primary cultures from newborn rat cerebra. Astrocyte purity of the glial cell cultures was demonstrated by glial fibrillary acidic protein and rat neural antigen-2 (Ran-2) immunopositive staining. The 435-Br1 cell line, which was derived from a brain metastases in a nude mouse, showed increased adhesion to astrocytes and enhanced growth in vitro in the presence of media from Con A-stimulated astrocytes, relative to the parental MDA-MB-435 and the lung metastasis-derived variant 435-Lung2. Furthermore, the growth-stimulatory effect was partially reversed by anti-IL-6, anti-transforming growth factor beta (anti-TGF beta), and anti-IGF-I antibodies, indicating that these metastatic cells use exogenous cytokines as paracrine growth factors. In an attempt to elucidate the role of several biologic-response modifiers produced by astrocytes, we tested the responses of MDA-MB-435 cells to purified cytokines and growth factors. We found that the addition of recombinant human or mouse IL-6 produced a variety of responses in the different 435 metastatic variants. Furthermore, IL-6 receptor (IL-6R) expression was slightly increased in the 435-Br1 cells, and exogenous IL-6 rescued 435-Br1 cells from apoptosis in serum-depleted cultures. No apoptotic protective effect was observed in either MDA-MB-435 parental cells or 435-Lung2 cells. Thus, responses to exogenous IL-6 might determine the differences among these metastatic variants by extending cell survival of selected subpopulations, giving them the opportunity to respond to growth factors or other favorable conditions that might be present. These results suggest that cytokines produced by glial cells in vivo may contribute, in a paracrine manner, to the development of brain metastases by breast cancer cells.

Topics: Adult; Animals; Animals, Newborn; Apoptosis; Astrocytes; Blotting, Northern; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Division; Culture Media, Conditioned; Cytokines; Dose-Response Relationship, Drug; Female; Humans; Insulin-Like Growth Factor I; Interleukin-6; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Rats; Rats, Wistar; Receptors, Interleukin-6; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Topics: Bone Marrow; Brain Neoplasms; Carcinoma; Cell Division; Culture Media; Dihydrotestosterone; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Tumor progression to the stage of metastasis may result in part from the selection of certain primary tumor cell clones which are phenotypically competent for survival, invasion, and growth at secondary sites. Selection for traits such as loss of growth inhibitory responses, acquisition of increased adhesiveness, increased local immunosuppression, and enhanced motility and collagenase activities likely contribute to cancer progression and may be regulated through the action of growth factors. The transforming growth factors (TGF-beta) family of growth factors has often been associated with these traits and tumor progression; therefore, elimination or subversion of TGF-beta-responsive pathways should be considered as a mechanistic framework for metastatic events. In this report, we have compared growth and extracellular matrix responses to TGF-beta in six metastatic and six primary tumor-derived cell lines in a mouse model of prostate cancer. We have found that tumor cell lines derived from focal pulmonary metastasis secreted relatively greater quantities of total TGF-betas, lost most or all TGF-beta1 growth inhibition, but responded to TGF-beta1 through induction of the type IV collagenase matrix metalloproteinase-9, whereas cell lines derived from tumors which proliferated at the primary site retained the growth inhibition but lacked collagenase activity. Synthesis of another extracellular matrix protein, plasminogen activator inhibitor 1, was stimulated by TGF-beta1 in both primary as well as metastatic tumors. These results suggest that acquisition of differential responses to the TGF-beta family could result in phenotypic traits which facilitate tumor metastasis from certain primary site clones.

Topics: Animals; Cell Division; Collagenases; Enzyme Induction; Gelatinases; Genes, myc; Genes, p53; Genes, ras; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Mice; Mice, Knockout; Molecular Weight; Neoplasm Metastasis; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

We have reported previously that highly metastatic LuM1 cells derived from colon carcinoma colon 26 secrete larger amounts of gelatinase B than NM11 cells with poor metastatic potential, and that an increase in this gelatinase B secretion can be induced by autocrine factors (Hyup et A, Cancer Res., 54: 3611-3616, 1994). In the present study, a partial characterization was achieved by comparison of the autocrine factor preparation (fraction G) from serum-free medium conditioned with metastatic LuM1 cells with soluble factors known to stimulate gelatinase B secretion. Secretion of gelatinase B by LuM1 cells was augmented by tumor necrosis factor alpha, transforming growth factor beta1 (TGF-beta1), interleukin 1beta, or epidermal growth factor, and specific neutralizing antibodies abolished the induced increases. Platelet-derived growth factor and insulin-like growth factor 1 had no effect on gelatinase B secretion by LuM1 cells. The enhancement of gelatinase B secretion by fraction G was partially inhibited by the antibody to TGF-beta1. TGF-beta1 was detected in both active and latent forms in serum-free medium conditioned with LuM1 or NM11 cells, with the amount of TGF-beta1 higher in the former case. Gelatinase B secretion by LuM1 cells was enhanced by the addition of TGF-beta1 to the culture medium, but that by NM11 cells was not seriously affected, although the latter bound more of the factor. These results indicate the involvement of this growth factor in the autocrine stimulation of gelatinase B secretion by LuM1 cells. However, the autocrine factor effect was not fully explained by TGF-beta1 in the medium, and the involvement of some other unknown factor(s) was thus indicated.

Topics: Animals; Carcinoma; Collagenases; Colonic Neoplasms; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Growth Substances; Interleukin-1; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Metalloendopeptidases; Mice; Neoplasm Metastasis; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2). Six- to 8-week-old female C57B1-6 mice were induced with murine B16 melanoma by tail vein injection. Therapy was started 48 h after tumor injections. Monoclonal anti-TGF beta antibody (2G7) was administered intraperitoneally (i.p.) at 500 micrograms every other day, and IL-2 at 10,000 U i.p. twice daily, for 21 days. A threefold decrease in the number of lesions in the anti-TGF beta/IL-2 treatment group compared with the control group was observed, a highly significant statistical difference (p = 0.002). No statistically significant differences were seen between the control group and other studied groups (IL-2 alone, anti-TGF beta alone). Analysis of TGF beta levels in plasma by the TGF beta-1 Quantikine assay indicated normal levels in the control and IL-2 groups, and significantly diminished levels in the two groups that received TGF beta antibody. However, acid-ethanol extraction of plasma (to reverse antibody binding before assay) showed normal plasma TGF beta levels in all groups, suggesting that the antibody may alter the availability of TGF beta in vivo. Microscopic analysis of metastases revealed a decrease in the average size of lesions in the groups treated with IL-2. Thus, combination therapy using anti-TGF beta antibody and IL-2 may be a novel, less toxic approach to tumor immunotherapy.

Topics: Animals; Antibodies, Monoclonal; Female; Immunotherapy; Interleukin-2; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Transforming Growth Factor beta

Blood levels of transforming growth factor beta 2 (TGF-beta 2) were measured in 20 patients with metastatic breast cancer before and during treatment with the antiestrogen tamoxifen, and in a control group of 7 patients with primary breast cancer before and during adjuvant tamoxifen treatment. The results of this study reveal typical time patterns for TGF-beta 2 in relation to the clinical outcome. Patients in remission showed a significant increase of TGF-beta 2 in the first 4-6 weeks of therapy, followed by a subsequent decrease. Patients who did not respond showed unchanged or diminished TGF-beta 2 values after start of therapy, followed by a later increase preceding the clinical manifestation of tumor progression. Thus, TGF-beta 2 blood levels after 4 weeks of tamoxifen treatment can be used as an early marker for prediction of response.

Topics: Breast Neoplasms; Estrogen Antagonists; Humans; Neoplasm Metastasis; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Transforming Growth Factor beta

Production of metalloproteinases such as collagenases has been reported to be involved in the metastasis of cancer cells. Granulocytic sarcoma in extramedullary sites can be formed by similar steps to other cancers. In this study, we have examined the secretion of type IV collagenases and a tissue inhibitor of metalloproteinase-1 (TIMP-1) in several human leukemia cell lines, including a granulocytic sarcoma-derived cell line established from a patient with granulocytic sarcomas in dermal tissues. We have also examined the invasive capacity of these leukemia cell lines into reconstituted basement membrane, Matrigel, which was used for in vitro invasion assay. Among the human leukemia cell lines used in this study, only the granulocytic sarcoma cell line was found to secrete type IV collagenase constitutively. Other myeloid leukemia cell lines such as HL-60 and U-937 produced type IV collagenase only after treatment with 12-O-tetradecanoylphorbol-13-acetate. All the cell lines secreted similar amounts of the tissue inhibitor of metalloproteinases. In vitro invasion assay revealed that the granulocytic sarcoma cell line showed higher invasive capacity than the other cell lines. These results suggest that the secretion of 92 kDa type IV collagenase plays a role in the leukemia cells' invasion of extramedullary tissues.

Topics: Collagenases; Glycoproteins; Humans; Interferon-alpha; Interleukin-1; Interleukin-6; Leukemia; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Sarcoma; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Monoclonal antibodies (mAb) were prepared against conjugated transforming growth factor beta 1 (TGF beta 1) peptides: amino acid positions 48-60 and positions 86-101. Two antibodies, mAb 16-3G1 [anti-(48-60)] and mAb 5-2G6 [anti-(86-101)] cross-reacted with native TGF beta 1, -beta 2 and -beta 3 (16-3G1) or only with native TGF beta 1 (5-2G6). Both mAb were used to characterize TGF beta-mediated effects on the metastatic potential in nude mice of human carcinoma cell line SLU-1 and its metastatic subline SLU-M1. Autocrine TGF beta 1-mediated up-regulation of cell proliferation and its suppression by anti-TGF beta antibodies in vitro was recorded for SLU-M1 cells whereas SLU-1 cell proliferation in vitro appeared to be refractory to anti-TGF beta antibodies and exogenous TGF-beta 1. However, the potential of s.c. tumours to develop distant metastases in nude mice was about the same for both cell lines. Development of primary tumours and distant metastases could be suppressed by treatment of mice with anti-TGF beta antibodies. Thus we assume that the metastatic potential of tumour cells is independent of TGF beta-mediated growth-regulation effects in vitro. The anti-TGF beta-induced suppression of tumour progression and metastasis in nude mice might rather result from stimulation of the immune surveillance. TGF beta-mediated autocrine down-regulation of MHC-unrestricted cytotoxicity of activated human monocytes and CD56+ LAK cells and its reversion by anti-TGF beta antibodies could be readily demonstrated. In all our experimental series, the neutralizing potential of both anti-TGF beta antibodies, though directed against opposite sites of the TGF beta 1 molecule, was very similar.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; CD56 Antigen; Cytotoxicity, Immunologic; Female; Humans; Killer Cells, Lymphokine-Activated; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Transplantation; Transforming Growth Factor beta; Transplantation, Heterologous

A transforming growth factor beta1 (TGF beta1) antisense expression plasmid under constitutive control of the Rous sarcoma virus promoter was introduced into the highly tumorigenic and invasive colon carcinoma U9A cell line, which uses its autocrine TGF beta1 as a growth-stimulating factor. Stable transfectants were infrequent, and only the K6 transfectant exhibited 39 and 33%, respectively, of the levels of TGF beta1 mRNA and active, secreted TGF beta1 protein of the parental line. K6 exhibited no change in TGF beta2 expression, and TGF beta3 expression was not detected in either parental or transfectant cells. Compared to the parental line, the K6 antisense transfectant exhibited a 3-fold increase in lag time in anchorage-dependent colony formation. The parental line was 44 times as invasive through a collagen l-coated polycarbonate membrane in vitro as K6 cells and, after s.c. injection at low-cell inocula, U9A cells induced tumors 75 times as large in vivo as did the K6 antisense transfectant. The decreases in in vitro invasion and anchorage-dependent colony formation seen in K6 cells were largely reversed by the addition of TGF beta1. Tumors that did arise from the K6 antisense transfectant cells had lost antisense TGF beta1 expression and expressed the same TGF beta1 mRNA levels as controls. U9A cells were more metastatic to the liver after intrasplenic injection than K6 cells. These findings demonstrate a role for autocrine TGE beta1 in colon cancer tumorigenicity and invasion. They also show that a relatively small decrease in TGF beta1 levels was enough to markedly decrease colon carcinoma cell aggressiveness. This is not unprecedented, as we had found in an earlier study that a small, 2-4-fold increase in TGF beta1 protein levels in human colon cancers correlated with disease progression to metastases (E. Friedman et al., Cancer Epidemiol, Biomarkers & Prev., 4:549-554, 1995).

Topics: Animals; Base Sequence; Cell Division; Cell Line; Cell Survival; Colonic Neoplasms; DNA Primers; DNA Probes; DNA, Antisense; Humans; Kinetics; Liver Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Plasmids; Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

A method has been developed to determine true plasma transforming growth factor beta (TGF-beta) levels by using the platelet alpha granule-specific marker, platelet factor 4, to correct for the TGF-beta contributed by platelets degranulated ex vivo. TGF-beta levels were measured on acid-ethanol extracts of human plasma using isoform-specific sandwich enzyme-linked immunosorbent assays. Normal human subjects had 4.1 +/- 2.0 ng/ml TGF-beta1 (range, 2.0-12.0; n = 42), <0.2 ng/ml TGF-beta2, and <0.1 ng/ml TGF-beta3 in their plasma. There were no significant changes with age or with hormonal status, but any given individual showed fluctuations of up to 3-fold in measured plasma TGF-beta levels due to unknown factors. Of 28 patients with advanced metastatic breast cancer, 2 had greatly elevated TGF-beta1 levels, while the rest were in the normal range. The presence of physiologically significant levels of TGF-beta1 in the plasmas of normal human subjects may indicate previously unsuspected endocrine roles for this peptide, while TGF-beta2 and TGF-beta3 appear to act only in a local autocrine/paracrine fashion.

Topics: Adult; Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Platelet Factor 4; Postmenopause; Pregnancy; Premenopause; Protein Isoforms; Reference Values; Reproducibility of Results; Transforming Growth Factor beta

Continuous local delivery of interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) via gene transfer appears to be more effective than systemic therapy in preventing the growth of human renal cell carcinoma (RCC) in vitro and in vivo. To understand further if cytokine-gene transfection of RCC could alter certain cellular properties that are associated with the invasive and metastatic potentials of tumor, the authors characterized six cell lines that produce IL-2 and/or IFN-alpha in their expression of intercellular adhesion molecule-1 (ICAM-1) and CD44; binding affinity to extracellular matrix (ECM) components (fibronectin, laminin, type IV collagen, and vitronectin); and preference in forming homotypic aggregation and mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), tumor transforming growth factor-beta (TGF-beta) and type IV collagenase. These six lines were compared with control vector transfected parental R11 line.. The expression of ICAM-1 and CD44 was determined by fluorescence-activated cell sorter (FACS) analysis, the tumor cell binding affinity to ECM components was measured by cell attachment assay, the degree of homotypic aggregation was quantified by cell aggregation assay, and the mRNA levels of c-myc, EGF-R, TGF-beta, and collagenase were analyzed by a quantitative polymerase chain reaction analysis.. Both IL-2-gene- and IFN-alpha-gene-modified R11 exhibited enhanced expression of ICAM-1, suppression of CD44, and decreased binding affinity to ECM components, when compared with the R11-control vector. All cytokine-producing tumor lines showed a decreased preference to form homotypic aggregation. Interferon-alpha gene transfer downregulated c-myc, EGF-R, and type IV collagenase mRNA expression, whereas only the higher producers of IL-2 downregulated TGF-beta mRNA expression. Exogenous IL-2 and/or IFN-alpha treatment of a IFN-alpha-resistant RCC enhanced both HLA class I antigen and ICAM-1 expression and suppressed CD44 expression, but had no effect on tumor growth rate.. The local production of high concentrations of IL-2 and IFN-a at the tumor site may directly alter tumor properties associated with invasive and metastatic phenotypes of RCC. Interleukin-2 and/or IFN-alpha gene therapy may be an effective strategy for treatment of patients with advanced renal cancer.

Topics: Base Sequence; Carcinoma, Renal Cell; Carrier Proteins; Cell Adhesion; Cell Adhesion Molecules; Cell Membrane; Collagenases; Down-Regulation; Drug Resistance; ErbB Receptors; Extracellular Matrix; Gene Transfer Techniques; Humans; Hyaluronan Receptors; Interferon-alpha; Interleukin-2; Kidney Neoplasms; Matrix Metalloproteinase 9; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; Receptors, Cell Surface; Receptors, Lymphocyte Homing; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF-beta) is a family of multifunctional signalling molecules that play a fundamental role in both normal and malignant cell behavior. Procedures that alter mouse TGF-beta 1 gene expression provide an important approach for analyzing the complex regulatory processes associated with this member of the growth factor family. Therefore, we have designed oligodeoxyribonucleotides (oligos) in an antisense orientation, which are complementary to regions of the TGF-beta 1 message, in an attempt to obtain an oligo sequence that specifically reduces TGF-beta 1 synthesis. We observed that oligos containing a mixture of phosphorothioate and phosphodiester linkages were less toxic and more specific when compared to those only containing phosphorothioate. A non-toxic sequence was identified that markedly reduced the levels of TGF-beta 1 in oligo-treated malignant mouse fibrosarcoma cells. The invasive and metastatic properties of these fibrosarcoma cells were also significantly decreased following treatment with the antisense oligo. The results indicate an important role for altered TGF-beta 1 expression in the regulation of malignant cell proliferation, invasion and metastasis. These results also indicate that this oligo sequence is a useful tool for studies directed towards understanding the complex relationships between TGF-beta 1 and cellular regulation.

Topics: Animals; Base Sequence; Fibrosarcoma; Gene Expression; Mice; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotides, Antisense; Transforming Growth Factor beta; Tumor Cells, Cultured

The aggressiveness of follicular thyroid cancer (FTC) varies widely, and metastasis is the primary cause of death. Uncontrolled proliferation of cancer cells may be associated with loss of growth factor control. We investigated the effects of stimulating (epidermal growth factor [EGF]; thyreotropin [TSH] in low concentrations) and inhibiting growth factors (transforming growth factor beta 1 [TGF beta 1]; TSH in high concentrations) on invasion and growth of FTC cell lines from the thyroid tumor (FTC133) and from the lymph node (FTC236) and lung (FTC238) metastases of the same patient. Invasion-penetration through an 8 microns pore membrane, covered by Matrigel (basement membrane)-and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of all FTC cell lines, but the amplitude of stimulation differed significantly. The parental cell line FTC133 was considerably more responsive to growth factor stimulation than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 8% (EGF; p < 0.02) and 8% (TSH; p < 0.01), and invasion of FTC238 by 9% (EGF; p < 0.02) and 8% (TSH; p < 0.01). Conversely, invasion and growth of FTC133 were significantly more inhibited by TGF beta 1 (10 ng/ml) and supraphysiologic concentrations of TSH (100 mU/ml) than the cell lines from the lymph node and lung metastases. At day 7, invasion of FTC133 was inhibited by 32% (TGF beta 1; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 18% (TGF beta 1; p < 0.02) and 11% (TSH; p < 0.01), and invasion of FTC238 by 16% (TGF beta 1; p < 0.02) and 12% (TSH; p < 0.01). Moreover, we analyzed growth factor independence in minimally supplemented or unsupplemented medium. Growth, but no invasion was evident when cells were cultured completely unsupplemented over 7 days. These results suggest that metastatic FTCs may have developed by escaping from the normal control of TSH and other growth factors.

Topics: Adenocarcinoma, Follicular; Cell Division; Culture Media; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Neoplasm Invasiveness; Neoplasm Metastasis; Thyroid Neoplasms; Thyrotropin; Transforming Growth Factor beta; Tumor Cells, Cultured

We have recently established a human renal cell carcinoma KG-2 line that is tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice but spontaneously metastasizes to the lung only after orthotopic implantation. KG-2 cells growing in the kidney (orthotopic) and lung metastases secreted higher levels of gelatinase than did cells growing in the subcutis (ectopic). We examined whether organ-specific fibroblasts play a role in the regulation of gelatinase production and invasion by renal carcinoma cells. The gelatinase level in the culture supernatants of KG-2 cells was increased by their cultivation with mouse kidney or lung fibroblasts. In contrast, cocultivation of KG-2 cells with mouse skin fibroblasts resulted in a significant reduction of gelatinase activity. Similar results were obtained by culturing KG-2 cells in the media conditioned by the different mouse fibroblasts. We, therefore, investigated effects on KG-2 cells of cytokines and growth factors known to be produced by fibroblasts of various origins. Of ten cytokines and growth factors tested, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-beta 1 (TGF-beta 1) stimulated gelatinase expression by the cultured KG-2 cells. Parallel immunohistochemical analyses revealed that mouse kidney and lung fibroblasts produced higher levels of TGF-beta 1 than did skin fibroblasts. These results indicate that gelatinase production by KG-2 renal cell carcinoma cells is influenced by the organ microenvironment. Specifically, organ-specific fibroblasts regulate the production of degradative enzymes by KG-2 cells and, hence, profoundly influence their invasive and metastatic capacity.

Topics: Animals; Carcinoma, Renal Cell; Fibroblasts; Gelatinases; Humans; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Organ Specificity; Skin Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

Topics: Animals; Antibodies; Breast Neoplasms; Cell Division; Collagen; Cytotoxicity, Immunologic; Factor VIII; Female; Humans; Immunoglobulin G; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Recombinant Proteins; Spleen; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

This study was designed to assess whether the excessive secretion of transforming growth factor-beta 1 (TGF-beta 1) by Chinese hamster ovary (CHO) cells transfected with TGF-beta 1 gene may be linked to the development of a metastatic phenotype. We observed large numbers of metastatic colonies in the lungs of nude mice inoculated with the transfected CHO cells. The tumors derived from these transfected cells demonstrated marked angiogenesis. We postulate that the overproduction of TGF-beta 1 by these tumors may participate in the metastatic progression following establishment of angiogenesis at the primary tumor site.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Female; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Phenotype; Transfection; Transforming Growth Factor beta

Tumor cell metastasis involves a complex series of events, including the adhesion, migration and invasive behavior of tumor cells on components of the extracellular matrix. Multiple cell surface receptors mediate interactions with the surrounding extracellular matrix and thereby influence cell adhesion, motility and invasion. We have previously described a cell surface CD44-related chondroitin sulfate proteoglycan on highly metastatic melanoma cells. CD44-chondroitin sulfate proteoglycan was shown to be important in melanoma cell motility and invasive behavior on type I collagen matrices. In our current studies, the role of cell surface CD44-chondroitin sulfate proteoglycan in collagen-mediated mouse melanoma cell migration and invasive behavior is further evaluated using transforming growth factor-beta 1. We report that transforming growth factor-beta 1 stimulates the migratory and invasive behavior of mouse melanoma cells on type I collagen. Transforming growth factor-beta 1 stimulated cell surface CD44-chondroitin sulfate proteoglycan synthesis in mouse melanoma cells, specifically through an upregulation of chondroitin sulfate production, while the expression of CD44-chondroitin sulfate proteoglycan core protein was not affected. Furthermore, transforming growth factor-beta 1-mediated enhancement of cell polarity, migration and invasive behavior on type I collagen gels was markedly inhibited in the presence of beta-D-xyloside, an agent that blocks chondroitin sulfate addition to the core protein. Collectively, our findings indicate that mouse melanoma cell surface CD44-chondroitin sulfate proteoglycan is required for transforming growth factor-beta 1-enhanced cell motility and invasion, and that CD44-chondroitin sulfate proteoglycan may play a role in forming and/or maintaining a dominant leading lamella, which is required for efficient locomotion.

Topics: Aggrecans; Animals; Antigens, Neoplasm; Cell Movement; Cell Polarity; Chondroitin Sulfate Proteoglycans; Collagen; Culture Media; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Glycoproteins; Glycosides; Hyaluronan Receptors; Lectins, C-Type; Melanoma, Experimental; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Proteoglycans; Receptors, Lymphocyte Homing; Transforming Growth Factor beta; Tumor Cells, Cultured

We characterized three clones of different metastatic capacity (MTC, MTLn2 and MTLn3) derived from the 13762NF rat mammary adenocarcinoma for their production and response to TGF-beta. All three clones expressed comparable amounts of TGF-beta 1 mRNA and secreted 100-300 pg/10(6) cells/24 h in a soluble latent form. TGF-beta was found in extracellular matrices produced by all three tumor clones. Addition of exogenous TGF-beta induced different responses. While the low metastatic clone MTC was highly sensitive to the growth inhibitory effect of TGF-beta (ID50 approximately 50 pg/ml), a 6-fold higher dose was necessary for the high metastatic clone MTLn3 (ID50 approximately 300 pg/ml). The clone with intermediate metastatic potential MTLn2 was unresponsive to TGF-beta (1 pg/ml to 3 ng/ml). Our data suggest that tumor cells can modulate their biological properties in an autocrine and/or paracrine fashion by virtue of expression of TGF-beta.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Division; Clone Cells; Dose-Response Relationship, Drug; Gene Expression; Liver; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Rats; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Low levels of tyrosine and phenylalanine alter the metastatic phenotype of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transforming growth factor beta-like (TGF beta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGF beta 1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF-like and TGF beta-like polypeptides as detected by Western blot analysis. An M(r) 29,000 bFGF-like form eluted from heparin-Sepharose by 0.6 M NaCl was found in extracts of both NDP and LTP cells. Elution at 0.6 M NaCl suggested that this M(r) 29,000 form might be more closely related to FGF-5 than to bFGF. In addition, cell extracts of LTP, but not NDP cells, contained an M(r) 47,000 monomeric bFGF-like form that was not retained on heparin-Sepharose. Three major specific immunoreactive forms of M(r) 44,000, 36,000, and 29,000 were present in conditioned medium from NDP cells. The M(r) 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharose. Only the M(r) 44,000 and 36,000 FGF-like molecules were detected in conditioned medium from LTP cells, and they were also not retained on heparin-Sepharose. Anti-TGF beta antibody that recognized both TGF beta 1 and TGF beta 2 detected 3 different TGF beta-like forms (M(r) 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium from NDP cells contained an M(r) 38,000 form of TGF beta; however, no immunoreactive forms were found in conditioned medium from LTP cells. Thus, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGF beta 1 on proliferation of LTP and NDP cells was determined by [methyl-3H]thymidine uptake. bFGF stimulated proliferation of NDP cells; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGF beta 1. Proliferation of NDP cells was inhibited more by this growth factor than was proliferation of LTP cells. When NDP and LTP cells were incubated with 5 ng/ml TGF beta 1 and various amounts of bFGF, the effect of TGF beta 1 was masked. Antibody depletion of bFGF-like molecules from NDP conditioned medium resulted in the decreased proliferation of NDP cells but not LTP cells. Depletion of TGF beta-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cell

Topics: Animals; Cell Division; Culture Media, Conditioned; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Metastasis; Neoplasm Proteins; Transforming Growth Factor beta

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Growth Substances; Neoplasm Metastasis; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

A long-term continuous exposure to epidermal growth factor (EGF) enhanced the tumorigenicity of spontaneously transformed cells arising in a clonal population of normal cultured rat liver epithelial cells propagated in a selective growth condition. Lengthy EGF exposure also induced the expression of several phenotypes that differed from the phenotypes of rat liver epithelial cells transformed spontaneously in the absence of EGF. Epidermal growth factor treatment caused consistently an enhancement of the constitutive mRNA expression of transforming growth factor-alpha (TGF-alpha), but not of the EGF receptor and transforming growth factor-beta. The overexpression of TGF-alpha persisted in cell lines derived from tumors formed by the EGF-treated transformed cells. These tumors also exhibited high metastatic incidence and ductal cell differentiation. In contrast, untreated spontaneously transformed cells formed non-metastatic tumors with hepatocellular differentiation. These results suggest that long-term, continuous exposure to EGF/TGF-alpha may modulate the phenotypic expressions of neoplastic transformation.

Topics: Animals; Blotting, Northern; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Epithelium; ErbB Receptors; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms; Neoplasm Metastasis; Phenotype; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Rats; Rats, Inbred F344; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.

Topics: Animals; Blotting, Northern; Gene Expression Regulation, Neoplastic; Hot Temperature; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We have shown in previous studies that metastatically-competent variant subpopulations (B5, C1) derived from a non-metastatic murine mammary adenocarcinoma (SP1) have a pronounced growth advantage over their non-metastatic tumor cell counterparts in primary tumors. As a result, primary tumors can be progressively overgrown by cells having the competence to spread elsewhere in the body. This occurs despite any evidence to indicate an intrinsic in vivo growth rate advantage of the metastatic cells when grown as isolated populations. This suggested that cell-cell interactions between metastatic and non-metastatic tumor populations may be involved in the metastatic cell growth dominance process. Evidence was therefore sought for growth factors released by SP1 cells which could preferentially stimulate the B5 or C1 variants and thereby mediate this cell-cell interaction process. We found that cocultures of SP1 and C1 or B5 cells with irradiated C1, B5, or SP1 "feeder" cells showed significant stimulation of C1 and B5 by SP1 "feeder" cells. Cell growth stimulation in response to EGF, TGF-alpha, TGF-beta 1, bFGF, PDGF, NGF, IGF-1, or IGF-2 demonstrated that only TGF-beta 1 could duplicate this effect. A repeat of the coculture experiment in the presence of specific neutralizing anti-TGF-beta antibodies was therefore undertaken and this was found to markedly reduce the stimulation of C1 or B5 cells by irradiated SP1 cells. Conditioned media from the SP1 and C1 cell lines was quantitated for TGF-beta activity and contained 4.5 ng/ml and 2.0 ng/ml, respectively. However, the majority of the TGF-beta released by SP1 cells was found to be spontaneously active, whereas 70% of the TGF-beta released by C1 cells was in its latent form. Scatchard analysis revealed approximately four times the number of TGF-beta receptors, of similar type and affinity, present on C1 as compared with SP1 cells. The in vitro results support the hypothesis that active TGF-beta released by SP1 cells may stimulate the proliferation of metastatic variant cells in a paracrine like fashion. In vivo evidence for this was obtained by showing that coinjection of irradiated SP1 cells could selectively stimulate tumor growth of viable C1 cells and this effect was markedly diminished by neutralizing polyclonal anti-TGF-beta antibodies. Taken together, the results suggest a novel role for TGF-beta in clonal evolution of malignant tumor growth and as a molecular mediator of tumor cell-tumor cell interacti

Topics: Adenocarcinoma; Animals; Antibodies; Cell Communication; Cell Division; Cell Line; Culture Media, Serum-Free; DNA Replication; Female; Growth Substances; Mammary Neoplasms, Experimental; Mice; Mice, Inbred DBA; Mice, Nude; Neoplasm Metastasis; Radioligand Assay; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Thymidine; Transforming Growth Factor beta

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma; Cell Line; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 3; Metalloendopeptidases; Mice; Mice, Inbred CBA; Neoplasm Metastasis; Protease Inhibitors; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Clone Cells; Growth Substances; Heparin; Heparitin Sulfate; Hormones; Humans; In Vitro Techniques; Insulin-Like Growth Factor II; Liver Neoplasms; Neoplasm Metastasis; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

Reduced expression of the nm23 gene in certain rodent model systems and human breast tumors has been correlated with high tumor metastatic potential. To investigate the functional effects of nm23 expression, we have transfected a constitutive murine nm23-1 expression construct into highly metastatic K-1735 TK murine melanoma cells. TK clones expressing the exogenous nm23-1 construct exhibited a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential independent of tumor cell growth, and altered responses to the cytokine transforming growth factor beta 1 in soft agar colonization assays, compared with control-transfected TK clones. In contrast, nm23-1-transfected TK clones exhibited no significant differences in intrinsic tumor cell growth, i.e., primary tumor size in vivo, anchorage-dependent growth rate in vitro, and anchorage-independent colony formation in soft agar in vitro. The data demonstrate a suppressive effect of nm23 on several aspects of the cancer process, including tumor metastasis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Neoplasm; Gene Expression; Genes, Tumor Suppressor; Melanoma; Mice; Molecular Sequence Data; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; Neoplasm Transplantation; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phenotype; Protein Biosynthesis; Proteins; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The experimental metastatic potential of 13762NF mammary adenocarcinoma clone MTLn3 was tested after pretreatment in serum-free medium containing transforming growth factor (TGF) beta 1 at 0-5000 pg/ml. Lung colonies were measured 2 weeks after inoculation in syngeneic F344 rats, and a bell-shaped dose-response curve with 2- to 3-fold increase in number of surface lung metastases was seen. Maximal enhancement occurred at the 50 pg/ml dose level. The effect was specific because addition of neutralizing anti-TGF-beta antibody blocked the stimulatory activity at all levels of TGF-beta 1 pretreatment, but when antibody was given alone, neutralizing anti-TGF-beta antibody had no effect on untreated cells. Increased metastatic potential appears to be from an increased propensity of cells to extravasate as tested in the membrane invasion culture system. MTLn3 cells penetrated reconstituted basement-membrane barriers 2- to 3.5-fold more than did untreated control cells, depending upon length of TGF-beta 1 exposure. Increased invasive potential is apparently due, in part, to a 2- to 6-fold increase in type IV collagenolytic (gelatinolytic) and a 2.4-fold increase in heparanase activity. TGF-beta 1 treatment of MTLn3 cells did not alter their growth rate or morphology in the presence of serum; however, growth was inhibited in serum-free medium. Likewise, adhesion to human umbilical vein endothelial cell monolayers or to immobilized reconstituted basement membrane or fibronectin matrices was unchanged. These results suggest that TGF-beta 1 may modulate metastatic potential of mammary tumor cells by controlling their ability to break down and penetrate basement-membrane barriers.

Topics: Adenocarcinoma; Animals; Antibodies; Cell Line; Female; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Neoplasm Metastasis; Platelet-Derived Growth Factor; Rats; Rats, Inbred F344; Transforming Growth Factor beta; Tumor Cells, Cultured