transforming-growth-factor-beta and Lung-Neoplasms

Lung cancer (LC) is the primary reason for cancer-associated fatalities globally. Due to both tumor-suppressing and tumor-promoting activities, the TGF-β family of growth factors is extremely essential to tumorigenesis. A non-coding single-stranded short RNA called microRNA (miRNA), which is made up of about 22 nt and is encoded by endogenous genes, can control normal and pathological pathways in various kinds of cancer, including LC. Recent research demonstrated that the TGF-β signaling directly can affect the synthesis of miRNAs through suppressor of mothers against decapentaplegic (SMAD)-dependent activity or other unidentified pathways, which could generate allostatic feedback as a result of TGF-β signaling stimulation and ultimately affect the destiny of cancer tissues. In this review, we emphasize the critical functions of miRNAs in lung cancer progression and, more critically, how they affect the TGF-β signaling pathway, and explore the role of both the TGF-β signaling pathway and miRNAs as potential therapeutic targets for improving the treatments of LC patients.

Topics: Humans; Lung; Lung Neoplasms; MicroRNAs; Signal Transduction; Transforming Growth Factor beta

Cancer immunotherapy using monoclonal antibodies targeting immune checkpoints has undoubtedly revolutionized the cancer treatment landscape in the last decade. Immune checkpoint inhibitors can elicit long-lasting, previously unheard-of responses in a number of tumor entities. Yet, even in such tumors as metastatic melanoma and non-small cell-lung cancer, in which immune checkpoint inhibition has become the first-line treatment of choice, only a minority of patients will benefit considerably from these treatments. This has been attributed to a number of factors, including an immune-suppressive tumor microenvironment (TME). Using different modalities to break these barriers is of utmost importance to expand the population of patients that benefit from immune checkpoint inhibition. The multifunctional cytokine transforming growth factor-β (TGF-β) has long been recognized as an immune-suppressive factor in the TME. A considerable number of drugs have been developed to target TGF-β, yet most of these have since been discontinued. The combination of anti-TGF-β agents with immune checkpoint inhibitors now has the potential to revive this target as a viable immunomodulatory therapeutic approach. Currently, a limited number of small molecular inhibitor and monoclonal antibody candidates that target TGF-β are in clinical development in combination with the following immune checkpoint inhibitors: SRK 181, an antibody inhibiting the activation of latent TGF-β1; NIS 793, a monoclonal antibody targeting TGF-β; and SHR 1701, a fusion protein consisting of an anti-PD-L1 monoclonal antibody fused with the extracellular domain of human TGF-β receptor II. Several small molecular inhibitors are also in development and are briefly reviewed: LY364947, a pyrazole-based small molecular inhibitor of the serine-threonine kinase activity of TGFβRI; SB-431542, an inhibitor targeting several TGF-β superfamily Type I activin receptor-like kinases as well as TGF-β1-induced nuclear Smad3 localization; and galunisertib, an oral small molecular inhibitor of the TGFβRI kinase. One of the most advanced agents in this area is bintrafusp alfa, a bifunctional fusion protein composed of the extracellular domain of TGF-β receptor II fused to a human IgG1 mAb blocking PD-L1. Bintrafusp alfa is currently in advanced clinical development and as an agent in this space with the most clinical experience, is a focused highlight of this review.

Topics: Antibodies, Monoclonal; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Immunotherapy; Lung Neoplasms; Recombinant Fusion Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with evidence suggesting they represent a heterogeneous population. This study summarises the prognostic role of all proteins characterised in CAFs with immunohistochemistry in non-small cell lung cancer thus far. The functions of these proteins in cellular processes crucial to CAFs are also analysed. Five databases were searched to extract survival outcomes from published studies and statistical techniques, including a novel method, used to capture missing values from the literature. A total of 26 proteins were identified, 21 of which were combined into 7 common cellular processes key to CAFs. Quality assessments for sensitivity analyses were carried out for each study using the REMARK criteria whilst publication bias was assessed using funnel plots. Random effects models consistently identified the expression of podoplanin (Overall Survival (OS)/Disease-specific Survival (DSS), univariate analysis HR 2.25, 95% CIs 1.80-2.82) and α-SMA (OS/DSS, univariate analysis HR 2.11, 95% CIs 1.18-3.77) in CAFs as highly prognostic regardless of outcome measure or analysis method. Moreover, proteins involved in maintaining and generating the CAF phenotype (α-SMA, TGF-β and p-Smad2) proved highly significant after sensitivity analysis (HR 2.74, 95% CIs 1.74-4.33) supporting attempts at targeting this pathway for therapeutic benefit.

Topics: Actins; Biomarkers, Tumor; Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Fibroblasts; Genetic Heterogeneity; Humans; Lung Neoplasms; Phenotype; Prognosis; Smad2 Protein; Transforming Growth Factor beta; Tumor Microenvironment

Diabetes mellitus (DM), is the most common metabolic disease and is characterized by sustained hyperglycemia. Accumulating evidences supports a strong association between DM and numerous lung diseases including chronic obstructive pulmonary disease (COPD), fibrosis, and lung cancer (LC). The global incidence of DM-associated lung disorders is rising and several ongoing studies, including clinical trials, aim to elucidate the molecular mechanisms linking DM with lung disorders, in particular LC. Several potential mechanisms, including hyperglycemia, hyperinsulinemia, glycation, inflammation, and hypoxia, are cited as plausible links between DM and LC. In addition, studies also propose a connection between the use of anti-diabetic medications and reduction in the incidence of LC. However, the exact cause for DM associated lung diseases especially LC is not clear and is an area under intense investigation. Herein, we review the biological links reported between DM and lung disorders with an emphasis on LC. Furthermore, we report common signaling pathways (eg: TGF-β, IL-6, HIF-1, PDGF) and miRNAs that are dysregulated in DM and LC and serve as molecular targets for therapy. Finally, we propose a nanomedicine based approach for delivering therapeutics (eg: IL-24 plasmid DNA, HuR siRNA) to disrupt signaling pathways common to DM and LC and thus potentially treat DM-associated LC. Finally, we conclude that the effective modulation of commonly regulated signaling pathways would help design novel therapeutic protocols for treating DM patients diagnosed with LC.

Topics: Antineoplastic Agents; Diabetes Mellitus; Humans; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-6; Lung Diseases; Lung Neoplasms; MicroRNAs; Nanomedicine; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

Malignant pleural mesothelioma (MPM) is a rare malignancy of mesothelial cells with increasing incidence, and in many cases, dismal prognosis due to its aggressiveness and lack of effective therapies. Environmental and occupational exposure to asbestos is considered the main aetiological factor for MPM. Inhaled asbestos fibres accumulate in the lungs and induce the generation of reactive oxygen species (ROS) due to the presence of iron associated with the fibrous silicates and to the activation of macrophages and inflammation. Chronic inflammation and a ROS-enriched microenvironment can foster the malignant transformation of mesothelial cells. In addition, MPM cells have a highly glycolytic metabolic profile and are positive in

Topics: Animals; Antineoplastic Agents; Asbestos; Cell Transformation, Neoplastic; Cisplatin; Humans; Loss of Function Mutation; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Oxidation-Reduction; Pleural Neoplasms; Reactive Oxygen Species; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins; Ubiquitin Thiolesterase

Lung cancer is one of the most malignant cancers around the world, with high morbidity and mortality. Metastasis is the leading cause of lung cancer deaths and treatment failure. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs), two groups of small non-coding RNAs (nc-RNAs), are confirmed to be lung cancer oncogenes or suppressors. Transforming growth factor-β (TGF-β) critically regulates lung cancer metastasis. In this review, we summarize the dual roles of miRNAs and lncRNAs in TGF-β signaling-regulated lung cancer epithelial-mesenchymal transition (EMT), invasion, migration, stemness, and metastasis. In addition, lncRNAs, competing endogenous RNAs (ceRNAs), and circular RNAs (circRNAs) can act as miRNA sponges to suppress miRNAs, thereby mediating TGF-β signaling-regulated lung cancer invasion, migration, and metastasis. Through this review, we hope to cast light on the regulatory mechanisms of miRNAs and lncRNAs in TGF-β signaling-regulated lung cancer metastasis and provide new insights for lung cancer treatment.

Topics: Animals; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Lung cancer is the most important cause of cancer-related deaths worldwide and the overall survival of patients with non-small cell lung cancer has not improved. Transforming growth factor beta or TGF-β is a polypeptide member of the transforming growth factor beta superfamily of cytokines, while far fewer clinical studies addressing the association between TGF-β expression and the disease prognosis have been reported up to now. Therefore, our meta-analysis aims to determine the prognostic significance of TGF-β expression in lung cancer patients.. PubMed, EMBASE, the Web of Science and China National Knowledge Infrastructure (CNKI) databases were searched for full-text literature citations. We applied the hazard ratio (HR) with 95% confidence interval (CI) as the appropriate summarized statistics. Q-test and I. Eight eligible studies involving 579 patients were selected for this meta-analysis. The combined HR for the eight eligible studies was 2.17 (95% CI: 1.71-2.77, P < 0.00001) and heterogeneity of overall prognosis was relatively low (I. The present evidence indicates that TGF-β expression can significantly predict the worse prognosis in patients with lung cancer. The findings of our meta-analysis may be confirmed in the future by the use of more updated review pooling and additional relevant investigations.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Female; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Prognosis; Transforming Growth Factor beta

Advanced non-small cell lung cancer (NSCLC) continues to be an incurable family of thoracic malignancies that is chronically managed with chemotherapy, targeted therapy, and immunotherapy. While the discovery of driver oncogenes and the advent of targeted and immunotherapies in the last decade have vastly improved clinical disease management for patients harboring druggable mutations, the mainstay treatment for the majority of NSCLC patients remains cytotoxic chemotherapy. The clinical efficacy of targeted, immune, and cytotoxic therapies is limited by the development of drug resistance. Transforming growth factor beta (TGFβ) signaling, a crucial mediator of embryonic development and peripheral immune tolerance, may be dysregulated in some malignant contexts, including lung cancer, and has been correlated with poor prognosis in advanced cancers. Aberrant upregulation of TGFβ expression in the tumor microenvironment has also been implicated in promoting NSCLC progression and metastasis, as well as driving the development of resistance to cytotoxic, targeted, and immunomodulatory therapeutic interventions. Here, we examine the mechanisms underlying TGFβ-mediated drug resistance in NSCLC, and consider TGFβ as a combinatorial therapeutic intervention to circumvent or delay the development of NSCLC treatment resistance.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Models, Biological; Molecular Targeted Therapy; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor (TGF)-β regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-β have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-β as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-β, in relation to crosstalk with some other signaling pathways, and the roles of TGF-β in lung and pancreatic cancers, in which TGF-β has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-β signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-β plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-β is produced as latent high molecular weight complexes, and the latent TGF-β complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-β activities through regulation of the latent TGF-β complex activation will be discussed.

Topics: Drug Discovery; Humans; Lung Neoplasms; Membrane Proteins; Pancreatic Neoplasms; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is an evolutionarily conserved pleiotropic factor that regulates a myriad of biological processes including development, tissue regeneration, immune responses, and tumorigenesis. TGF-β is necessary for lung organogenesis and homeostasis as evidenced by genetically engineered mouse models. TGF-β is crucial for epithelial-mesenchymal interactions during lung branching morphogenesis and alveolarization. Expression and activation of the three TGF-β ligand isoforms in the lungs are temporally and spatially regulated by multiple mechanisms. The lungs are structurally exposed to extrinsic stimuli and pathogens, and are susceptible to inflammation, allergic reactions, and carcinogenesis. Upregulation of TGF-β ligands is observed in major pulmonary diseases, including pulmonary fibrosis, emphysema, bronchial asthma, and lung cancer. TGF-β regulates multiple cellular processes such as growth suppression of epithelial cells, alveolar epithelial cell differentiation, fibroblast activation, and extracellular matrix organization. These effects are closely associated with tissue remodeling in pulmonary fibrosis and emphysema. TGF-β is also central to T cell homeostasis and is deeply involved in asthmatic airway inflammation. TGF-β is the most potent inducer of epithelial-mesenchymal transition in non-small cell lung cancer cells and is pivotal to the development of tumor-promoting microenvironment in the lung cancer tissue. This review summarizes and integrates the current knowledge of TGF-β signaling relevant to lung health and disease.

Topics: Animals; Asthma; Humans; Lung; Lung Diseases; Lung Neoplasms; Pulmonary Emphysema; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown etiology and dismal prognosis. IPF patients are known to have an increased risk of lung cancer and careful decision-making is required for the treatment of lung cancer associated with IPF. Transforming growth factor (TGF)-β signaling plays a central role in tissue fibrosis and tumorigenesis. TGF-β-mediated pathological changes that occur in IPF lung tissue may promote the process of field cancerization and provide the microenvironment favorable to cancer initiation and progression. This review summarizes the current knowledge related to IPF pathogenesis and explores the molecular mechanisms that underlie the occurrence of lung cancer in the background of IPF, with an emphasis on the multifaceted effects of TGF-β signaling.

Topics: DNA Methylation; Genetic Predisposition to Disease; Humans; Idiopathic Pulmonary Fibrosis; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) plays a crucial role and takes part in many processes in the human body both in physiology and pathology. This cytokine is involved in angiogenesis, regulates apoptosis and stimulates divisions of cells, such as hepatocytes, lymphocytes or hematopoietic cells. SMAD proteins family is a unique group of particles responsible for transducting the signal induced by TGF-β into the nucleus. This molecules, after receiving a signal from activated TGF-β, act on transcription factors in the nucleus, leading directly to the expression of the corresponding genes. According to current knowledge, disturbances in the functioning of SMAD proteins are present in a number of diseases. The reduced expression was observed, for example in cardiovascular diseases such as primary pulmonary hypertension or myocardial infarction, autoimmune diseases for instance systemic lupus erythematosus and multiple sclerosis, Alzheimer's disease or osteoporosis. The latest clinical data showed the presence of mutations in SMAD proteins in cancerogenesis. Mutation of SMAD-4 protein can be detected in half of the patients with pancreatic cancer, 20% of patients with colorectal cancer and 10% of patients with lung cancer. However, mutation in SMAD-2 protein was observed in 7% of both patients with colorectal cancer and lung cancer. On the basis of numerous works, SMAD protein expression would be valuable prognostic factor in some of neoplastic diseases.

Topics: Apoptosis; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Neoplasms; Neovascularization, Pathologic; Pancreatic Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

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

Transforming growth factor β (TGFβ) plays critical roles in regulating a plethora of physiological processes in normal organs, including morphogenesis, embryonic development, stem cell differentiation, immune regulation, and wound healing. Though considered a tumor suppressor, TGFβ is a critical mediator of tumor microenvironment, in which it likewise mediates tumor and stromal cell phenotype, recruitment, inflammation, immune function, and angiogenesis. The fact that activation of TGFβ is an early and persistent event in irradiated tissues and that TGFβ signaling controls effective DNA damage response provides a new means to manipulate tumor response to radiation. Here we discuss preclinical studies unraveling TGFβ effects in cancer treatment and review TGFβ biology in lung cancer as an example of the opportunities for TGFβ pathway inhibition as a pharmaceutical approach to augment radiation therapy.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; DNA Damage; Humans; Lung Neoplasms; Neovascularization, Pathologic; Oxidative Stress; Radiobiology; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Epithelial-mesenchymal transition (EMT) has been widely accepted as the early stage of tumor metastasis, which is accomplished by a group of transcription factors based on cancer genome. However, with the progress of epigenome profiling technique, it has been demonstrated that aberrant histone modifications especially acetylation play an important role in EMT and cancer metastasis. Besides this, numerous studies have elucidated the mechanisms of histone acetyltransferases and deacetylases involved in EMT. Moreover, the network of these histone-related proteins and those transcription factors that play key roles in EMT is under increasing investigation. In addition, the crosstalk among deoxyribonucleic acid methylation, histone acetylation and micro Ribonucleic acid, three major epigenetic modifications, is also an important part in tumor progression. Here, we explore the mechanisms of histone acetylation in EMT and discuss the potential clinical strategies using the epigenetic drugs.

Topics: Acetylation; Animals; Cell Hypoxia; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Histone Deacetylases; Histones; Humans; Lung Neoplasms; Protein Processing, Post-Translational; 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

The signaling pathway mediated by transforming growth factor-beta (TGF-beta) participates in various biologic processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. In the context of cancer, TGF-beta signaling can inhibit tumor growth in early-stage tumors. However, in late-stage tumors, the very same pathway promotes tumor invasiveness and metastasis. This paradoxical effect is mediated through similar to mothers against decapentaplegic or Smad protein dependent and independent mechanisms and provides an opportunity for targeted cancer therapy. This review summarizes the molecular process of TGF-beta signaling and the changes in inhibitory Smads that contribute to lung cancer progression. We also present current approaches for rational therapies that target the TGF-beta signaling pathway in cancer.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Signal Transduction; Smad Proteins, Inhibitory; Transforming Growth Factor beta

Lung cancer is the leading cause of death from cancer worldwide. First-line therapy is based on stage at diagnosis and can include chemotherapy, radiation, and surgery. Despite advances, the prognosis for advanced-stage lung cancer is very poor. Vaccines with the capability to activate the host immune system may have a role in second-line therapy. Advances in the understanding of cellular and molecular immunology are forming the basis for improving vaccine therapy. Most trials to date have demonstrated safety but inconsistent efficacy. Further research is needed to enhance this potential.

Topics: Antigens, Neoplasm; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Dendritic Cells; Genetic Therapy; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Factors; Lung Neoplasms; Neoplasm Proteins; Transforming Growth Factor beta

Although dysfunctional telomeres and oncogenic or stressful stimuli are known to trigger cellular senescence in normal human diploid cells, the molecules and signaling network involved in the cellular senescence program are not fully understood. We have been trying to identify cellular senescence-inducing factors by various means. First, we screened for an extrinsic signal that can induce cellular senescence in human lung adenocarcinoma cell line A549, and identified transforming growth factor-beta (TGF-beta) as the cellular senescence-inducing factor. Cancer cells senesced by treatment with TGF-beta impaired tumorigenicity both in vitro and in vivo, suggesting that cellular senescence functions as a tumor suppression mechanism. Next, we identified 86 independent senescence-associated genes by subtractive screening using A549-derived cell lines. Thirdly, we established novel cell lines (AST cells) from A549 cells exposed to mild oxidative stress. AST cells demonstrated functional impairment of telomerase due to perturbed subcellular localization of human telomerase reverse transcriptase, suggesting that mild oxidative stress might affect the cell fate of cancer cells. These results should provide insight into the molecular basis of the cellular senescence program.

Topics: Cellular Senescence; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Oxidative Stress; Telomerase; Telomere; Transforming Growth Factor beta

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

Molecular targeting is a promising option to increase the radiation response of tumours and to decrease normal tissue reactions, i.e. to achieve therapeutic gain. Molecular targeting substances in themselves are not curative while radiation is a highly efficient cytotoxic agent, with local recurrences often occurring from only few surviving clonogenic cells. High-dose radiotherapy therefore offers optimal conditions to evaluate the potential of specific biology-driven drugs for oncology. This review summarises the current status of preclinical and clinical research on combined radiation with examples of molecular targeting substances relevant for the treatment of NSCLC (EGFR, COX-2, VEGFR, KGF, TGF-beta, BBI).

Topics: Angiogenesis Inhibitors; Animals; Combined Modality Therapy; Cyclooxygenase Inhibitors; ErbB Receptors; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Humans; Lung Neoplasms; Transforming Growth Factor beta; Trypsin Inhibitor, Bowman-Birk Soybean

Clubbing was first described by Hippocrates more than 2.500 years ago. It may be seen alone or as part of an entity called hypertrophic osteoarthropathy which include periostitis, arthritis and sometimes thickening and edema of the skin around the affected joints. Pulmonary diseases such as cancer, abscess, empyema, bronchiectasis and cystic fibrosis are the major diseases known to be associate with hypertrophic osteoarthropathy. Digestive tract cancer, cyanogenic congenital heart disease are well known association. Many theories have attempted to explain the appearance of this sign but few have persisted. In this article, we review characteristics, relation with etiology and the basis of the pathophysiology of hypertrophic osteoarthropathy and particularly of clubbing.

Topics: Bronchiectasis; Causality; Cystic Fibrosis; Digestive System Neoplasms; Empyema; Ferritins; Heart Defects, Congenital; Humans; Lung Abscess; Lung Neoplasms; Osteoarthropathy, Secondary Hypertrophic; Platelet-Derived Growth Factor; Prostaglandins; Transforming Growth Factor beta

Topics: Animals; DNA-Binding Proteins; Drug Design; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Liver Neoplasms; Lung Neoplasms; Mammary Neoplasms, Animal; Melanoma, Experimental; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

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

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Combined Modality Therapy; Diagnosis, Differential; Humans; Lung Neoplasms; Prognosis; Pulse Therapy, Drug; Radiation Pneumonitis; Radiotherapy; Steroids; Transforming Growth Factor beta

Topics: Acute-Phase Reaction; Genes, p53; Genes, ras; Humans; Lung Neoplasms; Mutation; Precancerous Conditions; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Risk Factors; Smoking; Transforming Growth Factor beta

Radiation therapy (RT) is frequently used to treat patients with tumors in and around the thorax. Clinical radiation pneumonitis is a common side effect, occurring in 5% to 20% of patients. Efforts to identify patients at risk for pneumonitis have focused on physical factors, such as dose and volume. Recently, the underlying molecular biological mechanisms behind RT-induced lung injury have come under study. Improved knowledge of the molecular events associated with RT-induced lung injury may translate into a better ability to individualized therapy. This review discusses our current understanding of the physical and molecular factors contributing to RT-induced pulmonary injury.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Models, Biological; Radiation Pneumonitis; Radiotherapy Dosage; Risk Factors; Transforming Growth Factor beta

The maximum dose of radiation which can be delivered to a tumor is limited by the tolerance of the surrounding normal tissues. The ability to identify patients at high or low risk of injury from radiation therapy might enable the clinician to tailor radiation doses in order to maximize efficacy and minimize risk. The cytokine transforming growth factor beta 1 (TGF beta 1) has been implicated in the development of normal tissue injury after irradiation in several organs, including the lung. Herein, the evidence supporting the role of TGF beta 1 in radiation-induced lung injury is reviewed. Using the treatment of non-small cell lung cancer as a model, we also discuss how it may be possible to identify patients at risk for this complication using measurements of plasma TGF beta 1, and how this information may be used in the future to adjust doses of radiation in the treatment of lung cancer.

Topics: Animals; Biomarkers; Carcinoma, Non-Small-Cell Lung; Humans; Lung; Lung Neoplasms; Radiation Injuries; Radiotherapy; Transforming Growth Factor beta

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

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

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

An increased interest in the role of growth factors in the regulation of processes concerning normal and pathologic lung physiology has spurred a flurry of research in this area. Peptide growth factors are known to control not only cell proliferation but other events such as differentiation, chemotaxis, and matrix deposition as well. The transforming growth factor beta (TGF beta) family of regulatory peptides serves as a prime example to illustrate the multiplicity of effects elicited by peptide growth factors in various lung-derived cell types. At present, the TGF beta family consists of at least 17 proteins and, based on sequence analysis, they can be divided into two groups: a cluster that shows very high sequence similarity to TGF beta 1, the closely related group, and a cluster that shows weaker sequence similarity to TGF beta 1, the distantly related group. The purpose of this brief review is to summarize the salient features of TGF beta structure and regulatory abilities of the closely related group. In addition, we will outline the evidence suggesting a role for TGF beta in normal lung development and physiology. Emphasis will be placed on studies with the closely related members TGF beta 1 and TGF beta 2 because, until recently, purified protein was available only for these two proteins.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Extracellular Matrix; Humans; Lung; Lung Neoplasms; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The lung (in particular the bronchial epithelium) is a major site for tumor formation in humans. Environmental factors in conjunction with genetic factors are important determinants in this disease. The acquisition of defects in the control of proliferation and differentiation appears to constitute crucial steps in the transition of a normal to a neoplastic cell. Several factors have been identified that control positively or negatively the proliferation and differentiation of tracheobronchial epithelial cells. These factors include EGF/TGF alpha, TGF beta, insulin/IGFI, KGF, certain cytokines, retinoids, and activators of protein kinase C. Studies with neoplastic cells have identified several protooncogenes and tumor suppressor genes whose gene products are involved in the regulation of cell growth of normal tracheobronchial epithelial cells, and when mutated, lost, or activated, bring about a neoplastic phenotype. Future studies on the precise function of these genes will help to elucidate the mechanisms by which proliferation and differentiation in normal tracheobronchial epithelial cells are regulated and help to understand the molecular changes involved in diseases such as cancer.

Topics: Bronchi; Cell Division; Cell Transformation, Neoplastic; Epithelial Cells; Genes, Tumor Suppressor; Growth Substances; Humans; Lung Neoplasms; Proto-Oncogenes; Retinoids; Trachea; Transforming Growth Factor beta

NIS793 is a human IgG2 monoclonal antibody that binds to transforming growth factor beta (TGF-β). This first-in-human study investigated NIS793 plus spartalizumab treatment in patients with advanced solid tumors.. Patients received NIS793 (0.3-1 mg/kg every 3 weeks (Q3W)) monotherapy; following evaluation of two dose levels, dose escalation continued with NIS793 plus spartalizumab (NIS793 0.3-30 mg/kg Q3W and spartalizumab 300 mg Q3W or NIS793 20-30 mg/kg every 2 weeks [Q2W] and spartalizumab 400 mg every 4 weeks (Q4W)). In dose expansion, patients with non-small cell lung cancer (NSCLC) resistant to prior anti-programmed death ligand 1 or patients with microsatellite stable colorectal cancer (MSS-CRC) were treated at the recommended dose for expansion (RDE).. Sixty patients were treated in dose escalation, 11 with NIS793 monotherapy and 49 with NIS793 plus spartalizumab, and 60 patients were treated in dose expansion (MSS-CRC: n=40; NSCLC: n=20). No dose-limiting toxicities were observed. The RDE was established as NIS793 30 mg/kg (2100 mg) and spartalizumab 300 mg Q3W. Overall 54 (49.5%) patients experienced ≥1 treatment-related adverse event, most commonly rash (n=16; 13.3%), pruritus (n=10; 8.3%), and fatigue (n=9; 7.5%). Three partial responses were reported: one in renal cell carcinoma (NIS793 30 mg/kg Q2W plus spartalizumab 400 mg Q4W), and two in the MSS-CRC expansion cohort. Biomarker data showed evidence of target engagement through increased TGF-β/NIS793 complexes and depleted active TGF-β in peripheral blood. Gene expression analyses in tumor biopsies demonstrated decreased TGF-β target genes and signatures and increased immune signatures.. In patients with advanced solid tumors, proof of mechanism of NIS793 is supported by evidence of target engagement and TGF-β pathway inhibition.. NCT02947165.

Topics: Adult; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Humans; Kidney Neoplasms; Lung Neoplasms; Transforming Growth Factor beta

The safety and efficacy of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor β (TGF-β) receptor II (a TGF-β "trap") fused to a human immunoglobulin G1 antibody blocking programmed death-ligand 1 (PD-L1), was evaluated in patients with advanced NSCLC.. This expansion cohort of NCT02517398, an ongoing, phase 1, open-label trial, includes 80 patients with advanced NSCLC that progressed after platinum doublet therapy or after platinum-based adjuvant or neoadjuvant treatment and those who also have not received previous immunotherapy. Patients were randomized at a one-to-one ratio to receive either bintrafusp alfa 500 mg or the recommended phase 2 dosage of 1200 mg every 2 weeks. The primary end point was the best overall response (by Response Evaluation Criteria in Solid Tumors 1.1 as adjudicated by independent review committee) and was assessed by the objective response rate (ORR).. A total of 80 patients were randomized to receive bintrafusp alfa 500 or 1200 mg (n = 40 each). Median follow-up was 51.9 weeks (IQR, 19.6-74.0). The ORR in all patients was 21.3% (17 of 80). The ORR was 17.5% (seven of 40) and 25.0% (10 of 40) for the 500 mg dose and the 1200 mg dose (recommended phase 2 dose), respectively. At the 1200 mg dose, patients with PD-L1-positive and PD-L1-high (≥80% expression on tumor cells) had ORRs of 36.0% (10 of 27) and 85.7% (six of seven), respectively. Treatment-related adverse events occurred in 55 of the 80 patients (69%) and were graded as greater than or equal to 3 in 23 of the 80 patients (29%). Of the 80 patients, eight (10%) had a treatment-related adverse event that led to treatment discontinuation; no treatment-related deaths occurred.. Bintrafusp alfa had encouraging efficacy and manageable tolerability in patients with NSCLC previously treated with platinum.

Topics: Antibodies, Monoclonal; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Transforming Growth Factor beta

The aim of this study was to evaluate whether genetic variations in the transforming growth factor-β (TGF-β) pathway influenced clinical outcome of advanced lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations treated with gefitinib.. Two hundred six patients with advanced lung adenocarcinomas were enrolled in this study. EGFR mutation in these tumors was detected. Among them, 106 patients with EGFR mutation and 37 of 100 patients with wild type were treated with gefitinib. Genotype of 33 single-nucleotide polymorphisms (SNPs) from 13 genes involved in the TGF-β signaling pathway was determined, and their association with survival time was analyzed. Univariate and multivariate analyses were carried out to assess the role of biological/clinical parameters in progression-free survival (PFS) and overall survival (OS) using Pearson's χ(2) test, log-rank test, and Cox proportional hazards model.. Among SNPs analyzed, multivariate analysis showed the cytidylate and thymidine (CT) genotype of SMAD3: rs11632964 was associated with a longer OS and PFS when the entire cohort of 143 patients were included; the association was significant in the patients with EGFR mutant tumors (30.8 versus 17.5 months; log-rank P = 0.020; and 20.8 versus 9.4 months; log-rank P = 0.001), when compared with patients with wild-type EGFR tumors. In patients with mutant EGFR, the CT genotype of SMAD3: rs11071938 and the cytidylate and cytidylate genotype of SMAD3: rs6494633 were also found to be associated with better PFS. Dual luciferase reporter assays showed gefitinib-resistant PC9/G cells transfected with SMAD3: rs11632964T allelic reporter construct showed significantly lower luciferase activities compared with cells expression C allelic reporter construct. There was significantly decreased expression of SMAD3 and pi-SMAD3 in the PC-9/G cells compared with PC-9.. Among the candidate genes involved in the TGF-β pathway, the polymorphisms of SMAD3 appear to be highly predictive of outcome of patients with lung adenocarcinoma after gefitinib treatment, especially in those with EGFR mutations.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Antineoplastic Agents; Disease Progression; ErbB Receptors; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Polymorphism, Single Nucleotide; Prognosis; Quinazolines; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Treatment Outcome

The aim of the study was to observe the variation of CD4(+)CD25(+) regulatory T cells in periphery blood and tumor microenvironment of non-small cell lung cancer (NSCLC) patients and the effects of CpG ODN. The proportion of CD4(+)CD25(+) regulatory T cells, Foxp3 gene expression, levels of tumor growth factor-β (TGF-β) and immunoreactive fibronectin-γ (IFN-γ) in the periphery blood of 30 NSCLC patients and 30 healthy volunteers were compared. These indicators were compared before and after CpG ODN treatment. Foxp3 gene expression in the tumor microenvironment of NSCLC patients was also observed. The results showed CD4(+)CD25(+) regulatory T cell proportion, Foxp3 expression and TGF-β levels in the periphery blood of NSCLC patients were higher than those of healthy volunteers (p < 0.05), and these indicators of patients were significantly decreased after CpG ODN 2006 treatment (p < 0.05). Foxp3 expression in the metastatic lymph nodes was higher than that in the non-metastatic ones of NSCLC patients (p = 0.000). In conclusion, a rise in the proportion of CD4(+)CD25(+)Foxp3(+) regulatory T cells was demonstrated in the periphery blood and tumor microenvironments of NSCLC patients. CpG ODN 2006 downregulated the CD4(+)CD25(+)Foxp3(+) regulatory T cells proportion and TGF-β levels in the periphery blood of these patients.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Down-Regulation; Female; Forkhead Transcription Factors; Humans; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Lung Neoplasms; Lymph Nodes; Male; Middle Aged; Oligodeoxyribonucleotides; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Radiation induced lung toxicity (RILT) is the main adverse effect in the radiation therapy of lung cancer. However, the optimal management of RILT has not been defined. In this paper, we investigated the effects of rhubarb extract on RILT, pulmonary function (PF), transforming growth factor-beta-1 (TGF-beta1), and interleukin-6 (IL-6) in lung cancer patients treated with radiotherapy.. We conducted a randomized, double-blind, placebo-controlled trial. Eighty consecutive patients were randomly enrolled into two groups: trial group and control group. The trial group received three-dimensional conformal radiation therapy (3D-CRT) plus rhubarb (at a dose of 20 mg kg(-1) once a day) for 6 weeks. The control group received 3D-CRT plus a placebo containing starch for 6 weeks. Plasma TGF-beta1 and serum IL-6 were measured in all patients before, every 2 weeks during, and at 6 weeks after the completion of the treatment. RILT and PF were evaluated at 6 weeks and 6 months after the end of the treatment, respectively. The differences of TGF-beta1, IL-6, RILT, and PF between the two groups were analysed.. The incidence of RILT in the trial group was significantly lower than that in the control group at 6 weeks and 6 months after treatment (32.4% versus 56.7% at week 6, and 27.0% versus 52.8% at month 6, both P<0.05). The plasma TGF-beta1 levels in the trial group were significantly lower than that in the control group during and after the treatment (P<0.05 or 0.01, respectively). The serum IL-6 levels in the trial group were significantly lower than that in the control group during the treatment (all P<0.01). The forced vital capacity (FVC), forced expiratory volume at 1s (FEV1) at 6 weeks and the diffusion capacity for carbon monoxide (DLCO) at 6 months in the trial group were significantly improved compared to the control group (P<0.05 or 0.01, respectively).. The rhubarb extract significantly attenuated RILT and improved PF, probably by decreasing the level of TGF-beta1 and IL-6. These results may be of value for the prophylaxis of RILT, but the exact mechanisms underlying these prophylactic effects remain to be further explored.

Topics: Biomarkers, Tumor; Chromatography, High Pressure Liquid; Dose-Response Relationship, Radiation; Double-Blind Method; Female; Follow-Up Studies; Humans; Interleukin-6; Lung Neoplasms; Male; Middle Aged; Phytotherapy; Plant Preparations; Prospective Studies; Radiation Injuries; Radiotherapy, Conformal; Rheum; Transforming Growth Factor beta; Treatment Outcome

Belagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition.. We performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.25, 2.5, or 5.0 x 10(7) cells/injection) of belagenpumatucel-L on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety, and anticancer activity were monitored.. Seventy-five patients (two stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients) received a total of 550 vaccinations. No significant adverse events were observed. A dose-related survival difference was demonstrated in patients who received > or = 2.5 x 10(7) cells/injection (P = .0069). Focusing on the 61 late-stage (IIIB and IV) assessable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% and 52%, respectively for the higher dose groups combined and 39% and 20%, respectively, for the low-dose group. Immune function was explored in the 61 advanced-stage (IIIB and IV) patients. Increased cytokine production (at week 12 compared with patients with progressive disease) was observed among clinical responders (interferon gamma, P = .006; interleukin [IL] -6, P = .004; IL-4, P = .007), who also displayed an elevated antibody-mediated response to vaccine HLAs (P = .014). Furthermore, positive enzyme-linked immunospot reactions to belagenpumatucel-L showed a correlation trend (P = .086) with clinical responsiveness in patients achieving stable disease or better.. Belagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

Topics: Adult; Aged; Aged, 80 and over; Antibody Formation; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interferon-gamma; Lung Neoplasms; Male; Middle Aged; Oligonucleotides, Antisense; Survival Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta2; Treatment Outcome

To report the incidence of late complications in long-term survivors of very-high-dose thoracic radiotherapy (RT) treated on a prospective clinical trial.. Patients with locally advanced or medically inoperable non-small-cell lung cancer received three-dimensional conformal RT to the primary tumor and radiographically involved lymph nodes to a dose of 73.6 Gy at 1.6 Gy twice daily. If the plasma transforming growth factor-beta1 (TGF-beta1) level was normal after 73.6 Gy, additional twice-daily RT was delivered to successively higher total doses until the maximal tolerated dose was reached. Patients within a given dose level were followed for 6 months before escalation to the next dose level was permitted. Late complications were defined according to Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer criteria.. Thirty-eight patients were enrolled between 1996 and 1999. Twenty-four patients were not eligible for radiation dose escalation beyond 73.6 Gy because of persistently abnormal TGF-beta1 levels. Fourteen patients received dose escalation (80 Gy in 8; 86.4 Gy in 6). Grade 3 or greater late complications occurred in 4 of 24, 1 of 8, and 2 of 6 patients treated to 73.6, 80, and 86.4 Gy, respectively. The corresponding patient numbers with late Grade 4-5 toxicity were 3 of 24, 0 of 6, and 0 of 8. Overall, 7 (18%) of the 38 patients developed Grade 3-5 late toxicity. Nonpulmonary complications predominated (4 of 7). Five (71%) of seven serious complications developed within 11 months after RT; however, the remaining two complications (29%) occurred very late (at 43 and 62 months). The 5-year actuarial risk of late Grade 3-5 complications was 33%.. Long-term survivors of very-high-dose RT for non-small-cell lung cancer have a significant risk of severe treatment-related complications. At these high dose levels, the predominant toxicity may no longer be pulmonary. All Grade 4-5 complications occurred in patients whose dose was limited to 73.6 Gy because of a persistently elevated TGF-beta1. Thus, persistently elevated plasma TGF-beta1 levels toward the end of RT may identify patients at greatest risk of severe complications.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Disease-Free Survival; Dose-Response Relationship, Radiation; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prospective Studies; Radiotherapy, Conformal; Radiotherapy, High-Energy; Transforming Growth Factor beta; Transforming Growth Factor beta1

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

The ability to prescribe treatment based on relative risks for normal tissue injury has important implications for oncologists. In non-small-cell lung cancer, increasing the dose of radiation may improve local control and survival. Changes in plasma transforming growth factor beta (TGFbeta) levels during radiotherapy (RT) may identify patients at low risk for complications in whom higher doses of radiation could be safely delivered.. Patients with locally advanced or medically inoperable non-small-cell lung cancer received three-dimensional conformal RT to the primary tumor and radiographically involved nodes to a dose of 73.6 Gy (1.6 Gy twice daily). If the plasma TGFbeta level was normal after 73.6 Gy, additional twice daily RT was delivered to successively higher total doses. The maximum-tolerated dose was defined as the highest radiation dose at which < or = one grade 4 (life-threatening) late toxicity and < or = two grade 3 to 4 (severe life-threatening) late toxicities occurred.. Thirty-eight patients were enrolled. Median follow-up was 16 months. Twenty-four patients were not eligible for radiation dose escalation beyond 73.6 Gy because of persistently abnormal TGFbeta levels. Fourteen patients whose TGFbeta levels were normal after 73.6 Gy were escalated to 80 Gy (n = 8) and 86.4 Gy (n = 6). In the 86.4-Gy group, dose-limiting toxicity was reached because there were two (33%) grade 3 late toxicities.. It is feasible to use plasma TGFbeta levels to select patients for RT dose escalation for non-small-cell lung cancer. The maximum-tolerated dose using this approach is 86.4 Gy.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Patient Selection; Prospective Studies; Radiation Injuries; Radiotherapy Dosage; Radiotherapy, Conformal; Sensitivity and Specificity; Survival Rate; Transforming Growth Factor beta

Long non-coding RNAs (lncRNAs) participate in carcinogenesis and cancer malignancies. Transforming growth factor-β (TGF-β) is involved in various cellular processes including cancer progression. We performed comprehensive RNA sequencing analyses to identify lncRNAs regulated by TGF-β and found that lincNMR (long intergenic noncoding RNA-nucleotide metabolism regulator, also identified as MAP3K9-DT) was induced by TGF-β in various cell lines. There are several variants of lincNMR (hereafter lincNMRs) in the lincNMR/MAP3K9-DT locus, and their expression was increased by TGF-β. TGF-β-mediated induction of lincNMRs was decreased by depletion of Smad2/3 in Huh7, suggesting that the TGF-β-Smad pathway is involved in lincNMRs expression. We also found that APOBEC3B but not other APOBEC family members were a target gene of lincNMRs. APOBEC3B, a cytidine deaminase, promotes C to U mutation and highly expressed in various human cancers. Although it is associated with cancer progression, regulatory mechanisms of APOBEC3B expression have not been fully elucidated. We performed RNA immunoprecipitation assays and proved that lincNMRs bound to endogenous Smad2 in Huh7 cells. The increased activity of the promoter of APOBEC3B induced by overexpression of Smad2/3 was inhibited by depletion of lincNMRs. These data suggest that lincNMRs participate in APOBEC3B expression by collaborating with TGF-β-Smad pathway. High expression of lincNMRs was positively correlated with high expression of APOBEC3B in various cancer cell lines. Overexpression of APOBEC3B as well as lincNMR was found in human cancers such as hepatic and lung cancers and was associated with their poor prognosis, suggesting that lincNMR may contribute to tumor malignancy via enhanced expression of APOBEC3B.

Topics: Cell Line, Tumor; Cytidine Deaminase; Humans; Liver; Lung Neoplasms; MAP Kinase Kinase Kinases; Minor Histocompatibility Antigens; RNA, Long Noncoding; Transforming Growth Factor beta

Bone tissue is a common metastatic site of lung cancer, and bone metastasis is characterized by abnormal differentiation and malfunction of osteoclast, and the roles of exosomes derived from lung cancer have attracted much attention. In our study, we found that the level of HOTAIR expression in A549 and H1299 exosomes was higher than those of normal lung fibrocytes. Overexpression of HOTAIR in A549 and H1299 exosomes promoted osteoclast differentiation. Furthermore, A549-Exos and H1299-Exos targeted bone tissues, and bone formation was significantly inhibited in vivo. Mechanistically, exosomal lncRNA HOTAIR promoted bone resorption by targeting TGF-β/PTHrP/RANKL pathway.

Topics: Cell Differentiation; Exosomes; Humans; Lung Neoplasms; Osteoclasts; Parathyroid Hormone-Related Protein; RANK Ligand; RNA, Long Noncoding; Transforming Growth Factor beta

Immune-checkpoint inhibitors (ICIs) are an effective therapeutic strategy, improving the survival of patients with lung cancer compared with conventional treatments. However, novel predictive biomarkers are needed to stratify which patients derive clinical benefit because the currently used and highly heterogenic histological PD-L1 has shown low accuracy. Liquid biopsy is the analysis of biomarkers in body fluids and represents a minimally invasive tool that can be used to monitor tumor evolution and treatment effects, potentially reducing biases associated with tumor heterogeneity associated with tissue biopsies. In this context, cytokines, such as transforming growth factor-β (TGF-β), can be found free in circulation in the blood and packaged into extracellular vesicles (EVs), which have a specific delivery tropism and can affect in tumor/immune system interaction. TGF-β is an immunosuppressive cytokine that plays a crucial role in tumor immune escape, treatment resistance, and metastasis. Thus, we aimed to evaluate the predictive value of circulating and EV TGF-β in patients with non-small-cell lung cancer receiving ICIs.. Plasma samples were collected in 33 patients with advanced non-small-cell lung cancer before and during treatment with ICIs. EV were isolated from plasma by serial ultracentrifugation methods and circulating and EV TGF-β expression levels were evaluated by enzyme-linked immunosorbent assay.. Baseline high expression of TGF-β in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-β levels and tissue PD-L1 as a predictive biomarker.. If validated, EV TGF-β could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-β blockade.. Treatment with immune-checkpoint inhibitors (ICIs) has improved the survival of some patients with lung cancer. However, the majority of patients do not benefit from this treatment, making it essential to develop more reliable biomarkers to identify patients most likely to benefit. In this pilot study, the expression of transforming growth factor-β (TGF-β) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-β before treatment were able to determine which patients would benefit from treatment with ICIs and have a longer survival with higher accuracy than circulating TGF-β and tissue PD-L1, which is the currently used biomarker in clinical practice.

Topics: B7-H1 Antigen; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Extracellular Vesicles; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Lung Neoplasms; Pilot Projects; Transforming Growth Factor beta; Transforming Growth Factors

Epithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF-β-induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF-β/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP-glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF-β signaling and TGF-β-induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF-β signaling by promoting lipid raft localization of the TGF-β type I receptor (TβRI) and by increasing TβRI ubiquitination and degradation. Importantly, we identified ST3GAL5-synthesized a-series gangliosides as the main GSL subtype involved in inhibition of TGF-β signaling and TGF-β-induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis.

Topics: Animals; Catalysis; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gangliosides; Glycosphingolipids; Humans; Lung Neoplasms; Mice; Transforming Growth Factor beta; Zebrafish

Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor beta receptor II (a TGF-β "trap") fused to a human immunoglobulin G1 monoclonal antibody blocking programmed cell death 1 ligand 1 (PD-L1). We report the efficacy and safety in patients with non-small cell lung cancer (NSCLC) that progressed following anti-PD-(L)1 therapy.. In this expansion cohort of NCT02517398-a global, open-label, phase I trial-adults with advanced NSCLC that progressed following chemotherapy and was primary refractory or had acquired resistance to anti-PD-(L)1 treatment received intravenous bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. The primary endpoint was best overall response (by Response Evaluation Criteria in Solid Tumors version 1.1 adjudicated by independent review committee); secondary endpoints included safety.. Eighty-three eligible patients (62 [74.7%] treated with ≥3 prior therapies) received bintrafusp alfa. Four patients (3 primary refractory, 1 acquired resistant) had confirmed partial responses (objective response rate, 4.8%; 95% CI, 1.3%-11.9%), and 9 had stable disease. Tumor cell PD-L1 expression was not associated with response. Nineteen patients (22.9%) experienced grade ≥3 treatment-related adverse events, most commonly asthenia (3 [3.6%]) and fatigue, eczema, and pruritus (2 each [2.4%]). One patient had grade 4 amylase increased. One patient died during treatment for pneumonia before initiation of bintrafusp alfa.. Although the primary endpoint was not met, bintrafusp alfa showed some clinical activity and a manageable safety profile in patients with heavily pretreated NSCLC, including prior anti-PD-(L)1 therapy. Tumor responses occurred irrespective of whether disease was primary refractory or had acquired resistance to prior anti-PD-(L)1 therapy.

Topics: Adult; Antineoplastic Agents, Immunological; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Lung Neoplasms; Transforming Growth Factor beta

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGF

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Integrins; Ligands; Lung Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial-to-mesenchymal transition (EMT) of primary cancer contributes to the acquisition of lethal properties, including metastasis and drug resistance. Blocking or reversing EMT could be an effective strategy to improve cancer treatment. However, it is still unclear how to achieve complete EMT reversal (rEMT), as cancer cells often transition to hybrid EMT states with high metastatic potential. To tackle this problem, we employed a systems biology approach and identified a core-regulatory circuit that plays the primary role in driving rEMT without hybrid properties. Perturbation of any single node was not sufficient to completely revert EMT. Inhibition of both SMAD4 and ERK signaling along with p53 activation could induce rEMT in cancer cells even with TGFβ stimulation, a primary inducer of EMT. Induction of rEMT in lung cancer cells with the triple combination approach restored chemosensitivity. This cell-fate reprogramming strategy based on attractor landscapes revealed potential therapeutic targets that can eradicate metastatic potential by subverting EMT while avoiding hybrid states.. Network modeling unravels the highly complex and plastic process regulating epithelial and mesenchymal states in cancer cells and discovers therapeutic interventions for reversing epithelial-to-mesenchymal transition and enhancing chemosensitivity.

Topics: Cell Differentiation; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

We assessed the function and mechanism of RNA binding motif protein 15 (RBM15) silencing in lung cancer development.. The effects of RBM15 knockdown on A549 and H1299 cells were evaluated by MTT, EdU, wound healing, and transwell assay. We then detected the functions of RBM15 silencing on lipid peroxidation, labile iron pool (LIP), ferrous iron (Fe. We demonstrated that RBM15 silencing promoted ferroptosis in lung cancer cells by TGF-β/Smad2 pathway, thereby inhibiting lung cancer cell growth, which may provide new light for lung cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Ferroptosis; Humans; Lung Neoplasms; Mice; Mice, Knockout; RNA-Binding Proteins; Smad2 Protein; Transforming Growth Factor beta

TGF-β induces senescence in embryonic tissues. Whether TGF-β in the hypoxic tumor microenvironment (TME) induces senescence in cancer and how the ensuing senescence-associated secretory phenotype (SASP) remodels the cellular TME to influence immune checkpoint inhibitor (ICI) responses are unknown. We show that TGF-β induces a deeper senescent state under hypoxia than under normoxia; deep senescence correlates with the degree of E2F suppression and is marked by multinucleation, reduced reentry into proliferation, and a distinct 14-gene SASP. Suppressing TGF-β signaling in tumors in an immunocompetent mouse lung cancer model abrogates endogenous senescent cells and suppresses the 14-gene SASP and immune infiltration. Untreated human lung cancers with a high 14-gene SASP display immunosuppressive immune infiltration. In a lung cancer clinical trial of ICIs, elevated 14-gene SASP is associated with increased senescence, TGF-β and hypoxia signaling, and poor progression-free survival. Thus, TME-induced senescence may represent a naturally occurring state in cancer, contributing to an immune-suppressive phenotype associated with immune therapy resistance.

Topics: Animals; Cellular Microenvironment; Cellular Senescence; Disease Models, Animal; Humans; Lung Neoplasms; Mice; Phenotype; Transforming Growth Factor beta; Tumor Microenvironment

Forkhead box L2 (FOXL2) has been recognized as a transcription factor in the progression of many malignancies, but its role in non-small cell lung cancer (NSCLC) remains unclear. This research clarified on the role of FOXL2 and the specific molecular mechanism in NSCLC.. RNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting assays. Cell proliferation was examined by cell counting kit-8 (CCK-8) and clonogenic assays. Transwell and wound healing assays were used to detect cell invasion and migration. Cell cycle alterations were assessed by flow cytometry. The relationship between FOXL2 and miR-133b was verified by dual-luciferase reporter assays. In vivo metastasis was monitored in the tail vein-injected mice.. FOXL2 was upregulated in NSCLC cells and tissues. Downregulation of FOXL2 restrained cell proliferation, migration, and invasion and arrested the cell cycle of NSCLC cells. Moreover, FOXL2 promoted the epithelial-mesenchymal transition (EMT) process of NSCLC cells by inducing the transforming growth factor-β (TGF-β)/Smad signaling pathway. miR-133b directly targeted the 3'-UTR of FOXL2 and negatively regulated FOXL2 expression. Knockdown of FOXL2 blocked metastasis in vivo.. miR-133b downregulates FOXL2 by targeting the 3'-UTR of FOXL2, thereby inhibiting cell proliferation, EMT and metastasis induced by the TGF-β/Smad signaling pathway in NSCLC. FOXL2 may be a potential molecular target for treating NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice; MicroRNAs; Transforming Growth Factor beta

Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. Identifying key molecular targets related to the initiation, development, and metastasis of lung cancer is important for its diagnosis and target therapy. The ADAMTS families of multidomain extracellular protease enzymes have been reported to be involved in many physiological processes. In this study, we found that ADAMTS1 was highly expressed in NSCLC tissues, which promoted cell proliferation, migration, invasion, and epithelial to mesenchymal transition (EMT) of NSCLC cells. In the NSCLC tumor metastasis model involving nude mice, overexpression of ADAMTS1 promoted EMT and lung metastasis of tumor cells. Moreover, ADAMTS1 positively regulated TGF-β expression, and TGF-β was highly expressed in NSCLC tumor tissues. si-TGF-β or inhibition of TGF-β expression through the short peptide KTFR on ADAMTS1 protein could reverse the oncogenic effects of ADAMTS1 on lung cancer cells. Taken together, ADAMTS1 functioned as an oncogene in NSCLC cells by promoting TGF-β expression, indicating that ADAMTS1 has important regulatory roles in the progression of NSCLC.

Topics: ADAMTS1 Protein; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Transforming Growth Factor beta

Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the lung adenocarcinoma tissues examined in the present study, expression of claudin-2 was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells. The EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-β type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-β1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-β1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-β1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma.

Topics: A549 Cells; Adenocarcinoma of Lung; Claudin-2; Down-Regulation; Epidermal Growth Factor; Humans; Lung Neoplasms; Tight Junctions; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

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

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-β target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-β-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-β/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-β type I receptor (TβRI). LITATS1 interacts with TβRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-β/SMAD signaling and EMT.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Plasticity; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Receptor, Transforming Growth Factor-beta Type I; RNA, Long Noncoding; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

An emerging view regarding cancer-associated fibroblast (CAF) is that it plays a critical role in tumorigenesis and immunosuppression in the tumor microenvironment (TME), but the clinical significance and biological functions of CAFs in non-small cell lung cancer (NSCLC) are still poorly explored. Here, we aimed to identify the CAF-related signature for NSCLC through integrative analyses of bulk and single-cell genomics, transcriptomics, and proteomics profiling. Using CAF marker genes identified in weighted gene co-expression network analysis (WGCNA), we constructed and validated a CAF-based risk model that stratifies patients into two prognostic groups from four independent NSCLC cohorts. The high-score group exhibits a higher abundance of CAFs, decreased immune cell infiltration, increased epithelial-mesenchymal transition (EMT), activated transforming growth factor beta (TGFβ) signaling, and a limited survival rate compared with the low-score group. Considering the immunosuppressive feature in the high-score group, we speculated an inferior clinical response for immunotherapy in these patients, and this association was successfully verified in two NSCLC cohorts treated with immune checkpoint blockades (ICBs). Furthermore, single-cell RNA sequence datasets were used to clarify the molecular mechanisms underlying the aggressive and immunosuppressive phenotype in the high-score group. We found that one of the genes in the risk model, filamin binding LIM protein 1 (FBLIM1), is mainly expressed in fibroblasts and upregulated in CAFs compared to fibroblasts from normal tissue. FBLIM1-positive CAF subtype was correlated with increased TGFβ expression, higher mesenchymal marker level, and immunosuppressive tumor microenvironment. Finally, we demonstrated that FBLIM1 might serve as a poor prognostic marker for immunotherapy in clinical samples. In conclusion, we identified a novel CAF-based classifier with prognostic value in NSCLC patients and those treated with ICBs. Single-cell transcriptome profiling uncovered FBLIM1-positive CAFs as an aggressive subtype with a high abundance of TGFβ, EMT, and an immunosuppressive phenotype in NSCLC.

Topics: Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Cell Adhesion Molecules; Cytoskeletal Proteins; Humans; Immunotherapy; Lung Neoplasms; Prognosis; Single-Cell Gene Expression Analysis; Transforming Growth Factor beta; Tumor Microenvironment

Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) have been the focus of numerous studies, and some circRNAs have been linked to the development of multiple malignant tumors, including NSCLC. Nevertheless, the functional role and mechanisms of circRNAs in NSCLC remain largely unknown. The primary objective of this study was to screen the associated circRNA in NSCLC and investigate its mechanism. CircRNA microarray was used to identify circRNAs that were abnormally expressed in NSCLC tissue samples. Expression of hsa_circRNA_0088036 was validated in NSCLC tissues and cell lines after the correlation between hsa_circRNA_0088036 and prognosis was determined. We then used a series of function gain-and-loss assays to determine the role of hsa_circ_0088036 in NSCLC progression. RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and RNA interference assays were used to assess the interaction between hsa_circ_0088036 and miR-1343-3p/Bcl-3 axis. Moreover, mechanistic assays were applied to investigate the involved signaling pathway regulated by the hsa_circ_0088036/miR-1343-3p/Bcl-3 axis. Microarray analysis and reverse transcription polymerase chain reaction confirmed the presence of a circRNA termed hsa_circ 0088036 that was upregulated in NSCLC tissue samples and cell lines and indicated a positive association with patient prognosis. Functionally, hsa_circ_0088036 silencing inhibited proliferative, invasive, and migrative potential of NSCLC cells as well as epithelial-mesenchymal transition (EMT)-related proteins by sponging miR-1343-3p to inhibit Bcl-3. Furthermore, mechanistic experiments demonstrated that hsa_circ_0088036 promoted NSCLC progression by activating the TGFβ/Smad3/EMT signaling pathway via miR-1343-3p/Bcl-3 axis. In conclusion, hsa_circ_0088036 functions as an oncogene by targeting the miR-1343-3p/Bcl-3 axis via TGFβ/Smad3/EMT signaling pathway.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; MicroRNAs; RNA, Circular; Transforming Growth Factor beta

IFNγ-mediated signaling in tumor cells can induce immunosuppressive responses and cause tumor resistance to immunotherapy. Blocking TGFβ promotes T lymphocyte infiltration and turns immunologically cold tumors into hot tumors, thereby improving the efficacy of immunotherapy. Several studies have shown that TGFβ inhibits IFNγ signaling in immune cells. We thus sought to determine whether TGFβ affects IFNγ signaling in tumor cells and plays a role in the development of acquired resistance to immunotherapy. TGFβ stimulation of tumor cells increased SHP1 phosphatase activity in an AKT-Smad3-dependent manner, decreased IFNγ-mediated tyrosine phosphorylation of JAK1/2 and STAT1, and suppressed the expression of STAT1-dependent immune evasion-related molecules, e.g., PD-L1, IDO1, herpes virus entry mediator (HVEM), and galectin-9 (Gal-9). In a lung cancer mouse model, dual blockade of TGFβ and PD-L1 led to superior antitumor activity and prolonged survival compared with anti-PD-L1 therapy alone. However, prolonged combined treatment resulted in tumor resistance to immunotherapy and increased expression of PD-L1, IDO1, HVEM, and Gal-9. Interestingly, after initial anti-PD-L1 monotherapy, dual TGFβ and PD-L1 blockade promoted both immune evasion gene expression and tumor growth compared with that in tumors treated with continuous PD-L1 monotherapy. Alternatively, treatment with JAK1/2 inhibitor following initial anti-PD-L1 therapy effectively suppressed tumor growth and downregulated immune evasion gene expression in tumors, indicating the involvement of IFNγ signaling in immunotherapy resistance development. These results demonstrate an unappreciated effect of TGFβ on the development of IFNγ-mediated tumor resistance to immunotherapy.. Blocking TGFβ facilitates IFNγ-mediated resistance to anti-PD-L1 therapy due to the role of TGFβ in inhibiting IFNγ-induced immunoevasion by increasing SHP1 phosphatase activity in tumor cells.

Topics: Adenocarcinoma of Lung; Animals; B7-H1 Antigen; Cell Line, Tumor; Immune Evasion; Immunotherapy; Interferon-gamma; Lung Neoplasms; Mice; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta

As the most classic and extensively studied transcription factor in response to environmental toxic chemicals, the human aryl hydrocarbon receptor (AHR) has been implicated in mediating some oncogenic responses also. Limited information is available, however, on whether arsenic, a widely presented environmental carcinogen, can regulate AHR to exert its carcinogenic activity. Through chromatin immunoprecipitation and sequencing (ChIP-seq), CRISPR-Cas9 gene editing, RNA-seq, and immunohistochemistry (IHC), in this report we provided evidence showing that arsenic enforces TGFβ and other oncogenic signaling pathways in bronchial epithelial cells through disrupting the tumor suppressor-like activity of AHR. AHR is normally enriched on a number of oncogenic genes in addition to the known phase I/II enzymes, such as genes in TGFβ and Nrf2 signaling pathways and several known oncogenes. Arsenic treatment substantially reduced the binding of AHR on these genes followed by an increased expression of these genes. CRISPR-Cas9-based knockout of AHR followed by RNA-seq further demonstrated increased expression of the TGFβ signaling and some oncogenic signaling pathway genes in the AHR knockout cells. IHC studies on human tissue samples revealed that normal human lung tissues expressed high level of AHR. In contrast, the AHR expression was diminished in the lung cancer tissues. Accordingly, the data from this study suggest that AHR has tumor suppressor-like activity for human lung cancer, and one of the carcinogenic mechanisms of arsenic is likely mediated by the inhibition of arsenic on the tumor suppressor-like activity of AHR.

Topics: Arsenic; Epithelial Cells; Humans; Lung Neoplasms; Receptors, Aryl Hydrocarbon; Transforming Growth Factor beta

ING5 belongs to the inhibitor of growth (ING) candidate tumor suppressor family, which is involved in multiple cellular functions, such as cell cycle regulation, apoptosis, and chromatin remodelling. Previously, we reported that ING5 overexpression inhibits EMT by regulating EMT-related molecules, including Snail1, at the mRNA and protein levels. However, the mechanisms remain unclear. In the current study, we identify that ING5 overexpression induces the upregulation of miR-34c-5p. The expression levels of both ING5 and miR-34c-5p in NSCLC tissues from the TCGA database are decreased compared with that in adjacent tissues. Higher expression levels of both ING5 and miR-34c-5p predict longer overall survival (OS). Snail1 is the target gene of miR-34c-5p, as predicted by an online database, which is further verified by a dual-luciferase reporter assay. The expression level of Snail1 in NSCLC cells is markedly reduced following miR-34c-5p overexpression, leading to the inactivation of the Snail1 downstream TGF-β/Smad3 signaling pathway. The TGF-β signaling-specific inhibitor LY2157299 reverses the enhanced EMT, proliferation, migration, and invasion abilities induced by the miR-34c-5p inhibitor. Furthermore, tail vein injection of miR-34c-5p agomir inhibits xenografted tumor metastasis. Overall, this study concludes that miR-34c-5p, induced by ING5 overexpression, is a tumor suppressor that targets Snail1 and mediates the inhibitory effects of ING5 on the EMT and invasion of NSCLC cells. These results provide a novel mechanism mediating the antitumor effects of ING5.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

Lung adenocarcinoma (LUAD) is highly malignant and associated with poor prognoses in patients worldwide. There has been widespread recognition that lncRNAs are tightly linked to LUAD tumorigenesis and development. Here, we identified that the LINC00621 level was increased in LUAD tissues and concerned with the poor prognoses in LUAD patients.. Bioinformatical analysis and RT-qPCR determined the level of LINC00621 in LUAD tissues and cell lines. The admeasurement of the proliferation, migration, and invasion abilities of LUAD cells was utilized in the CCK8 and Transwell formulas. Luciferase reporter assay was used to corroborate the downstream target genes of LINC00621. The phosphorylated SMAD3 protein was tested by Western blotting assay. The impression of LINC00621 knockdown on LUAD tumor growth and metastasis put into effect by murine models. ChIP-qPCR assay was carried out to verify the transcriptional regulation by FOXA1 on LINC00621.. In vitro, the knockdown of LINC00621 significantly reduced the proliferative, migrating, and invasive abilities, the same was true for tumorigenesis and metastasis in vivo. MiR-34a-5p as a straight target of LINC00621 was ascertained, and LUAD patients with inferior miR-34a-5p levels had undesirable prognoses. Furthermore, TGFBR1 is an immediate and functional connection site of miR-34a-5p. Collectively, LINC00621 can sponge miR-34a-5p and upregulate TGFBR1 levels, which further sensitized TGF-β signaling pathway. Finally, it was revealed that FOXA1 transcriptionally upregulated LINC00621.. This study uncovered that FOXA1-induced LINC00621 promotes LUAD progression via the miR-34a-5p/TGFBR1/TGF-β axis, and is one novel therapeutic target that may be used in LUAD treatment.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Mice; MicroRNAs; Receptor, Transforming Growth Factor-beta Type I; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Dendritic cells (DCs) are difficult to evaluate in lung regional lymph nodes because of region-specific structures, such as abundant trabeculae connecting the medullary and subcapsular sinuses, the latter of which contains few anthracotic macrophages. Therefore, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DCsign)-positive DCs and CD68-positive macrophages are unlikely to show a typical distribution. The present study therefore explored quantitative factors connecting the nodal DC morphology to the patient outcome.. Lymph nodes from 34 non-small-cell lung cancer patients who underwent complete resection were used for immunohistochemical assessments of DCsign and CD68 and terminal deoxynucleotidyl transferase dUTP nick-end labeling. Preoperative patient blood samples were used for the quantitative evaluation of monocytes.. The nodal DCs showed a complementary distribution with macrophages, thus few DCs were seen in clusters of macrophages. DCs often presented as a mesh-like rosette that was solitary or connected to a DC cluster. DCs disappeared, and some macrophages were apoptotic when surrounded by cancer cells that have metastasized to lymph nodes. The proportional area of a DC cluster was significantly associated with the histological differentiation of cancer (p=0.013), with a higher ratio tending to lead to a better overall survival (p=0.059), and significantly so in adenocarcinoma (p=0.007). The rosette number was significantly correlated with the smoking index and blood monocyte number (p=0.013 and p=0.005, respectively).. The nodal DC morphology appears useful as a prognostic factor and may lead to a new phase of clinicopathological studies of solid cancers.

Topics: Carcinoma, Non-Small-Cell Lung; Clinical Relevance; Dendritic Cells; Humans; Lung Neoplasms; Lymph Nodes; Macrophages; Monocytes; Transforming Growth Factor beta

Recent advancements in immunotherapy led to the development of Chimeric antigen receptor (CAR) T-cell therapy. CAR-T cell therapy in non-small cell lung cancer (NSCLC) is hindered by overexpression of transforming growth factor (TGFβ) in the cancer cells that have a negative regulatory role on T-cells activity. This study characterized CAR-T with overexpression of mothers against decapentaplegic homologue 7 (SMAD), a negative regulator of TGFβ downstream signalling.. We have generated three types of CAR-T: epidermal growth factor receptor (EGFR)-CAR-T, EGFR-dominant-negative TGFbeta receptor 2 (DNR)-CAR-T, and EGFR-SMAD7-CAR-T by transducing human T-cells with the lentivirus constructs. We characterized the proliferation, expression of proinflammatory cytokines, activation profile, and lysis capacity in co-cultures with A549 lung carcinoma cells with and without TGFβ neutralizing antibodies. We also tested the therapeutic potential of EGFR-SMAD7-CAR-T in the A549 cells tumour-bearing mice model.. Both EGFR-DNR-CAR-T and EGFR-SMAD7-CAR-T demonstrated a higher proliferation rate and lysis capacity to A549 than traditional EGFR-CAR-T. Neutralization of TGFβ by the antibodies resulted in increased performance of EGFR-CAR-T. In vivo, both EGFR-DNR-CAR-T and EGFR-SMAD7-CAR-T resulted in complete tumour resorption by day 20, whereas conventional CAR-T only has a partial effect.. We demonstrated the high efficacy and resistance to negative TGFβ regulation of EGFR-SMAD7-CAR-T comparable with EGFR-DNR-CAR-T and without the systemic effect of TGFβ inhibition.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Humans; Lung Neoplasms; Mice; Receptors, Chimeric Antigen; Smad7 Protein; T-Lymphocytes; Transforming Growth Factor beta

The determination of the programmed death ligand-1 (PD-L1) expression is part of the diagnostic algorithm for advanced non-small cell lung cancer (NSCLC) patients. We aimed to analyze the diagnostic performance of EBUS-TBNA performed as first-choice nodal staging procedure for the determination of PD-L1 expression in NSCLC patients.. Longitudinal-prospective study including NSCLC patients diagnosed between January 2018 and October 2019, for whom a primary tumor biopsy sample and an EBUS-TBNA cytological malignant sample were available. Samples with fewer than 100 malignant cells were considered inadequate. PDL-1 IHC 22C3 pharmDx antibody was used. The percentage of tumor cells expressing PD-L1, setting 1% and 50% as cutoff points, was collected. The weighted kappa coefficient was used to assess the concordance of PD-L1 expression. The PD-L1 expression was compared in precision terms.. From a total of 43 patients, 53 pairs of samples were obtained, of which 23 (43.4%) were adequate and included for analysis. The weighted kappa coefficient for PD-L1 expression was 0.41 (95% CI 0.15-0.68) and 0.56 (95% CI 0.23-0.9) for cutoff values ≥ 1% and ≥ 50%, respectively. In advanced stages, the weighted kappa coefficient was 0.6 (95% CI 0.3-0.9) and 1 (95% CI 1-1) for PD-L1 expression cutoff values ≥ 1% and ≥ 50%, respectively. EBUS-TBNA showed a sensitivity, specificity, positive predictive value, and negative predictive value of 1 to detect PDL-1 expression ≥ 50% in advanced stages.. EBUS-TBNA performed as first nodal staging procedure in advanced NSCLC patients provides reliable specimens for the detection of PD-L1 expression ≥ 50% and could guide immunotherapy.

Topics: B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Endoscopic Ultrasound-Guided Fine Needle Aspiration; Humans; Lung Neoplasms; Neoplasm Staging; Prospective Studies; Retrospective Studies; Transforming Growth Factor beta

Tumour-reactive T cells producing the B-cell attractant chemokine CXCL13, in solid tumours, promote development of tertiary lymphoid structures (TLS) and are associated with improved prognosis and responsiveness to checkpoint immunotherapy. Cancer associated fibroblasts are the dominant stromal cell type in non-small cell lung cancer (NSCLC) where they co-localise with T cells and can influence T cell activation and exhaustion. We questioned whether CAF directly promote CXCL13-production during T cell activation.. We characterised surface markers, cytokine production and transcription factor expression in CXCL13-producing T cells in NSCLC tumours and paired non-cancerous lung samples using flow cytometry. We then assessed the influence of human NSCLC-derived primary CAF lines on T cells from healthy donors and NSCLC patients during activation. CAFs significantly increased the production of CXCL13 by both CD4. Promoting CXCL13 production represents a newly described immune-regulatory function of CAF with the potential to shape the immune infiltrate of the tumour microenvironment both by altering the effector-function of tumour infiltrating T-cells and their capacity to attract B cells and promote TLS formation.

Topics: Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; CD8-Positive T-Lymphocytes; Chemokine CXCL13; Humans; Interleukin-2; Lung Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-β increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-β decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.

Topics: Activating Transcription Factor 3; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Humans; Inflammation; Interleukin-10; Lung Neoplasms; Macrophage Colony-Stimulating Factor; Macrophages; MicroRNAs; Transcription Factor 3; Transforming Growth Factor beta

Tumour differentiation is an important index for adjuvant therapy in many cancers; however, non-small cell lung cancer (NSCLC) is an exception. Furthermore, postoperative radiotherapy (PORT) is controversial in patients with NSCLC with N0-1 and N2 disease. We aimed to evaluate the impact of tumour-related factors on overall survival (OS), cancer-specific survival (CSS), and distant control (DC) in patients with completely resected stage IIIA NSCLC.. Patients with stage IIIA non-metastatic NSCLC who underwent complete resection and adjuvant chemotherapy were identified from the Taiwan Cancer Registry (January 2007-December 2017). Logistic regression analysis was performed to determine the factors associated with PORT. Survival and relapse outcomes were compared using log-rank tests and Cox regression analysis. Sensitivity analysis was performed using propensity score-matched pairs.. In total, 1,897 patients were included and stratified according to PORT use (PORT vs. non-PORT). After adjusting for covariates, PORT was not found to be associated with improved survival outcomes. In patients with poorly differentiated tumours and N2 disease, absolute benefits for OS (adjusted hazard ratio [aHR] 0.76), CSS (aHR 0.80), and DC (aHR 0.74) were observed. Multivariable hazard models of propensity score-matched pN2 disease and poorly differentiated tumour subgroups also showed significant survival benefit with PORT treatment.. Patients with poorly differentiated tumours and receiving PORT for pN2 disease showed a lower risk of distant recurrence and more favourable survival outcomes in stage IIIA NSCLC with R0 resection.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Chemotherapy, Adjuvant; Humans; Lung Neoplasms; Transforming Growth Factor beta

Non-small cell lung cancer (NSCLC) accounts for ~85% of all lung cancer cases. Neferine is used as a traditional Chinese medicine with many pharmacological effects, including antitumor properties; however, it has not been reported whether neferine plays an anticancer role by causing pyroptosis in NSCLC cells. We used two typical lung cancer cell lines, A549 and H1299, and 42 lung cancer tissue samples to investigate the regulatory effects of neferine on TGF-β and MST1. We also treated lung cancer cells with different concentrations of neferine to study its effects on lung cancer cell survival, migration, invasion, and epithelial-mesenchymal transition (EMT) as well as on pyroptosis. Lentivirus-mediated gain-of-function studies of TGF-β and MST1 were applied to validate the roles of TGF-β and MST1 in lung cancer. Next, we used murine transplanted tumor models to evaluate the effect of neferine treatment on the metastatic capacity of lung cancer tissues. With increasing neferine concentration, the viability, migration, invasion, and EMT capacity of A549 and H1299 cells decreased, whereas pyroptosis increased. Neferine repressed TGF-β expression to modulate the induction of reactive oxygen species (ROS) by MST1. Overexpression of TGF-β in either in vitro or mouse-transplanted A549 cells restored the inhibitory effect of neferine on tumor development. Overexpression of MST1 clearly enhanced pyroptosis. Neferine contributed to pyroptosis by regulating MST1 expression through downregulation of TGF-β to induce ROS formation. Therefore, our study shows that neferine can serve as an adjuvant therapy for NSCLC patients.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Mice; Pyroptosis; Reactive Oxygen Species; Transforming Growth Factor beta

Occupational and environmental exposure of various toxins or cigarette smoke causes non-small cell lung carcinoma (NSCLC); a devastating disease with a very low survival rate after metastasis. Increased activity of plasmin is a hallmark in NSCLC metastasis. It is accepted that metastatic cells exhibit higher plasmin activity than cells from primary tumors. Mechanisms behind this elevation, however, are barely understood. We compared plasmin activity and cell migration of A549 cells derived from a primary lung tumor with metastatic H1299 lung cells isolated from lymph nodes. Surprisingly, we found higher plasmin activity and migration for A549 cells. mRNA levels of the plasminogen activator inhibitor-1 (PAI-1) were higher in H1299 cells and activity of extracellular-regulated kinases-1/2 (ERK-1/2) was increased. An inhibitor of ERK-1/2 decreased PAI-1 mRNA levels and increased plasmin activity or cell migration in H1299 cells. Transforming growth factor-β (TGF-β) decreased plasmin activity and migration in A549 cells but enhanced both in H1299 cells. The cytokine massively increased PAI-1 and decreased urokinase plasminogen activator (uPA) levels in A549 cells but strongly induced uPA and only weakly PAI- 1 expression in H1299 cells. Consequently, TGF-β enhanced plasmin activity and cell migration in H1299. Additionally, TGF-β activated ERK-1/2 stronger in H1299 than in A549 cells. Accordingly, an ERK-1/2 inhibitor completely reversed the effects of TGF-β on uPA expression, plasmin activity and migration in H1299 cells. Hence, we provide first data indicating TGF-β-promoted increased plasmin activity and suggest that blocking TGF-β-promoted ERK-1/2 activity might be a straightforward approach to inhibit NSCLC metastasis.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Movement; Fibrinolysin; Humans; Lung Neoplasms; Plasminogen Activator Inhibitor 1; RNA, Messenger; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

We aimed to explore the roles of T helper 17 (Th17) cells and cytokines interleukin 17 (IL-17), IL-6, IL-4, transforming growth factor β (TGF-β) and γ-interferon (IFN-γ) in the peripheral blood of patients with non-small cell lung cancer (NSCLC) of different clinical stages, pathological types, and differentiation degrees.. A total of 120 NSCLC patients admitted and 80 healthy people receiving physical examinations from June 2019 to October 2021 were enrolled as the case group (TNM stage: I, II, III and IV; pathological type: adenocarcinoma and squamous cell carcinoma; differentiation degree: low and moderate-high) and the control group, respectively. The serum levels of IL-17, IL-6, IL-4, TGF-β and IFN-γ were measured using an enzyme-linked immunosorbent assay. Th17 cells were counted by flow cytometry.. The case group had higher levels of Th17 cells, IL-17, IL-6, IL-4, and TGF-β and lower IFN-γ level in the peripheral blood than those of the control group (p < 0.05). NSCLC patients with different TNM stages had significantly different levels of Th17 cell, IL-17, IL-6, TGF-β, and IFN-γ (p < 0.05). The expression levels of Th17 and IL-6 in patients with lowly differentiated NSCLC were lower than those of patients with moderately and highly differentiated NSCLC, while the IFN-γ expression level followed an opposite trend (p < 0.05). The count of Th17 cells in the peripheral blood of NSCLC patients had significantly positive correlations with IL-17, IL-6, IL-4, and TGF-β levels, but negative correlation with IFN-γ level (p < 0.05).. The count of Th17 cells increases significantly in NSCLC patients and has correlations with TNM stage and differentiation degree.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Humans; Interferon-gamma; Interleukin-17; Interleukin-4; Interleukin-6; Lung Neoplasms; Th17 Cells; Transforming Growth Factor beta

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

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

Obstructive sleep apnea (OSA) is associated with severity of pneumonia; however, the mechanism by which OSA promotes lung cancer progression is unclear.. Twenty-five lung cancer patients were recruited to investigate the relationship between OSA and cancer-associated fibroblast (CAFs) activation. Lung cancer cells (A549) and WI38 fibroblast cells were used to explore the hypoxia-induced TGFβ expression using qPCR, Western blot, and ELISA. Wound healing and transwell assays were performed to evaluate cancer cell migration and invasion. A549 or A549-Luc + WI38 xenograft mouse models were established to detect the intermittent hypoxia (IH) associated with lung tumor growth and epithelial-mesenchymal transition (EMT). OSA promotes CAF activation and enrichment in lung cancer patients. Hypoxia (OSA-like treatment) activated TGFβ signaling in both lung cancer cells and fibroblasts, which promoted cancer cell migration and invasion, and enriched CAFs. IH promoted the progression and EMT process of lung cancer xenograft tumor. Co-inoculation of lung cancer cells and fibroblast cells could further promote lung cancer progression.. IH promotes lung cancer progression by upregulating TGFβ signaling, promoting lung cancer cell migration, and increasing the CAF activation and proportion of lung tumors.

Topics: Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Humans; Hypoxia; Lung Neoplasms; Mice; Neoplasm Invasiveness; Sleep Apnea, Obstructive; Transforming Growth Factor beta

Inhibitors of poly(ADP)-ribose polymerase (PARP) exploit defective DNA repair pathways existing in several forms of cancer, such as those with BRCA mutations, and have proven clinical efficacy as chemosensitizers. However, platinum-based chemopotentiation by PARP inhibitors (PARPi), particularly for non-small cell lung cancer (NSCLC), has only been confirmed in a few preclinical models and the molecular mechanisms that drive PARPi combinatorial synergy with chemotherapeutics remains poorly defined. To better understand these mechanisms, we characterized cisplatin and veliparib efficacy in A549 and Calu6 NSCLC

Topics: Adenosine Diphosphate; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cholesterol; Cisplatin; Humans; Integrins; Lung Neoplasms; Platinum; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Ribose; Transcriptome; Transforming Growth Factor beta

Non-small cell lung carcinoma (NSCLC) is one of the most common malignant tumors worldwide with high incidence and mortality. Long non-coding RNAs (lncRNAs) have been reported to affect human cancer progression. The present study aimed to investigate the regulatory role and mechanism of long intergenic non-protein coding RNA 1232 (LINC01232) in NSCLC cells. RT-qPCR results revealed that LINC01232 expression was high in NSCLC cells. Flow cytometry and sphere formation assays indicated that LINC01232 significantly promoted NSCLC cell stemness. Luciferase reporter assay and ChIP assay validated that forkhead box P3 (FOXP3) could bind to LINC01232 promoter and activate LINC01232 transcription. Further, LINC01232 was certified to activate TGF-β signaling pathway through regulating transforming growth factor beta receptor 1 (TGFBR1). After RIP and RNA pull down assays, insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was proven as the RNA-binding protein (RBP) for LINC01232. LINC01232 promoted TGFBR1 mRNA stability via recruiting IGF2BP2. Subsequently, LINC01232 was verified to accelerate NSCLC cell stemness and induce macrophage M2 polarization via upregulating TGFBR1. Taken together, FOXP3 activated-LINC01232 accelerated NSCLC cell stemness by activating TGF-β signaling pathway and recruiting IGF2BP2 to stabilize TGFBR1, which might offer a rationale for lncRNA-based treatment to NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Receptor, Transforming Growth Factor-beta Type I; RNA-Binding Proteins; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

To investigate regulatory function and underlying mechanism of TRIM66 in non-small cell lung cancer (NSCLC).. TRIM66 and MMP9 expression in NSCLC cells and tissues was assayed via qRT-PCR and western blot. CCK-8, colony formation, Transwell and flow cytometry assays were conducted to measure cell functional alternations in NSCLC. Western blot was employed to measure expression as well as phosphorylation levels of epithelial-mesenchymal transition-(EMT) and TGF-β/SMAD pathways-related proteins. Co-immunoprecipitation (Co-IP) assay was done to probe interaction between TRIM66 and MMP9. Xenograft in vivo experiment and tumor metastasis model in nude mice were utilized to investigate effects of TRIM66 on tumor growth of NSCLC.. TRIM66 and MMP9 were conspicuously highly expressed in NSCLC cells and tissues. High TRIM66 level was markedly correlated with metastasis. Silencing TRIM66 prominently repressed the proliferation, migration and invasion of transfected cells, while inducing cell apoptosis. Whereas forced expression of TRIM66 exerted the opposite effect. The aberrant expression of TRIM66 modulated EMT pathway. TRIM66 also regulated MMP9 expression, and the interaction between them was validated by Co-IP assay. Overexpression of MMP9 could activate TGF-β/SMAD pathway. Rescue experiments manifested that si-MMP9 or SB431542 could partially reverse phenotypes induced by TRIM66. In vivo experiments revealed that silencing TRIM66 could hamper NSCLC tumor growth and metastasis.. TRIM66 and MMP9 were up-regulated in NSCLC. TRIM66 facilitated the malignant progression of NSCLC through modulating MMP9-mediated TGF-β/SMAD pathway.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Nude; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFβ withdrawal and correlates with metastatic colonization. Whether TGFβ promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFβ induced EMT, generating RFP

Topics: Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Phenotype; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

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

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

To analyze the expression of CEACAM1 in serum of patients with nonsmall cell lung cancer (NSCLC) and to explore the correlation and clinical significance between the expression of CEACAM1 and pathological parameters of NSCLC tissue.. A total of 100 patients with NSCLC who underwent tumor resection were screened. Another 100 healthy patients in physical examination department were selected as control group. Venous blood and cancer tissue samples were collected. The expression of CEACAM1, TGF-. The results of various indicators in the lung cancer group were much higher than those in the healthy group; CEACAM1 was significantly positively correlated with TGF-. The expression level of CEACAM1 in the serum of NSCLC patients is strongly correlated with TGF-

Topics: Antigens, CD; Carcinoma, Non-Small-Cell Lung; Cell Adhesion Molecules; Humans; Interleukin-8; Lung Neoplasms; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Cancer metastasis accounts for most of the mortality associated with solid tumors. However, antimetastatic drugs are not available on the market. One of the important biological events leading to metastasis is the epithelial to mesenchymal transition (EMT) induced by cytokines, namely transforming growth-factor-β (TGF-β). Although several classes of inhibitors targeting TGF-β and its receptor have been developed, they have shown profound clinical side effects. We focused on our synthetic compound, HPH-15, which has shown anti-fibrotic activity via the blockade of the TGF-β Smad-dependent signaling. In this study, 10 μM of HPH-15 was found to exhibit anti-cell migration and anti-EMT activities in non-small-cell lung cancer (NSCLC) cells. Although higher concentrations are required, the anti-EMT activity of HPH-15 has also been observed in 3D-cultured NSCLC cells. A mechanistic study showed that HPH-15 inhibits downstream TGF-β signaling. This downstream inhibition blocks the expression of cytokines such as TGF-β, leading to the next cycle of Smad-dependent and -independent signaling. HPH-15 has AMPK-activation activity, but a relationship between AMPK activation and anti-EMT/cell migration was not observed. Taken together, HPH-15 may lead to the development of antimetastatic drugs with a new mechanism of action.

Topics: AMP-Activated Protein Kinases; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors

Despite advances in immunotherapy and targeted therapy, platinum-based chemotherapy remains crucial for many patients with advanced non-small cell lung cancer (NSCLC). Resistance to platinum chemotherapy is common, and predictive biomarkers are needed to tailor treatment to patients likely to respond. In vitro evidence implicates the transforming growth factor-β (TGF-β) superfamily ligands activin-A and growth differentiation factor 11 (GDF-11) in innate platinum resistance. We performed a validation study to assess their utility as predictive biomarkers of platinum chemotherapy response in advanced NSCLC.. Our study included 123 adult patients with advanced NSCLC without a driver mutation treated with platinum chemotherapy. 98 patients were from a retrospective cohort and 25 from a prospective cohort. We performed immunohistochemistry staining for Activin-A, GDF-11 and TGF-β on tumour samples for each patient and analysed IHC expression with objective radiological response and overall survival.. The overall median survival was 14.8 months. We performed statistical analysis around a cytoplasmic score of 8/18 for Activin-A and GDF-11 based on previously published work, and 110/30 for TGF-β based on a calculated cutpoint for significance. No survival difference was detected between these groups for Activin-A (p=0.35), GDF-11 (p=0.57) or TGF-β (p=0.34). There was no association between rates of progressive disease and high Activin-A expression (p=0.43), high GDF-11 expression (p=1.0) or high TGF-β expression p=0.89).. Within the confines of our study, Activin-A, GDF-11 and TGF-β expression was not a predictor of objective radiological response to chemotherapy or overall survival.

Topics: Activins; Adult; Biomarkers; Bone Morphogenetic Proteins; Carcinoma, Non-Small-Cell Lung; Growth Differentiation Factors; Humans; Lung Neoplasms; Organoplatinum Compounds; Platinum; Prospective Studies; Retrospective Studies; Transforming Growth Factor beta; Transforming Growth Factors

Transforming growth factor beta (TGFβ) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFβ signaling is regulated by the endocytosis of cell surface receptors and their subcellular trafficking into the endo-lysosomal system. Here we investigated how autophagy, a cellular quality control network that delivers material to lysosomes, regulates TGFβ signaling pathways that induce EMT and cell migration. We impaired autophagy in non-small cell lung cancer cells using chloroquine, spautin-1, ULK-101, or small interfering RNA (siRNA) targeting autophagy-related gene (ATG)5 and ATG7 and observed that inhibiting autophagy results in a decrease in TGFβ1-dependent EMT transcription factor and cell marker expression, as well as attenuated stress fiber formation and cell migration. This correlated with decreased internalization of cell surface TGFβ receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFβ signaling were investigated by Smad2/Smad3 phosphorylation and cellular localization using western blotting, subcellular fractionation, and immunofluorescence microscopy. We observed that inhibiting autophagy decreased the amount and timeframe of Smad2/Smad3 signaling. Taken together, our results suggest that inhibiting autophagy attenuates pro-tumorigenic TGFβ signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.

Topics: Autophagy; Carcinoma, Non-Small-Cell Lung; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Transforming Growth Factor beta

Transforming growth factor (TGF)-β signaling functions importantly in regulating tumor microenvironment (TME). This study developed a prognostic gene signature based on TGF-β signaling-related genes for predicting clinical outcome of patients with lung adenocarcinoma (LUAD).. TGF-β signaling-related genes came from The Molecular Signature Database (MSigDB). LUAD prognosis-related genes were screened from all the genes involved in TGF-β signaling using least absolute shrinkage and selection operator (LASSO) Cox regression analysis and then used to establish a risk score model for LUAD. ESTIMATE and CIBERSORT analyzed infiltration of immune cells in TME. Immunotherapy response was analyzed by the TIDE algorithm.. A LUAD prognostic 5-gene signature was developed based on 54 TGF-β signaling-related genes. Prognosis of high-risk patients was significantly worse than low-risk patients. Both internal validation and external dataset validation confirmed a high precision of the risk model in predicting the clinical outcomes of LUAD patients. Multivariate Cox analysis demonstrated the model independence in OS prediction of LUAD. The risk model was significantly related to the infiltration of 9 kinds of immune cells, matrix, and immune components in TME. Low-risk patients tended to respond more actively to anti-PD-1 treatment, while high-risk patients were more sensitive to chemotherapy and targeted therapy.. The 5-gene signature based on TGF-β signaling-related genes showed potential for LUAD management.

Topics: Adenocarcinoma of Lung; Biomarkers, Tumor; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Lung Neoplasms; Prognosis; Transforming Growth Factor beta; Tumor Microenvironment

TGFβ contributes to chemoresistance in advanced colorectal cancer (CRC) via diverse immune-microenvironment mechanisms. Here, we found that cancer cell autonomous TGFβ directly triggered tumor programmed cell death 1 ligand 1 (PD-L1) upregulation, resulting in resistance to chemotherapy. Inhibition of tumor PD-L1 expression sensitized cancer cells to chemotherapy, reduced lung metastasis and increased the influx of CD8

Topics: B7-H1 Antigen; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Macrophages; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor beta; Tumor Microenvironment

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFβ). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFβ. We observed that culture media conditioned with A549 in the presence of TGFβ induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizin

Topics: Cancer-Associated Fibroblasts; Cannabidiol; Cannabinoid Receptor Agonists; Culture Media; Dronabinol; Humans; Lung Neoplasms; Transforming Growth Factor beta; Tumor Microenvironment

Bintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-β (TGF-β) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-β trap (TGF-βRII) to the TME, enabling it to sequester TGF-β in the tumor more effectively than systemic TGF-β blockade, thereby enhancing antitumor activity.. Multiple technologies were used to characterize the TGF-β trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-β trap or the pan-TGF-β antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-β and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-β were investigated in vitro.. BA and fresolimumab had comparable intrinsic binding to TGF-β1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-β-dependent and PD-L1-expressing cells more potently than TGF-β trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-β trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-β-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-β1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-β1 co-expression was also seen in human tumors. Finally, BA induced TGF-β1 internalization and degradation in the lysosomes.. BA more effectively blocks TGF-β by targeting TGF-β trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.

Topics: B7-H1 Antigen; Humans; Immunologic Factors; Lung Neoplasms; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

C-C motif chemokine ligand 20 (CCL20) participates in multiple oncogenic processes, but its role in lung adenocarcinoma (LUAD) is unclear. Herein, we explored the mechanism by which CCL20 works in LUAD progression. We performed bioinformatical analyses based on the complete transcriptome sequencing data from 1544 LUAD cases in 4 independent cohorts to evaluate signaling pathways regulated by CCL20. We established A549 and H358 cell lines with CCL20 knockdown to explore how CCL20 promotes tumor progression

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL20; Chemokines; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; Transforming Growth Factor beta; Urinary Bladder Neoplasms

Impairing autophagy disrupts transforming growth factor beta 1 (TGFβ1) signalling and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Since autophagy and proteasome-mediated degradation are interdependent, we investigated how prolonged downregulation of proteasomal catalytic activity affected TGFβ1-dependent signalling and EMT. Proteasome-dependent degradation was inhibited in A549 and H1299 NSCLC cells using MG132 and lactacystin, which are reversible and irreversible proteasome inhibitors, respectively. We observed that inhibiting proteasomal activity for 24 h decreased TGFβ-dependent nuclear accumulation of Smad2/3. Time course studies were then carried out to characterize the time frame of this observation. Short-term (< 8 h) proteasome inhibition resulted in increased receptor regulated Smad (R-Smad) phosphorylation and steady-state TGFβ receptor type II (TGFβRII) levels. However, prolonged (8-24 h) proteasome inhibition decreased TGFβ1-dependent R-Smad phosphorylation and steady-state TGFβRI and TGFβRII levels. Furthermore, proteasome inhibition blunted TGFβ-dependent E- to N-Cadherin shift, stress fiber formation, and increased cellular apoptosis via the TAK-1-TRAF6-p38 MAPK pathway. Interestingly, proteasome inhibition also increased autophagic flux, steady-state microtubule-associated protein light chain 3B-II and active uncoordinated 51-like autophagy activating kinase 1 levels, and co-localization of lysosomes with autophagy cargo proteins and autophagy-related proteins. Finally, we observed that proteasome inhibition increased TGFβRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFβ signalling outcomes through altered TGFβ receptor trafficking.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Lysosomes; Proteasome Endopeptidase Complex; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Regulator of G-protein signaling 6 (RGS6) is a newly discovered tumor suppressor that has been shown to be protective in development of various cancers such as breast cancer and bladder cancer. But the mechanisms underlying these tumor-suppressing functions of RGS6 are not fully understood. Here, we discover a novel function of RGS6 in suppressing TGF-β-induced epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells and in vivo NSCLC metastasis. Using both bioinformatics and experimental tools, we showed that RGS6 was downregulated in lung cancer tissues compared to noncancerous counterparts, and low expression of RGS6 was associated with poor survival of lung cancer patients. Overexpression of RGS6 suppressed TGF-β-induced EMT in vitro and TGF-β-promoted metastasis in vivo, by impairing gene expression of downstream effectors induced by the canonical TGF-β-SMAD signaling. The ability of RGS6 to suppress TGF-β-SMAD-mediated gene expression relied on its binding to SMAD4 to prevent complex formation between SMAD4 and SMAD2/3, but independent of its regulation of the G-protein signaling. Interaction between RGS6 and SMAD4 caused less nuclear entry of p-SMAD3 and SMAD4, resulting in inefficient SMAD3-mediated gene expression. Taken together, our findings reveal a novel and noncanonical role of RGS6 in regulation of TGF-β-induced EMT and metastasis of NSCLC and identify RGS6 as a prognostic marker and a potential novel target for NSCLC therapy.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Checkpoint Kinase 1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; RGS Proteins; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

Lung adenocarcinoma (LUAD) is the leading cause of death worldwide. However, the roles of long noncoding RNAs (lncRNAs) hijacked by super-enhancers (SEs), vital regulatory elements of the epigenome, remain elusive in the progression of LUAD metastasis.. SE-associated lncRNA microarrays were used to identify the dysregulated lncRNAs in LUAD. ChIP-seq, Hi-C data analysis, and luciferase reporter assays were utilized to confirm the hijacking of LINC01977 by SE. The functions and mechanisms of LINC01977 in LUAD were explored by a series of in vitro and in vivo assays.. We found that LINC01977, a cancer-testis lncRNA, was hijacked by SE, which promoted proliferation and invasion both in vitro and in vivo. LINC01977 interacted with SMAD3 to induce its nuclear transport, which facilitated the interaction between SMAD3 and CBP/P300, thereby regulating the downstream target gene ZEB1. Additionally, SMAD3 up-regulated LINC09177 transcription by simultaneously binding the promoter and SE, which was induced by the infiltration of M2-like tumor-associated macrophages (TAM2), subsequently activating the TGF-β/SMAD3 pathway. Moreover, LINC01977 expression was positively correlated with TAM2 infiltration and SMAD3 expression, especially in early-stage LUAD. Higher chromatin accessibility in the SE region of LINC01977 was observed with high expression of TGF-β. Early-stage LUAD patients with high LIN01977 expression had a shorter disease-free survival.. TAM2 infiltration induced a rich TGF-β microenvironment, activating SMAD3 to bind the promoter and the SE of LINC01977, which up-regulated LINC01977 expression. LINC01977 also promoted malignancy via the canonical TGF-β/SMAD3 pathway. LINC01977 hijacked by SE could be a valuable therapeutic target, especially for the treatment of early-stage LUAD.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Male; RNA, Long Noncoding; Smad3 Protein; Transforming Growth Factor beta; Tumor Microenvironment

Transforming growth factor β (TGF-β) is a well-known key mediator for the progression and metastasis of lung carcinoma. However, cost-effective anti-TGF-β therapeutics for lung cancer remain to be explored. Specifically, the low efficacy in drug delivery greatly limits the clinical application of small molecular inhibitors of TGF-β. In the present study, specific inhibitor of Smad3 (SIS3) is developed into a self-carried nanodrug (SCND-SIS3) using the reprecipitation method, which largely improves its solubility and bioavailability while reduces its nephrotoxicity. Compared to unmodified-SIS3, SCND-SIS3 demonstrates better anti-cancer effects through inducing tumor cell apoptosis, inhibiting angiogenesis, and boosting NK cell-mediated immune responses in syngeneic Lewis Lung Cancer (LLC) mouse model. Better still, it could achieve comparable anti-cancer effect with just one-fifth the dose of unmodified-SIS3. Mechanistically, RNA-sequencing analysis and cytokine array results unveil a TGF-β/Smad3-dependent immunoregulatory landscape in NK cells. In particular, SCND-SIS3 promotes NK cell cytotoxicity by ameliorating Smad3-mediated transcriptional inhibition of Ndrg1. Furthermore, improved NK cell cytotoxicity by SCND-SIS3 is associated with higher expression of activation receptor Nkp46, and suppressed levels of Trib3 and TSP1 as compared with unmodified-SIS3. Taken together, SCND-SIS3 possesses superior anti-cancer effects with enhanced bioavailability and biocompatibility, therefore representing as a novel therapeutic strategy for lung carcinoma with promising clinical potential.

Topics: Animals; Carcinoma; Cell Line, Tumor; Isoquinolines; Lung Neoplasms; Mice; Nanoparticles; Pyridines; Pyrroles; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Lung cancer is one of the leading causes of cancer-related death, with a five-year survival rate of 18%. It is a priority for us to understand the underlying mechanisms affecting lung cancer therapeutics' implementation and effectiveness. In this study, we combine the power of Bioinformatics and Systems Biology to comprehensively uncover functional and signaling pathways of drug treatment using bioinformatics inference and multiscale modeling of both scRNA-seq data and proteomics data. Based on a time series of lung adenocarcinoma derived A549 cells after DEX treatment, we first identified the differentially expressed genes (DEGs) in those lung cancer cells. Through the interrogation of regulatory network of those DEGs, we identified key hub genes including TGFβ, MYC, and SMAD3 varied underlie DEX treatment. Further gene set enrichment analysis revealed the TGFβ signaling pathway as the top enriched term. Those genes involved in the TGFβ pathway and their crosstalk with the ERBB pathway presented a strong survival prognosis in clinical lung cancer samples. With the basis of biological validation and literature-based curation, a multiscale model of tumor regulation centered on both TGFβ-induced and ERBB-amplified signaling pathways was developed to characterize the dynamic effects of DEX therapy on lung cancer cells. Our simulation results were well matched to available data of SMAD2, FOXO3, TGFβ1, and TGFβR1 over the time course. Moreover, we provided predictions of different doses to illustrate the trend and therapeutic potential of DEX treatment. The innovative and cross-disciplinary approach can be further applied to other computational studies in tumorigenesis and oncotherapy. We released the approach as a user-friendly tool named BIMM (Bioinformatic Inference and Multiscale Modeling), with all the key features available at https://github.com/chenm19/BIMM.

Topics: Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Lung Neoplasms; Proteomics; Single-Cell Analysis; Transforming Growth Factor beta

Crizotinib is a clinically approved tyrosine kinase inhibitor for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring EML4-ALK fusion. Crizotinib was originally developed as an inhibitor of MET (HGF receptor), which is involved in the metastatic cascade. However, little is known about whether crizotinib inhibits tumor metastasis in NSCLC cells. In this study, we found that crizotinib suppressed TGFβ signaling by blocking Smad phosphorylation in an ALK/MET/RON/ROS1-independent manner in NSCLC cells. Molecular docking and in vitro enzyme activity assays showed that crizotinib directly inhibited the kinase activity of TGFβ receptor I through a competitive inhibition mode. Cell tracking, scratch wound, and transwell migration assays showed that crizotinib simultaneously inhibited TGFβ- and HGF-mediated NSCLC cell migration and invasion. In addition, in vivo bioluminescence imaging analysis showed that crizotinib suppressed the metastatic capacity of NSCLC cells. Our results demonstrate that crizotinib attenuates cancer metastasis by inhibiting TGFβ signaling in NSCLC cells. Therefore, our findings will help to advance our understanding of the anticancer action of crizotinib and provide insight into future clinical investigations.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Crizotinib; Humans; Lung Neoplasms; Molecular Docking Simulation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Transforming Growth Factor beta

Transforming Growth Factor β (TGFβ) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFβ-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFβ-induced EMT is largely unkonwn.. Real-time qPCR analyses were performed to determine the effect of TGFβ1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFβ1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFβ1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFβ1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFβ1 on E-cadherin and mesenchymal genes in the cells.. Data provided suggest that TGFβ1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFβ1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFβ1-associated EMT in tumor cells.

Topics: A549 Cells; Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dual-Specificity Phosphatases; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Pancreatic Neoplasms; RNA, Small Interfering; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

A cluster of differentiation 47 (CD47) and immune-modulatory protein for myeloid cells has been implicated in cisplatin (CDDP) resistance. Exosome delivery of drugs has shown great potential for targeted drug delivery in the treatment of various diseases. In the current study, we explored the approach of co-delivering CDDP and CD47 antibody with MDA-MB-231 cell-derived exosome 231-exo (CaCE) and assessed the phagocytosis activity of bone marrow flow cytometry derived macrophages (BMDM) against co-cultured A549 cells. CD8

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; CD47 Antigen; Cell Line, Tumor; Cisplatin; Cytokines; Exosomes; Humans; Interferons; Lung; Lung Neoplasms; Mice; Transforming Growth Factor beta

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

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

Lung adenocarcinoma (LUAD) is one of the main causes of cancer-related mortality, with a strong tendency to metastasize early. Transforming growth factor-β (TGF-β) signaling is a powerful regulator to promote metastasis of LUAD. Here, we screened long non-coding RNAs (lncRNAs) responsive to TGF-β and highly expressed in LUAD cells, and finally obtained our master molecular LINC00152. We proved that the TGF-β promoted transcription of LINC00152 through the classical TGF-β/SMAD3 signaling pathway and maintained its stability through the RNA-binding protein HuR. Moreover, LINC00152 increased ZEB1, SNAI1 and SNAI2 expression via increasing the interactions of HuR and these transcription factors, ultimately promoting epithelial-mesenchymal transition of LUAD cell and enhancing LUAD metastasis in vivo. These data provided evidence that LINC00152 induced by TGF-β promotes metastasis depending HuR in lung adenocarcinoma. Designing targeting LINC00152 and HuR inhibitors may therefore be an effective therapeutic strategy for LUAD treatment.

Topics: Adenocarcinoma of Lung; Cell Line, Tumor; ELAV-Like Protein 1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; RNA, Long Noncoding; Transforming Growth Factor beta

The abnormal modification of chondroitin sulfate is one of the leading causes of disease, including cancer progression. During chondroitin sulfate biosynthesis, the CHST11 enzyme plays a vital role in its modification, but its role in cancer is not fully understood. Therefore, understanding the relationship between CHST11 and pulmonary-related diseases through clinically relevant information may be useful for diagnosis or treatment.. A variety of pulmonary fibrosis clinical gene expression omnibus (GEO) datasets were used to assess the association between CHST11-related manifestations and fibrosis. Multiple lung cancer-related databases, including The Cancer Genome Atlas, GEO datasets, UCSC Xena, GEPIA2, Cbioportal and ingenuity pathway analysis were used to evaluate the clinical correlation between CHST11 and lung cancer and potential molecular mechanisms. For drug repurposing prediction, the molecules that correlated with CHST11 were subjected to the LINCS L1000 algorithm. A variety of in vitro assays were performed to evaluate the in-silico models, including RNA and protein expression, proliferation, migration and invasion.. Clinical analyses indicate that the levels of CHST11 are significantly elevated in cases of pulmonary-related diseases, including fibrosis and lung cancer. According to multiple lung cancer cohorts, CHST11 is the only member of the carbohydrate sulfotransferase family associated with overall survival for lung adenocarcinomas, and it is highly related to smoking-induced lung cancer patients. Based on the results of in vitro experiments, CHST11 expression contributes to tumor malignancy and promotes multiple fibrotic activators. Correlation-based ingenuity pathway analysis indicated that CHST11-related molecules contributed to pulmonary fibrosis or lung adenocarcinomas via similar upstream stimulators. Based on known molecular regulatory relationships, CHST11 has been associated with the regulation of TGF-β and INFγ as important molecules contributing to fibrosis and cancer progression. Interestingly, WordCloud analysis revealed that CHST11-related molecules are involved in regulation primarily by integrin signaling, and these relationships were consistently reflected in the analysis of cell lines and the clinical correlation. A CHST11 signature-based drug repurposing analysis demonstrated that the CHST11/integrin axis could be targeted by AG-1478 (Tyrphostin AG 1478), brefeldin A, geldanamycin and importazole.. This study provides the first demonstration that CHST11 may be used as a biomarker for pulmonary fibrosis or lung cancer, and the levels of CHST11 were increased by TGF-β and INFγ. The molecular simulation analyses demonstrate that the CHST11/integrin axis is a potential therapeutic target for treating lung cancer.

Topics: Adenocarcinoma of Lung; Chondroitin Sulfates; Humans; Integrins; Lung Neoplasms; Pulmonary Fibrosis; Sulfotransferases; Transforming Growth Factor beta

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.

Topics: Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Methylation; Methyltransferases; RNA, Messenger; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factors

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

Stereotactic body radiotherapy (SBRT) demonstrates excellent local control in early stage lung cancer, however a quarter of patients develop recurrence or distant metastasis. Transforming growth factor-beta (TGF-β) supports metastasis and treatment resistance, and angiotensin receptor blockade (ARB) indirectly suppresses TGF-β signaling. This study investigates whether patients taking ARBs while undergoing SBRT for early stage lung cancer exhibited improved overall survival (OS) or recurrence free survival (RFS) compared to patients not taking ARBs. This was a single institution retrospective analysis of 272 patients treated with SBRT for early stage lung cancer between 2009 and 2018. Patient health data was abstracted from the electronic medical record. OS and RFS were assessed using Kaplan-Meier method. Log-rank test was used to compare unadjusted survival between groups. Univariable and multivariable Cox proportional hazard regression models were used to estimate hazard ratios (HRs). Of 247 patients analyzed, 24 (10%) patients took ARBs for the duration of radiotherapy. There was no difference in mean age, median tumor diameter, or median biologic effective dose between patients taking ARBs or not. Patients taking ARBs exhibited increased OS (ARB = 96.7 mo.; no ARB = 43.3 mo.; HR = 0.25 [95% CI: 0.10 to 0.62,

Topics: Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Biological Products; Carcinoma, Non-Small-Cell Lung; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lung Neoplasms; Radiosurgery; Receptors, Angiotensin; Retrospective Studies; Transforming Growth Factor beta; Transforming Growth Factors; Treatment Outcome

Tumor microenvironment has attracted more and more attention in oncology. Alisol B23 acetate (AB23A) inhibits the proliferation of tumor cells including non-small cell lung cancer (NSCLC) cells. However, whether AB23A plays a role in the tumor microenvironment of NSCLC still remains obscure.. After THP-1 cells were polarized to M0 type by PMA, M0 macrophages were differentiated into M1 by LPS and IFNγ, and were differentiated into M2 by IL-4 and IL-13. The differentiation of THP-1 cells was detected by flow cytometry. After AB23A was given to macrophage RT-qPCR and ELISA detected the expressions of IL-6, IL-1β, IL-10 and TGF-β. Western blot and RT-qPCR detected the expressions of CD11b and CD18 at both mRNA and protein levels. Lung cancer cell A549 cells were induced by above related macrophage culture medium. Cell proliferation was detected by CCK-8. Tunel, wound healing and Transwell detected the apoptotic, migration and invasion capabilities. Next, M0 and M1-type macrophages were cultured in the cell culture medium of conventional A549 cells, to which AB23A was added. Subsequently, cell differentiation and inflammatory response were measured. Finally, the expression of CD18 in A549 cells was knocked down to construct NSCLC tumor-bearing mice and AB23A was applied for intragastric administration. Immunohistochemistry detected the polarization of macrophages in tumor tissues. Western blot detected the expressions of CD11b, CD18, invasion-, migration- and apoptosis-related proteins.. AB23A promoted the polarization of macrophages towards M1, thus promoting the apoptosis and inhibiting the invasion and migration of A549 cells. The tumor cell culture medium induced M0 macrophages to M2, while AB23A reversed this effect. AB23A targeted CD11b/CD18 and improved the polarization of macrophages, thereby affecting tumor invasion, migration and apoptosis.. AB23A affected the polarization of tumor-associated macrophages through the targeted regulation of CD11b/CD18, thus inhibiting the development of lung cancer.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cholestenones; Interleukin-10; Interleukin-13; Interleukin-4; Interleukin-6; Lung Neoplasms; Macrophages; Mice; RNA, Messenger; Transforming Growth Factor beta; Tumor Microenvironment

Nowadays, the impact of the tumor-immune microenvironment (TME) in non-small-cell lung cancer (NSCLC) prognosis and treatment response remains unclear. Thus, we evaluated the expression of PD-L1, tumor-infiltrating lymphocytes (TILs), and transforming growth factor beta (TGF-β) in NSCLC to identify differences in TME, detect possible new prognostic factors, and assess their relationship. We retrospectively analyzed 55 samples from patients who underwent NSCLC surgery and had over a 5-year follow-up. PD-L1 expression was determined by immunohistochemistry following standard techniques. The presence of TILs was evaluated at low magnification and classified into two categories, “intense” and “non-intense”. Cytoplasmic TGF-β staining visualization was divided into four categories, and unequivocal nuclear staining in >1% of viable tumor cells was defined as “present” or “absent”. Our aim was to identify differences in disease-free survival (DFS) and overall survival (OS). Tumor stage was the only objective prognostic factor for OS. PD-L1 expression and the presence of TILs had no prognostic impact, neither their combination. There seems to be a lower expression of PD-L1 and a higher expression of TILs in early stages of the disease. Our TGF-β nuclear staining analysis was promising, since it was associated with worse DFS, revealing this protein as a possible prognostic biomarker of recurrence for resectable NSCLC.

Topics: B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Recurrence; Retrospective Studies; Risk Factors; Small Cell Lung Carcinoma; Staining and Labeling; Transforming Growth Factor beta; Tumor Microenvironment

TGF-β is secreted in the tumour microenvironment in a latent, inactive form bound to latency associated protein and activated by the integrin α

Topics: Animals; Antigens, CD; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Humans; Immunotherapy; Integrin alpha Chains; Integrin alphaV; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta; Tumor Microenvironment

Epithelial membrane protein 3 (EMP3) is a transmembrane glycoprotein that contains a peripheral myelin protein 22 domain. EMP3 first received attention as a tumor suppressor, but accumulating evidence has since suggested that it may exhibit a tumor‑promoting function. Nonetheless, the biological function of EMP3 remains largely unclear with regards to its role in cancer. Herein, it was shown that EMP3 expression is upregulated in non‑small cell lung cancer (NSCLC) cells overexpressing aldehyde dehydrogenase 1 (ALDH1). EMP3 was shown to be involved in cell proliferation, the formation of cancer stem cells (CSCs) and in epithelial‑mesenchymal transition (EMT). The ability to resist irradiation, one of the characteristics of CSCs, decreased when the EMP3 mRNA expression was knocked down using small interfering RNA. In addition, when EMP3 knockdown reduced the migratory ability of cells, a characteristic of EMT. Additionally, it was shown that the TGF‑β/Smad signaling axis was a target of EMP3. EMP3 was found to interact with TGF‑β receptor type 2 (TGFBR2) upon TGF‑β stimulation in lung CSCs (LCSC). As a result, binding of EMP3‑TGFBR2 regulates TGF‑β/Smad signaling activation and consequently affects CSCs and EMT. Kaplan‑Meier analysis results confirmed that patients with high expression of EMP3 had poor survival rates. Taken together, these findings showed that EMP3 may be a potential target for management of LCSCs with high expression of ALDH1, and that EMP3 is involved in TGF‑β/Smad signaling activation where it promotes acquisition of cancerous properties in tumors.

Topics: Aldehyde Dehydrogenase 1 Family; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Membrane Glycoproteins; Neoplastic Stem Cells; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

PD-L1 is abnormally regulated in many cancers and is critical for immune escape. Fully understanding the regulation of PD-L1 expression is vital for improving the clinical efficacy of relevant anticancer agents. TGF-β plays an important role in the low reactivity of PD-1/PD-L1 antibody immunotherapy. However, it is not very clear whether and how TGF-β affects PD-L1 expression. In the present study, we show that TGF-β upregulates the expression of the transcriptional coactivator MRTF-A in non-small-cell lung cancer cells, which subsequently interacts with NF-κB/p65 rather than SRF to facilitate the binding of NF-κB/p65 to the PDL1 promoter, thereby activating the transcription and expression of PD-L1. This leads to the immune escape of NSCLC cells. This process is dependent on the activation of the TGF-β signaling pathway. In vivo, inhibition of MRTF-A effectively suppresses the growth of lung tumor syngrafts with enrichment of NK and T cells in tumor tissue. Our study defines a new signaling pathway that regulates the transcription and expression of PD-L1 upon TGF-β treatment, which may have a significant impact on research into the application of immunotherapy in treating lung cancer.

Topics: Animals; B7-H1 Antigen; Biomarkers; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Models, Animal; Disease Susceptibility; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Immune Evasion; Lung Neoplasms; Mice; Models, Biological; Proteasome Endopeptidase Complex; Signal Transduction; Trans-Activators; Transcription Factor RelA; Transforming Growth Factor beta; Ubiquitin

Inhibition of mammalian target of rapamycin complex 1 (mTORC1) with rapamycin in the absence of transforming growth factor-β (TGFβ) signaling induces apoptosis in many cancer cell lines. In the presence of TGFβ, rapamycin induces G

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Drug Resistance, Neoplasm; E2F Transcription Factors; Humans; Lung Neoplasms; Male; Mutation; Phosphorylation; Prostatic Neoplasms; Retinoblastoma Protein; Signal Transduction; Sirolimus; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Topics: Biopsy; Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Fibroblast Growth Factor 7; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Precision Medicine; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation

Fluoroquinolones (FQs) are potent antimicrobials with multiple effects on host cells and tissues. Although FQs can attenuate cancer invasion and metastasis, the underlying molecular mechanisms remain unclear. Matrix metalloproteinase-9 (MMP-9) has functional roles in tumor angiogenesis, invasion, and metastasis, and is associated with cancer progression and poor prognosis, suggesting that inhibitors of MMP-9 activity and transcription are prime candidates for cancer therapy. Despite numerous preclinical data supporting the use of MMP-9 inhibitors as anticancer drugs, the few available examples are not therapeutically useful due to low specificity and off-target effects. We examined the effects of FQs on MMP-9 production in cancer cells following transforming growth factor beta (TGF-β) and phorbol 12-myristate 13-acetate (PMA) stimulation.. Using confluent cultures of HepG2 and A549 cells, the effects of FQs (ciprofloxacin, levofloxacin, clinafloxacin, gatifloxacin, and enrofloxacin) on TGF-β and PMA-induced MMP-9 mRNA expression and production were studied in RNA extracts and culture supernatants, respectively. FQs specifically abrogated TGF-β and PMA-induced MMP-9 levels and activity in a concentration and time-dependent manner, without affecting other MMPs or proteins involved in epithelial-mesenchymal transition. Additionally, FQs inhibited TGF-β and PMA-induced cell migration via p38 and cyclic AMP signaling pathways.. Overall, we demonstrated that FQs inhibit cancer cell migration and invasion by downregulating MMP-9 expression and revealed the cellular mechanisms underlying their potential value in cancer treatment.

Topics: A549 Cells; Anti-Bacterial Agents; Cell Line, Tumor; Cell Movement; Down-Regulation; Drug Repositioning; Epithelial-Mesenchymal Transition; Fluoroquinolones; Hep G2 Cells; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; p38 Mitogen-Activated Protein Kinases; Phosphorylcholine; Polymethacrylic Acids; Quinolones; Signal Transduction; Transforming Growth Factor beta

Ginsenoside CK is the main metabolite of protopanaxadiol saponins in intestinal bacteria. Previous studies have shown that ginsenoside CK can affect many aspects of tumor development through a variety of mechanisms. However, few studies have reported the antimetastatic effects of ginsenoside CK in non-small-cell lung cancer (NSCLC). In this study, we explored the effect of ginsenoside CK on epithelial-mesenchymal transition (EMT) induced by TGF-

Topics: A549 Cells; Animals; Cadherins; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Ginsenosides; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Sirtuin 1; Transforming Growth Factor beta; Up-Regulation; Vimentin

LINC00941 is a novel lncRNA that has been found to exhibit protumorigenic and prometastatic behaviors during tumorigenesis. However, its role in metastatic CRC remains unknown. We aimed to investigate the functions and mechanisms of LINC00941 in CRC metastasis. LINC00941 was shown to be upregulated in CRC, and upregulated LINC00941 was associated with poor prognosis. Functionally, LINC00941 promoted migratory and invasive capacities and accelerated lung metastasis in nude mice. Mechanistically, LINC00941 activated EMT in CRC cells, as indicated by the increased expression of key molecular markers of cell invasion and metastasis (Vimentin, Fibronectin, and Twist1) and simultaneous decreased expression of the main invasion suppressors E-cadherin and ZO-1. LINC00941 was found to activate EMT by directly binding the SMAD4 protein MH2 domain and competing with β-TrCP to prevent SMAD4 protein degradation, thus activating the TGF-β/SMAD2/3 signaling pathway. Our data reveal the essential role of LINC00941 in metastatic CRC via activation of the TGF-β/SMAD2/3 axis, which provides new insight into the mechanism of metastatic CRC and a novel potential therapeutic target for advanced CRC.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; RNA, Long Noncoding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Vimentin

The acquisition of chemoresistance remains a major cause of cancer mortality due to the limited accessibility of targeted or immune therapies. However, given that severe alterations of molecular features during epithelial-to-mesenchymal transition (EMT) lead to acquired chemoresistance, emerging studies have focused on identifying targetable drivers associated with acquired chemoresistance. Particularly, AXL, a key receptor tyrosine kinase that confers resistance against targets and chemotherapeutics, is highly expressed in mesenchymal cancer cells. However, the underlying mechanism of AXL induction in mesenchymal cancer cells is poorly understood. Our study revealed that the YAP signature, which was highly enriched in mesenchymal-type lung cancer, was closely correlated to AXL expression in 181 lung cancer cell lines. Moreover, using isogenic lung cancer cell pairs, we also found that doxorubicin treatment induced YAP nuclear translocation in mesenchymal-type lung cancer cells to induce AXL expression. Additionally, the concurrent activation of TGFβ signaling coordinated YAP-dependent AXL expression through SMAD4. These data suggest that crosstalk between YAP and the TGFβ/SMAD axis upon treatment with chemotherapeutics might be a promising target to improve chemosensitivity in mesenchymal-type lung cancer.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Axl Receptor Tyrosine Kinase; Humans; Lung Neoplasms; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Transcription Factors; Transforming Growth Factor beta; YAP-Signaling Proteins

Benzo[a]pyrene [BP] is one of the major carcinogenic precursors of cigarette smoke that primary affects the lung at its first proximity. The goal of the current research was to elucidate new mechanisms underlying the tumorigenic impact of oral BP in the lung of mice, with focus on immunosuppressive effects and cancer stemming properties.. Female albino mice (n = 44) were divided into 2 groups: normal control and BP group. BP was administered orally to mice (50 mg/kg body weight), twice a week for four weeks in succession. At the end of experiment (22 weeks), gene expression were measured for transforming growth factor-β (TGF-β), cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death ligand 1(PD-L1), forkhead box protein P3 (FOXP3) and interleukin 12 (IL-12) and CD83. The results indicated the tumorigenic role of BP in the lung which was evidenced by histopathological examination. BP group also showed immunosuppressive role which evidenced by increased expression of lung TGF-β, CTLA-4, PD-L1, FOXP3 genes and decreased expression of lung IL-12 gene compared with normal control group. BP group also showed decreased CD83. Our findings indicated that BP has immunosuppressive role in lung cancer besides increasing the percentage of cancer stem like cells.

Topics: Animals; Antigens, CD; B7-H1 Antigen; Benzo(a)pyrene; Carcinogenesis; CTLA-4 Antigen; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Immunosuppressive Agents; Interleukin-12; Lung Neoplasms; Mice; Survival Analysis; Transforming Growth Factor beta; Tumor Burden

Serpin family E member 1 (SERPINE1), a serine proteinase inhibitor, serves as an important regulator of extracellular matrix remodeling. Emerging evidence suggests that SERPINE1 has diverse roles in cancer and is associated with poor prognosis. However, the mechanism via which SERPINE1 is induced in cancer has not been fully determined. In order to examine the molecular mechanism of SERPINE1 expression, the present study took advantage of the isogenic pair of lung cancer cells with epithelial or mesenchymal features. Using genetic perturbation and following biochemical analysis, the present study demonstrated that SERPINE1 expression was upregulated in mesenchymal lung cancer cells and promoted cellular invasiveness. Yes‑associated protein (YAP)‑dependent SERPINE1 expression was modulated by treatment with a Rho‑associated protein kinase inhibitor, Y27632. Moreover, TGFβ treatment supported YAP‑dependent SERPINE1 expression, and an enhanced TGFβ response in mesenchymal lung cancer cells promoted SERPINE1 expression. TGFβ‑mediated SERPINE1 expression was significantly attenuated by knockdown of YAP or transcriptional co‑activator with PDZ‑binding motif, suggesting that crosstalk between the TGFβ and YAP pathways underlies SERPINE1 expression in mesenchymal cancer cells.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Cell Proliferation; Epithelial-Mesenchymal Transition; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Mesenchymal Stem Cells; Plasminogen Activator Inhibitor 1; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transforming Growth Factor beta; Up-Regulation; YAP-Signaling Proteins

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Lymph Nodes; Neoplasm Staging; Transforming Growth Factor beta

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

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

Topics: Alternative Splicing; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Etoposide; Female; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Protein-Arginine N-Methyltransferases; RNA-Binding Proteins; RNA, Messenger; Signal Transduction; Smad7 Protein; Small Cell Lung Carcinoma; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Cell Survival; Chromones; Coordination Complexes; Copper; DNA; DNA Cleavage; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Membrane Potential, Mitochondrial; Transforming Growth Factor beta

The interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment.. Human non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data.. TGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer.. TGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response.

Topics: B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Forkhead Transcription Factors; Humans; Lung Neoplasms; Machine Learning; Neoplasm Staging; Phosphorylation; Retrospective Studies; Signal Transduction; Smad3 Protein; Survival Analysis; Tissue Array Analysis; 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

Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-β (TGF-β) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMT-associated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 μmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 μmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-β (P < 0.05), while HSP70 induced by 50 μmol/L GGA did not. The results of the wound healing assay indicated that 200 μmol/L GGA significantly inhibited A549 cell migration induced by TGF-β. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-β induced EMT process and changed the cell morphology and migratory ability induced by TGF-β in A549 cells.

Topics: A549 Cells; Cadherins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; HSP70 Heat-Shock Proteins; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

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

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

Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-β (TGF-β) in cancer metastasis. TGF-β-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-β-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-β-stimulated cancer cells, suggesting that TGF-β might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Blood Platelets; Cell Movement; Cytokines; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Histocytological Preparation Techniques; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages

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

Quaking (QKI) proteins belong to the signal transduction and activation of RNA (STAR) family of RNA-binding proteins that have multiple functions in RNA biology. Here, we show that QKI-5 is dramatically decreased in metastatic lung adenocarcinoma (LUAD). QKI-5 overexpression inhibits TGF-β-induced epithelial-mesenchymal transition (EMT) and invasion, whereas QKI-5 knockdown has the opposite effect. QKI-5 overexpression and silencing suppresses and promotes TGF-β-stimulated metastasis in vivo, respectively. QKI-5 inhibits TGF-β-induced EMT and invasion in a TGFβR1-dependent manner. KLF6 knockdown increases TGFβR1 expression and promotes TGF-β-induced EMT, which is partly abrogated by QKI-5 overexpression. Mechanistically, QKI-5 directly interacts with the TGFβR1 3' UTR and causes post-transcriptional degradation of TGFβR1 mRNA, thereby inhibiting TGF-β-induced SMAD3 phosphorylation and TGF-β/SMAD signaling. QKI-5 is positively regulated by KLF6 at the transcriptional level. In LUAD tissues, KLF6 is lowly expressed and positively correlated with QKI-5 expression, while TGFβR1 expression is up-regulated and inversely correlated with QKI-5 expression. We reveal a novel mechanism by which KLF6 transcriptionally regulates QKI-5 and suggest that targeting the KLF6/QKI-5/TGFβR1 axis is a promising targeting strategy for metastatic LUAD.

Topics: Adenocarcinoma of Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; RNA-Binding Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Immunologic Factors; Lung Neoplasms; Transforming Growth Factor beta

Topics: B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Humans; Immunologic Factors; Lung Neoplasms; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT), transforming growth factor β(TGF-β) and reactive oxygen species(ROS) plays a central role in cancer metastasis. Moreover, nicotinamide adenine dinucleotide phosphate 4(NOX4) is one of the main sources of ROS in lung cancer cells suggesting that NOX4 is associated with tumor cell migration. NF-κB(Nuclear factor-Kappa-B) is known to regulate ROS-mediated EMT process by activating Snail transcription factor in A549 cells. The purpose of this study was to explore the relationship between NF-κB and NOX4 in ROS production during TGF-β induced EMT process. Several fractions have been pooled to evaluates the EMT process on lung cancer cells through real-time PCR, Western Blot and flow cytometry with DCFH-DA probe etc. Cells proliferation and migration activities were monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and wound healing assay respectively. The result showed that TGF-β induction decreased the expression of E-cadherin, increased the Vimentin and the EMT transcription factor Snail in A549 cells. DPI (Diphenyleneiodonium chloride, an inhibitor of NOX4) inhibited the NOX4 expression and reduced ROS production induced by TGF-β, but didn't affect the activation of NF-κB induced by TGF-β (P > 0.05). BAY11-7082 (an inhibitor of NF-κB) inhibited the NF-κB (p65) expression and prevented the increase of NOX4 expression and ROS production induced by TGF-β (P < 0.001), which has also verified reduced TGF-β induced cell migration by inhibiting the EMT process, and also reduced cell proliferation of A549 cells (P < 0.001). The current research confirmed the TGF-β mediated EMT process via NF-κB/NOX4/ROS signaling pathway, NF-κB and NOX4 are likely to be the potential therapeutic targets for lung cancer metastasis.

Topics: A549 Cells; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; NADPH Oxidase 4; NF-kappa B; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

TGF-β/Smad signaling pathway plays an important role in EMT during cancer progression. Long non-coding RNAs (lncRNAs) are involved in various behaviors of cancer cells, including EMT. Here, we report a novel lncRNA adjacent to Smad3, named Smad3-associated long non-coding RNA (SMASR). SMASR is downregulated by TGF-β via Smad2/3 in lung cancer cells. Knockdown of SMASR induces EMT and increases the migration and invasion of lung cancer cells. Moreover, knockdown of SMASR promotes the phosphorylation of Smad2/3. Mechanistically, SMASR interacts with Smad2/3 and inhibits the expression of TGFBR1, the TGF-β type I receptor responsible for phosphorylation of Smad2/3, thus leading to inactivation of TGF-β/Smad signaling pathway. Clinically, SMASR is downregulated in lung cancer tissues. Collectively, our findings prove a critical role of SMASR in EMT of lung cancer by forming a negative feedback loop with TGF-β/Smad signaling pathway.

Topics: Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Phosphorylation; Receptor, Transforming Growth Factor-beta Type I; RNA, Long Noncoding; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor beta (TGFβ) receptor trafficking regulates many TGFβ-dependent cellular outcomes including epithelial to mesenchymal transition (EMT). EMT in A549 non-small cell lung cancer (NSCLC) cells has recently been linked to the regulation of cellular autophagy. Here, we investigated the role of the autophagy cargo receptor, p62/sequestosome 1 (SQSTM1), in regulating TGFβ receptor trafficking, TGFβ1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFβ receptors in the late endosome. Small interfering RNA (SiRNA)-mediated silencing of p62/SQSTM1 resulted in an attenuated time-course of Smad2 phosphorylation but did not alter Smad2 nuclear translocation. However, p62/SQSTM1 silencing promoted TGFβ1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFβ1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFβ-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFβ1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.

Topics: A549 Cells; Autophagy; Carcinoma, Non-Small-Cell Lung; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Sequestosome-1 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

The monofunctional platinum(II) complex, phenanthriplatin, acts by blocking transcription, but its regulatory effects on long-noncoding RNAs (lncRNAs) have not been elucidated relative to traditional platinum-based chemotherapeutics, e.g., cisplatin. Here, we treated A549 non-small cell lung cancer and IMR90 lung fibroblast cells for 24 h with either cisplatin, phenanthriplatin or a solvent control, and then performed microarray analysis to identify regulated lncRNAs. RNA22 v2 microRNA software was subsequently used to identify microRNAs (miRNAs) that might be suppressed by the most regulated lncRNAs. We found that miR-25-5p, -30a-3p, -138-5p, -149-3p, -185-5p, -378j, -608, -650, -708-5p, -1253, -1254, -4458, and -4516, were predicted to target the cisplatin upregulated lncRNAs, IMMP2L-1, CBR3-1 and ATAD2B-5, and the phenanthriplatin downregulated lncRNAs, AGO2-1, COX7A1-2 and SLC26A3-1. Then, we used qRT-PCR to measure the expression of miR-25-5p, -378j, -4516 (A549) and miR-149-3p, -608, and -4458 (IMR90) to identify distinct signaling effects associated with cisplatin and phenanthriplatin. The signaling pathways associated with these miRNAs suggests that phenanthriplatin may modulate Wnt/β-catenin and TGF-β signaling through the MAPK/ERK and PTEN/AKT pathways differently than cisplatin. Further, as some of these miRNAs may be subject to dissimilar lncRNA targeting in A549 and IMR90 cells, the monofunctional complex may not cause toxicity in normal lung compared to cancer cells by acting through distinct lncRNA and miRNA networks.

Topics: beta Catenin; Cell Line, Tumor; Cisplatin; Down-Regulation; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Lung Neoplasms; MicroRNAs; Organoplatinum Compounds; Phenanthridines; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation; Wnt Signaling Pathway

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-β, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Bacillus; Bacteriocins; Fishes; Humans; Lung Neoplasms; Magainins; Molecular Weight; Receptors, TNF-Related Apoptosis-Inducing Ligand; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Transforming Growth Factor beta

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC. In the present study, we demonstrated that RhoJ knockdown accelerated TGF-βmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-β-Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Invasiveness; rho GTP-Binding Proteins; 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

Epithelial-mesenchymal transition (EMT)-a fundamental process in embryogenesis and wound healing-promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.

Topics: Amino Acids; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Humans; Lung Neoplasms; Metabolomics; Mice; Procollagen-Proline Dioxygenase; Transforming Growth Factor beta

Metastasis is the major cause of treatment failure and cancer-related deaths in prostate cancer (PCa) patients. Our previous study demonstrated that a CD44. In this study, we established PCa models via the orthotopic and subcutaneous implantation of different human PCa cancer cell lines, and compared the metastatic efficacy, after which process function analysis of target genes was pinpointed.. Several novel differentially expressed genes (DEGs) between orthotopic and ectopic tumours were identified. Among them, human homeobox B9 (HOXB9) transcription factor was found to be essential for PCa metastasis, as evidenced by the diminished number of lung metastatic foci derived from orthotopic implantation with HOXB9-deficient CWR22 cells, compared with the control. In addition, HOXB9 protein expression was upregulated in PCa tissues, compared with paracancer and benign prostate hyperplasia tissues. It was also positively correlated with Gleason scores. Gain- and loss-of-function assays showed that HOXB9 altered the expression of various tumour metastasis- and cancer stem cell (CSC) growth-related genes in a transforming growth factor beta (TGFβ)-dependent manner. Moreover, HOXB9 was overexpressed in an ALDH. Taken together, our study identified HOXB9 as a critical regulator of metastatic mPCSC behaviour. This occurs through altering the expression of a panel of CSC growth- and invasion/metastasis-related genes via TGFβ signalling. Thus, targeting HOXB9 is a potential novel therapeutic PCa treatment strategy.

Topics: Animals; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Lung Neoplasms; Male; Mice; Neoplasm Grading; Neoplasm Transplantation; Neoplastic Stem Cells; Prostatic Neoplasms; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor beta (TGF-β) is a typical immuno-inhibitory cytokine and highly secreted by lung cancer cells. It was supposed that its immunosuppressive effects to NK cell might be related with the altered expression of activating and inhibitory molecules in lung cancer cells. In this study, we examined the expression of NKG2DLs, PD-L1 and PD-L2 in lung cancer cells after treatment of TGF-β and a TGF-β inhibitor, Galunisertib (LY2157299).. TGF-β reduced the level of surface proteins of five NKG2DLs without altered transcription levels in lung cancer cells. Galunisertib reversed the effect of TGF-β on the expression of NKG2DLs. Since MMP inhibitors, MMPi III and MMP2 inhibitor I, restored the reduced expression of NKG2DLs after treatment of TGF-β, it was thought that TGF-β induced the expression of MMP2 which facilitated the shedding of the NKG2DLs in cancer cells. However, the expression of PD-L1, L2 were not changed by treatment with TGF-β or Galunisertib.. Therefore, inhibition of TGF-β might reverse the immunosuppressive status on immune cells and restore NK cell mediated anticancer immune responses by upregulation of NKG2DLs in cancer cells.

Topics: A549 Cells; Adenocarcinoma, Bronchiolo-Alveolar; Cytotoxicity, Immunologic; Down-Regulation; GPI-Linked Proteins; Humans; Immune Tolerance; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Killer Cells, Natural; Lung Neoplasms; Pyrazoles; Quinolines; Transforming Growth Factor beta; Tumor Escape

Tumor-associated macrophages (TAMs), one of the most common cell components in the tumor microenvironment, have been reported as key contributors to cancer-related inflammation and enhanced metastatic progression of tumors. To explore the underlying mechanism of TAM-induced tumor progression, TAMs were isolated from colorectal cancer patients, and the functional interaction with colorectal cancer cells was analyzed. Our study found that coculture of TAMs contributed to a glycolytic state in colorectal cancer, which promoted the stem-like phenotypes and invasion of tumor cells. TAMs produced the cytokine transforming growth factor-β to support hypoxia-inducible factor 1α (HIF1α) expression, thereby upregulating Tribbles pseudokinase 3 (TRIB3) in tumor cells. Elevated expression of TRIB3 resulted in activation of the β-catenin/Wnt signaling pathway, which eventually enhanced the stem-like phenotypes and cell invasion in colorectal cancer. Our findings provided evidence that TAMs promoted colorectal cancer progression in a HIF1α/TRIB3-dependent manner, and blockade of HIF1α signals efficiently improved the outcome of chemotherapy, describing an innovative approach for colorectal cancer treatment.

Topics: Animals; Cell Cycle Proteins; Cell Proliferation; Coculture Techniques; Colorectal Neoplasms; Disease Progression; Female; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplastic Stem Cells; Phenotype; Protein Serine-Threonine Kinases; Repressor Proteins; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages; Up-Regulation; Wnt Signaling Pathway

Tumor cells metastasis as well as proliferation are important factors that can substantially determines the prognosis of cancer. In particular, epithelial-mesenchymal transition (EMT) is key phenomena which can cause tumor cell transition into other organs by promoting the disruption of the cell-cell junctions. Because oxymatrine (OMT) have been reported to attenuate the tumor growth, we investigated whether OMT can down-regulate EMT process in tumor cells. We also focused on transforming growth factor-β (TGF-β)-induced EMT process because EMT process can be significantly induced by this growth factor.. The cell viability was measured by MTT and real time cell analysis (RTCA) assay. The expression levels of various proteins involved in the regulation of EMT and Akt/mTOR/PI3K signaling pathway were evaluated by Western blot analysis. mRNA levels of several important EMT markers were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The effects of OMT on the cellular invasion and migration were evaluated by RTCA, wound healing assay, and boyden chamber assays.. OMT suppressed the expression of both constitutive and TGF-β-induced mesenchymal markers, such as fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail, but induced the levels of epithelial markers. Moreover, OMT down-regulated oncogenic PI3K/Akt/mTOR pathways which lead to a significant attenuation of invasive and migratory potential of lung cancer cells.. Overall, our study established a novel anti-metastatic role of OMT against human lung cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Alkaloids; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Models, Biological; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolizines; Ribosomal Protein S6 Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Topics: Adult; Aged; Animals; Area Under Curve; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Early Detection of Cancer; Female; Gene Expression Regulation, Neoplastic; Heterografts; Hexosaminidases; Humans; Leukocytes; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Middle Aged; Orosomucoid; ROC Curve; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcriptome; Transforming Growth Factor beta

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

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

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

Recently, long noncoding RNAs (lncRNAs) have been widely reported to play pivotal roles in the regulation of human cancers. Although the oncogenic property of lncRNA small nucleolar RNA host gene 3 (SNHG3) has been revealed in a variety of cancers, functions and regulatory mechanism of SNHG3 in non-small-cell lung cancer (NSCLC) remain to be investigated. In this study, we detected the upregulated expression of SNHG3 in NSCLC tissues as well as cells through quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Using Kaplan-Meier analysis, we determined that a high-level of SNHG3 was associated with a low overall survival rate of patients with NSCLC. Through gain and loss of function experiments, we demonstrated that SNHG3 had a significantly positive effect on NSCLC cell proliferation and migration. Mechanistic investigations revealed that SNHG3 was a predicted direct transcriptional target of E2F1. We observed that the transcriptional activation of SNHG3 could be induced by E2F1. To explore the mechanism, rescue experiments were carried out, which revealed that the cotreatment with SB-431542, JSI-124, or JSI-124 + SB-431542 rescued the effects brought by the overexpression of SNHG3 on NSCLC cell proliferation, migration, and epithelial-mesenchymal transition process. Our results suggested that E2F1 activated SNHG3 and promoted cell proliferation and migration in NSCLC via transforming growth factor-β pathway and interleukin-6/janus-activated kinase 2/signal transducer and activator of transcription 3 pathway, which implied that SNHG3 may be a biomarker for the treatment of patients with NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; E2F1 Transcription Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Janus Kinase 2; Lung Neoplasms; Mice; RNA, Long Noncoding; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta; Up-Regulation

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Transforming Growth Factor beta

Circular RNAs (circRNAs) are novel RNA molecules that play important roles in chemoresistance in different cancers, including breast and gastric cancers. However, whether circRNAs are involved in the response to chemotherapy in small cell lung cancer (SCLC) remains largely unknown. In this study, we observed that cESRP1 (circular RNA epithelial splicing regulatory protein-1) expression was significantly downregulated in the chemoresistant cells compared with the parental chemosensitive cells. cESRP1 enhanced drug sensitivity by repressing miR-93-5p in SCLC. Cytoplasmic cESRP1 could directly bind to miR-93-5p and inhibit the posttranscriptional repression mediated by miR-93-5p, thereby upregulating the expression of the miR-93-5p downstream targets Smad7/p21(CDKN1A) and forming a negative feedback loop to regulate transforming growth factor-β (TGF-β) mediated epithelial-mesenchymal transition. Furthermore, cESRP1 overexpression and TGF-β pathway inhibition both altered tumour responsiveness to chemotherapy in an acquired chemoresistant patient-derived xenograft model. Importantly, cESRP1 expression was downregulated in SCLC patient tissues and was associated with survival. Our findings reveal, for the first time, that cESRP1 plays crucial a role in SCLC chemosensitivity by sponging miR-93-5p to inhibit the TGF-β pathway, suggesting that cESRP1 may serve as a valuable prognostic biomarker and a potential therapeutic target in SCLC patients.

Topics: Animals; Base Sequence; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; RNA, Circular; Signal Transduction; Smad7 Protein; Small Cell Lung Carcinoma; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Lung cancer is one of the cancers with highest morbidity and mortality rates and the metastasis of lung cancer is a leading cause of death. Mechanisms of lung cancer metastasis are yet to be fully understood. Herein, we demonstrate that mice deficient for REGγ, a proteasome activator, exhibited a significant reduction in tumor size, numbers, and metastatic rate with prolonged survival in a conditional Kras/p53 mutant lung cancer model. REGγ enhanced the TGFβ-Smad signaling pathway by ubiquitin-ATP-independent degradation of Smad7, an inhibitor of the TGFβ pathway. Activated TGFβ signaling in REGγ-positive lung cancer cells led to diminished expression of E-cadherin, a biomarker of epithelial-mesenchymal transitions (EMT), and elevated mesenchymal markers compared with REGγ-deficient lung cancer cells. REGγ overexpression was found in lung cancer patients with metastasis, correlating with the reduction of E-Cadherin/Smad7 and a poor prognosis. Overall, our study indicates that REGγ promotes lung cancer metastasis by activating TGF-β signaling via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for lung cancers with poor prognosis.

Topics: A549 Cells; Animals; Antigens, CD; Cadherins; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis-Associated Proteins; Smad7 Protein; Transforming Growth Factor beta

Topics: A549 Cells; Animals; Cell Membrane; Cell Movement; Epithelial-Mesenchymal Transition; Female; Graphite; Hep G2 Cells; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Particle Size; PC-3 Cells; Signal Transduction; Surface Properties; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

The aim of the present study was to investigate whether miR‑203 can inhibit transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT), and the migration and invasion ability of non‑small cell lung cancer (NSCLC) cells by targeting SMAD3. In the present study, the expression levels of miR‑203, SMAD3 mRNA and protein in NSCLC tissues were examined, as well as their corresponding paracancerous samples. The miR‑203 mimics and miR‑203 inhibitor were transfected into the H226 cell line. RT‑qPCR was used to assess the expression levels of E‑cadherin, Snail, N‑cadherin and vimentin mRNA, and western blotting was performed to detect the expression levels of p‑SMAD2, SMAD2, p‑SMAD3, SMAD3 and SMAD4. The cell migration and invasion abilities were detected by Transwell assays. The target site of SMAD3 was predicted by the combined action between miR‑203 and dual luciferase. The results revealed that the RNA levels of miR‑203, compared with paracancerous tissues, were decreased in NSCLC tissues, while SMAD3 mRNA and protein levels were upregulated, and miR‑203 inhibited SMAD3 expression. Induction of TGF‑β led to decreased E‑cadherin mRNA levels, upregulation of Snail, N‑cadherin and vimentin mRNA levels (P<0.05), and significant increase in cell migration and invasion, whereas transfection of miR‑203 mimics reversed the aforementioned results (P<0.05). Conversely, miR‑203 inhibitor could further aggravate the aforementioned results (P<0.05). Western blot results revealed that transfection of miR‑203 mimics significantly reduced the protein expression of SMAD3 and p‑SMAD3 (P<0.05). Furthermore, the results of the Dual‑Luciferase assay revealed that miR‑203 inhibited SMAD3 expression by interacting with specific regions of its 3'‑UTR. Overall, a novel mechanism is revealed, in which, miR‑203 can inhibit SMAD3 by interacting with specific regions of the 3'‑UTR of SMAD3, thereby restraining TGF‑β‑induced EMT progression and migration and invasion of NSCLC cells.

Topics: Adult; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-β-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-β-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-β-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-β-induced EMT of lung cancer cells through the regulation of JUNB.

Topics: Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Methyltransferases; Phenotype; RNA Stability; Transcription Factors; Transforming Growth Factor beta

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, and metastasis is the major cause of CRC-related mortality. Transforming growth factor-beta (TGF-β) has a central role not only in the regulation of the normal colon but also in the development and metastasis of CRC. However, TGF-β is not considered an ideal therapeutic target because it shows both pro-tumorigenic and anti-tumorigenic activity, depending on the tumor stage. Therefore, it is important to find a downstream signaling component of TGF-β that can be targeted to impair CRC metastasis. Here, we show that TGF-β promotes CRC migration and upregulates the expression of long-noncoding RNA Taurine Upregulated Gene 1 (TUG1). TUG1 knockdown inhibited migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells in vitro, and reduced CRC lung metastasis in vivo. TGF-β induced metastasis, and TUG1 knockdown inhibited these effects. In addition, TGF-β could not reverse the anti-metastasis effects of TUG1 knockdown. These data demonstrate that TUG1 is a downstream molecular of TGF-β. Moreover, TWIST1 expression was increased with TGF-β treatment, and TUG1 knockdown decreased TWIST1 expression in CRC cells. TWIST1 knockdown inhibited invasion and EMT in CRC cells; these effects were not changed by simultaneous TUG1 knockdown, indicating that TWIST1 is a downstream mediator of TUG1. Moreover, TUG1 was significantly overexpressed in CRC patients. In conclusion, TGF-β promotes metastasis of CRC via a TUG1/TWIST1/EMT signaling pathway. TUG1 may be a promising drug target to inhibit TGF-β pathway activation in the treatment of CRC.

Topics: Animals; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nuclear Proteins; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Twist-Related Protein 1

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

Myocardin (MYOCD) promotes Smad3-mediated transforming growth factor-β (TGF-β) signaling in mouse fibroblast cells. Our previous studies show that TGF-β/SMADs signaling activation enhances epithelial-mesenchymal transition (EMT) in human non-small cell lung cancer (NSCLC) cells. However, whether and how MYOCD contributes to TGF-β-induced EMT of NSCLC cells are poorly elucidated. Here, we found that TGF-β-induced EMT was accompanied by increased MYOCD expression. Interestingly, MYOCD overexpression augmented EMT and invasion of NSCLC cells induced by TGF-β, whereas knockdown of MYOCD expression attenuated these effects. Overexpression and knockdown of MYOCD resulted in the upregulation and downregulation of TGF-β-induced Snail mRNA, respectively. Moreover, MYOCD overexpression promoted TGF-β-stimulated NSCLC cell metastasis in vivo. MYOCD was highly expressed and positively correlated with Snail in metastatic NSCLC tissues. Mechanistically, MYOCD directly interacted with SMAD3 and sustained the formation of TGF-β-induced nuclear SMAD3/SMAD4 complex, facilitating TGF-β/SMAD3-induced transactivation of Snail. Importantly, MYOCD was transcriptionally activated by TGF-β-induced SMAD3/SMAD4 complex and CRISPR/Cas9-mediated silencing of SMAD3/SMAD4 led to a reduction in MYOCD mRNA expression. Taken together, our findings indicate that MYOCD promotes TGF-β-induced EMT and metastasis of NSCLC and identify a positive feedback loop between MYOCD and SMAD3/SMAD4 driving TGF-β-induced EMT.

Topics: A549 Cells; Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Nuclear Proteins; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

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

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

Gefitinib is a first tyrosine kinase inhibitor (TKI) designed with an EGFR tyrosine kinase for lung cancer targeted therapy. However, some lung cancer patients with wild-type EGFR (wtEGFR) or acquired secondary EGFR mutation showed lower response rate of gefitinib. In this study, we examined the efficacy of fucoidan on altering gefitinib-sensitivity on TKI-resistant lung cancer A549 and H1975 cells. We found that the simultaneous administration of fucoidan and gefitinib synergistically inhibited lung cancer cell viability via activating apoptotic response. Moreover, we found that fucoidan effectively downregulated expressions of mesenchymal-like molecules. Mechanistically, we demonstrated that fucoidan altered the gefitinib-inhibitory rate may result from induction of proteasome-dependent Slug degradation. Abolishment of TGFβ signaling enhanced gefitinib-inhibited cell viability and reduced N-cadherin, Twist and Slug levels. Moreover, knockdown of Slug contributed the increasing the gefitinib-sensitivity of H1975 cells. Our study is the first to find that fucoidan alters the gefitinib-sensitive of TKI-resistant cells by reduction of TGFβ receptor-mediated expressions of mesenchymal-like molecules and induction of Slug degradation. Together, our current results indicate that combination of fucoidan and gefitinib may be a potential and effective therapeutic strategy in gefitinib non-sensitive lung cancer.

Topics: A549 Cells; Animals; Drug Resistance, Neoplasm; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Neoplasm Proteins; Polysaccharides; Snail Family Transcription Factors; Transforming Growth Factor beta

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

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

To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism.. H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing. Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the

Topics: Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Movement; Cell Proliferation; Fibromodulin; Gene Expression; Gene Silencing; Humans; Lung Neoplasms; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

Poria cocos (Polyporacea), is a fungus used in traditional Chinese medicine. A study of the valuable sulfated polysaccharides (SPS) with the structure and pharmaceutical benefits from the mycelial culture conditions of P. cocos was attempted. The SPSs were fractionated by gel filtration chromatography to give a fucose-containing mannoglucan polysaccharide (denoted as FMGP): The main skeleton was a 1,4-α-Man-interlaced-1,3-β-glucan with interlaced 6-O-α-l-fucosyl 1,4-α-Glc and 1,4-α-Gal branches. FMGP dramatically inhibited cell migration in the highly metastatic human lung cancer cell line CL1-5 cells. Mechanistically, FMGP dramatically downregulated the expression of TGFβRI and inhibited phosphorylation of FAK and AKT. Moreover, FMGP reduced the metastasis-related protein, Slug, expression. This is the first paper reporting a branched 1,3-β-mannoglucan from P. cocos and its anti-lung cancer CL1-5 cells migration activities.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Survival; Chemical Fractionation; Dose-Response Relationship, Drug; Down-Regulation; Fucose; Humans; Lung Neoplasms; Phosphorylation; Polysaccharides; Poria; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

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

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

Gefitinib is currently the preferred treatment for non‑small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR)‑activating mutation. However, some patients gradually develop acquired resistance after receiving treatment. In addition to secondary T790M mutation, the remaining mechanisms contributing to non‑T790M mutations need to be explored. In the present study, NSCLC‑derived HCC827 and PC‑9 cells and the corresponding gefitinib‑resistant cell lines (HCC827GR and PC9GR) were utilized. Next‑generation DNA sequencing was performed on the HCC827GR and PC9GR cells. Under AXL receptor tyrosine kinase (AXL) knockdown or miR‑625‑3p overexpressing conditions, a cell growth inhibition assay was performed to evaluate gefitinib sensitivity. Wound healing and Transwell assays were used to examine the migratory and invasive abilities of the cells. Moreover, we also carried out western blot analysis to detect the altered downstream signaling pathway. Our study revealed markedly decreased miR‑625‑3p expression in the HCC827GR cell line, while its overexpression partly reversed gefitinib resistance. Integrated analysis based on Targetscan website showed that AXL can be potentially targeted by miR‑625‑3p and we further verified the hypothesis via dual‑luciferase reporter assays. Mechanistic analysis revealed that TGF‑β1‑induced EMT may contribute to the miR‑625‑3p/AXL axis‑mediated gefitinib resistance. Our data demonstrated that miR‑625‑3p contributes to the acquired resistance of gefitinib, which may provide novel insight to combat resistance to EGFR‑TKIs.

Topics: Axl Receptor Tyrosine Kinase; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Lung Neoplasms; MicroRNAs; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Sequence Analysis, DNA; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Pneumonectomy; Transforming Growth Factor beta

Activation of oncogenic KRAS is the most common driving event in lung adenocarcinoma development. Despite the existing rationale for targeting activated KRAS and its downstream effectors, the failure of clinical trials to date indicates that the mechanism of KRAS-driven malignancy remains poorly understood. Here we report that histone deacetylase 10 (HDAC10) might function as a putative tumor suppressor in mice carrying a spontaneously activated oncogenic

Topics: Adenocarcinoma of Lung; Animals; Carcinogenesis; Cell Line, Tumor; Disease Models, Animal; Gene Deletion; Genes, ras; Germ-Line Mutation; Histone Deacetylases; Lung Neoplasms; Macrophages; Mice; Neoplastic Stem Cells; Sequence Analysis, RNA; SOX9 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Suppressor Proteins

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

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

Transforming growth factor-β (TGF-β) promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT). EMT is often related with acquisition of stemness characteristics. The objective of this study was to determine whether EMT and stemness characteristics induced by TGF-β might be associated with epigenetic regulation in lung cancer. A human normal lung epithelial cell line and four lung cancer cell lines were treated with TGF-β. Transcriptome analysis of BEAS-2B and A549 cells incubated with TGF-β were analyzed through next-generation sequencing (NGS). Western blotting was carried out to investigate expression levels of epithelial and mesenchymal markers. Wound healing and Matrigel invasion assay, sphere formation assay, and in vivo mice tumor model were performed to evaluate functional characteristics of EMT and stemness acquisition. To investigate whether activation of EMT and stem cell markers might be involved in epigenetic regulation of lung cancer, experiment using a DNA methyltransferase inhibitor (5-azacytidine, AZA), methylation-specific PCR (MSP) and bisulfite sequencing were performed. NGS revealed changes in expression levels of EMT markers (E-cadherin, N-cadherin, fibronectin, vimentin, slug and snail) and stem cell markers (CD44 and CD87) in both BEAS-2B and A549 cells. Functional analysis revealed increased migration, invasion, sphere formation, and tumor development in mice after TGF-β treatment. Expression of slug and CD87 genes was activated following treatment with AZA and TGF-β. MSP and bisulfite sequencing indicated DNA demethylation of slug and CD87 genes. These results suggest that TGF-β induced EMT and cancer stemness acquisition could be associated with activation of slug and CD87 gene by their promoter demethylation.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Epigenesis, Genetic; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Promoter Regions, Genetic; Stem Cells; Transforming Growth Factor beta; Wound Healing

Early detection of cancer is essential for effective intervention. Urine has been used to reflect early changes in various tumor-bearing models. However, urine has not been used to predict whether tumors will form in animal models. In this study, a cancer model was established by tail vein injection of 2 million NuTu-19 tumor cells. Urine samples were randomly selected from tumor-forming and non-tumor-forming rats on day 0/12/27/39/52 and were analyzed by label-free and parallel reaction monitoring targeted proteomic quantitative analyses. In tumor-forming rats, differential proteins were associated with tumor cell migration, TGF-β signaling and the STAT3 pathway. A total of 9 urinary proteins showed significant changes in the early phase of lung tumor formation in all eight tumor-bearing rats. Differential proteins in non-tumor-forming rats were associated with glutathione biosynthesis, IL-12 signaling and vitamin metabolism. A total of 12 urinary proteins changed significantly in the early phase in all seven non-tumor-forming rats. Our small-scale pilot study indicated that (1) the urinary proteome reflects early changes during lung tumor formation and that (2) the urinary proteome can distinguish early tumor-forming rats from non-tumor-forming rats.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Early Detection of Cancer; Female; Lung Neoplasms; Ovarian Neoplasms; Pilot Projects; Proteome; Proteomics; Rats; Rats, Wistar; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

Malignant pleural effusion (MPE) is defined as the presence of tumor cells in pleural fluid and it is a fatal complication of advanced lung adenocarcinoma (LAC). To understand the immune response to the tumor in MPE, we compared the concentration of immunomodulatory factors in MPE of LAC and pleural effusion of heart failure (HF) patients by ELISA, and the proliferation and cytotoxic phenotype of T cells stimulated in the presence of LAC and HF pleural fluids by cytometry. Platelet factor 4 (PF4), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and P-selectin levels were higher in LAC than in HF pleural fluids. However, plasmatic PF4 and P-selectin levels were similar in LAC and HF. VEGF positively correlated with TGF-β and sPD-L1 in LAC but not in HF pleural fluids. LAC pleural fluids also inhibited T lymphocyte proliferation and cytotoxicity and reduced IL-17 production. PF4 levels inversely correlated with T cell function. The high content of PF4 in MPE was associated with poor prognosis. Our findings suggest that an impaired response of T lymphocytes induced by PF4 provides a significant advantage for tumor progression.

Topics: Adenocarcinoma of Lung; Aged; Aged, 80 and over; Female; Heart Failure; Humans; Lung Neoplasms; Lymphocyte Activation; Male; Middle Aged; Platelet Factor 4; Pleural Effusion, Malignant; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors.. A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-β-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed.. TGF-β treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-β-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-β-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor.. Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.

Topics: Antifungal Agents; Carcinoma, Non-Small-Cell Lung; Cell Survival; Drug Repositioning; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Lung Neoplasms; Monensin; Protein Kinase Inhibitors; Proton Ionophores; Transforming Growth Factor beta

Despite advances in cancer treatment, drug resistance and metastasis continue to contribute to treatment failure. Since drug resistance and metastasis in cancer are features that often occur toward the late stages in the disease after withstanding numerous selective pressures, they may rely on a shared adaptive mechanism in order to persist. The heat shock response is one of the most well conserved adaptive responses to cellular stress found in nature. A major player in the heat shock response is HSP90, with some studies suggesting that it can facilitate the molecular evolution of drug resistance and metastasis in cancer. Non-small cell lung cancers (NSCLCs) are strongly associated with drug resistance and metastasis either at the time of diagnosis or early in the treatment process.. We explored the role of HSP90 in the evolution of metastatic and drug resistant features in NSCLC by treating A549 cells with AUY922, a clinically relevant HSP90 inhibitor, and inducing metastatic and drug resistant phenotypes via treatment with TGF-β and paclitaxel, respectively. We measured phenotypic plasticity in E-Cadherin, a marker for epithelial to mesenchymal transition and two ABC transporters associated with drug resistant lung cancers.. We found that metastatic and efflux dependent drug resistant features negatively correlated with AUY922 treatment. We followed our results with functional assays relevant to metastasis and ABC transporters to confirm our results. Specifically we found the expression of E-cadherin was significantly increased in A549 cultures pretreated with AUY922 prior to exposure to paclitaxel, while expression of the drug transporters ABCB1 and ABCC1 was significantly reduced under similar conditions.. Together our data indicates that HSP90 inhibition with AUY922 can limit the acquisition of metastatic and drug resistant phenotypes in A549 cells at low, clinically appropriate doses.

Topics: A549 Cells; Antigens, CD; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Plasticity; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Paclitaxel; Resorcinols; Signal Transduction; Transforming Growth Factor beta

Regulation of the stemness factor, SOX2, by cytokine stimuli controls self-renewal and differentiation in cells. Activating mutations in EGFR are proven therapeutic targets for tyrosine kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKIs inevitably occurs. The mechanism by which stemness and differentiation signaling emerge in lung cancers to affect TKI tolerance and lung cancer dissemination has yet to be elucidated. Here, we report that cross-talk between SOX2 and TGFβ signaling affects lung cancer cell plasticity and TKI tolerance. TKI treatment favored selection of lung cancer cells displaying mesenchymal morphology with deficient SOX2 expression, whereas SOX2 expression promoted TKI sensitivity and inhibited the mesenchymal phenotype. Preselection of EGFR-mutant lung cancer cells with the mesenchymal phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2 silencing induced vimentin, but suppressed BCL2L11, expression and promoted TKI tolerance. TGFβ stimulation downregulated SOX2 and induced epithelial-to-mesenchymal transdifferentiation accompanied by increased TKI tolerance, which can interfere with ectopic SOX2 expression. SOX2-positive lung cancer cells exhibited a lower dissemination capacity than their SOX2-negative counterparts. Tumors expressing low SOX2 and high vimentin signature were associated with worse survival outcomes in patients with EGFR mutations. These findings provide insights into how cancer cell plasticity regulated by SOX2 and TGFβ signaling affects EGFR-TKI tolerance and lung cancer dissemination. SIGNIFICANCE: These findings suggest the potential of SOX2 as a prognostic marker in EGFR-mutant lung cancer, as SOX2-mediated cell plasticity regulated by TGFβ stimulation and epigenetic control affects EGFR-TKI tolerance and cancer dissemination.

Topics: Adenocarcinoma of Lung; Antineoplastic Agents; Biomarkers, Tumor; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Signal Transduction; SOXB1 Transcription Factors; Transforming Growth Factor beta; Vimentin

Phosphoglycerate mutase 1 (PGAM1) is a recently identified key catalytic enzyme in aerobic glycolysis. Recent literature has documented that dysregulated PGAM1 expression is associated with tumorigenesis in various cancers. However, the expression status and biological function of PGAM1 in non-small-cell lung cancer (NSCLC) are poorly elucidated. In this study, we found that PGAM1 was overexpressed in NSCLC tissues and that high expression of PGAM1 was associated with poor prognosis in NSCLC patients. Functionally, gain- and loss-of-function analysis showed that PGAM1 promoted proliferation and invasion in vitro, and facilitated tumor growth in vivo. Mechanistically, the transforming growth factor-β (TGF-β) signaling pathway was also markedly impaired in response to PGAM1 silencing. Additionally, we verified that PGAM1 was inhibited by miR-3614-5p via direct targeting of its 3'-untranslated regions in a hypoxia-independent manner. Furthermore, overexpression of miR-3614-5p attenuated NSCLC cell proliferation and invasion, and these effects could be partially reversed by reintroduction of PGAM1. Conclusively, our results suggest that the miR-3614-5p/PGAM1 axis plays a critical role during the progression of NSCLC, and these findings may provide a potential target for the development of therapeutic strategies for NSCLC patients.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; China; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Oncogenes; Phosphoglycerate Mutase; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factors

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

Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-β binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-β activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-β signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.

Topics: Adenocarcinoma; Aged; Animals; Antigens, CD; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; Disease Models, Animal; Female; GPI-Linked Proteins; Humans; Latent TGF-beta Binding Proteins; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Prognosis; RNA, Small Interfering; Transfection; Transforming Growth Factor beta

MiR-133a has been confirmed to be involved in the development of multiple cancers including non-small cell lung cancer (NSCLC). However, the precise molecular mechanism has not yet been fully elucidated. The purpose of this study was to investigate the functional role and underlying mechanism of miR-133a in the progression of NSCLC.. Quantitative real-time PCR (qRT-PCR) was performed to measure miR-133a and LASP1 expression in NSCLC tissues and cells. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to detect cell viability. The protein levels were measured by western blot. The tumor growth was measured by xenograft tumor formation assay.. miR-133a was significantly decreased while LASP1 was increased in NSCLC tissues and cells compared with control groups. Moreover, overexpression of miR-133a suppressed cell viability, whereas miR-133a knockdown enhanced the viability of A549 cells. More importantly, LASP1 was verified as a direct target of miR-133a. Moreover, overexpression of miR-133a inhibited the epithelial-mesenchymal transition (EMT) and TGF-β/Smad3 pathways by regulating LASP1 in vitro. In addition, miR-133a mimic suppressed tumor growth by modulating the TGF-β/Smad3 pathway in vivo.. In conclusion, miR-133a acted as a tumor suppressor in lung cancer progression by regulating the LASP1 and TGF-β/Smad3 signaling pathway.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Animals; Cell Proliferation; Cytoskeletal Proteins; Disease Progression; Heterografts; Humans; LIM Domain Proteins; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Antitumor immune responses induced by immune checkpoint inhibitors anti-PD-1 or anti-PD-L1 have been used as therapeutic strategies in advanced non-small cell lung cancer (NSCLC) patients over the last decade. Favorable antitumor activity to immune checkpoint inhibitors is correlated with high PD-L1 expression, increased tumor-infiltrating lymphocytes, and decreased suppressive immune cells including Treg cells, myeloid-derived suppressor cells, or tumor-associated macrophages in various cancer types. In this study, we investigated the potential correlation between clinical outcomes and peripheral blood immune cell profiles, specifically focused on FoxP3

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Carcinoma, Non-Small-Cell Lung; Cohort Studies; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Immune Checkpoint Inhibitors; Lung Neoplasms; Male; Middle Aged; Nivolumab; Survival Analysis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Treatment Outcome; Tumor Microenvironment

Transforming growth factor beta (TGFβ) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFβ may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFβ1 modulates autophagy related (

Topics: Autophagosomes; Autophagy; Autophagy-Related Proteins; Biomarkers; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Lysosomes; Microtubule-Associated Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

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

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

Stimulator of interferon genes (STING) controlled innate immune pathway is essential for host defense against pathogenic infection and effective anti-tumor adaptive immunity initiation. Although macrophages transformed across diverse phenotypes play crucial roles in anti-tumor immune response, events determining this transformation and the host-intrinsic role of STING in this process remain controversial. Here we report how STING signaling acts as a key switch to dominate the gene expression patterns of macrophage transformation for promoting priming and releasing immunosuppression. Furthermore, polyphyllin VII, a potential STING agonist, exerts anti-tumor efficacy upon macrophages priming and subsequent cytotoxic T lymphocytes intratumoral infiltration. Meanwhile, the simultaneous PD-L1 amplification on macrophages in response to PP VII is also ruled by STING, thus PP VII may benefit immune-checkpoint blockade therapy for combining. Moreover, PP VII suppresses carcinogenesis upon restraining the immunosuppressed macrophage transformation. This is due to the boosted STING that negatively regulates a STAT3 propagated crosstalk between immune cells and tumor cells. Overall, PP VII-stimulated STING in macrophages provides a paradigm for anti-tumor, and if possible, anti-infection immunotherapy.

Topics: Animals; CD8-Positive T-Lymphocytes; Immunohistochemistry; Interleukin-10; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Saponins; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

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

The purpose of this study was to investigate the anti-cancer effect of melittin on growth, migration, invasion, and apoptosis of non-small-cell lung cancer (NSCLC) cells. This study also explored the potential anti-cancer mechanism of melittin in NSCLC cells. The results demonstrated that melittin suppressed growth, migration, and invasion, and induced apoptosis of NSCLC cells in vitro. Melittin increased pro-apoptotic caspase-3 and Apaf-1 gene expression. Melittin inhibited tumor growth factor (TGF)-β expression and phosphorylated ERK/total ERK (pERK/tERK) in NSCLC cells. However, TGF-β overexpression (pTGF-β) abolished melittin-decreased TGF-β expression and pERK/tERK in NSCLC cells. Treatment with melittin suppressed tumor growth and prolonged mouse survival during the 120-day observation in vivo. Treatment with melittin increased TUNEL-positive cells and decreased expression levels of TGF-β and ERK in tumor tissue compared to the control group. In conclusion, the findings of this study indicated that melittin inhibited growth, migration, and invasion, and induced apoptosis of NSCLC cells through down-regulation of TGF-β-mediated ERK signaling pathway, suggesting melittin may be a promising anti-cancer agent for NSCLC therapy.

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Movement; Down-Regulation; Gene Expression Regulation, Neoplastic; Lung Neoplasms; MAP Kinase Signaling System; Melitten; Mice; Neoplasm Invasiveness; Transforming Growth Factor beta

Lung cancer causes approximately one fifth of all cancer deaths. Tumour cells actively communicate with the surrounding microenvironment to support malignant progression. Extracellular vesicles (EVs) play a pivotal role in intercellular communication and modulate recipient cells by delivering their contents, including proteins and nucleic acids such as microRNAs (miRNAs). We isolated EVs from the conditioned medium (CM) of human lung cancer cell lines and plasma of lung cancer patients and cancer-free smokers using an ultracentrifugation method. A significant increase in bronchial HBEC-KRAS

Topics: A549 Cells; Aged; Apoptosis; Cell Proliferation; Extracellular Vesicles; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Tomography, Emission-Computed; Transforming Growth Factor beta; Tumor Microenvironment

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFβ) and Hedgehog (Hh) signal transduction cascades often associate with poor prognosis and aggressive disease progression in NSCLC, as these signals can drive cell proliferation, angiogenesis, metastasis, immune evasion and emergence of drug resistance. Therefore, simultaneous inhibition of TGFβ and Hh signaling, by a single agent, or in combination with other drugs, could yield therapeutic benefits in NSCLC and other cancers. In the current study, we report on the biological and pharmacological evaluation of Oxy210, an oxysterol-based dual inhibitor of TGFβ and Hh signaling. In NSCLC cells, Oxy210 inhibits proliferation, epithelial-mesenchymal transition (EMT) and invasive activity. Combining Oxy210 with Carboplatin (CP) increases the anti-proliferative response to CP and inhibits TGFβ-induced resistance to CP in A549 NSCLC cells. In addition, Oxy210 displays encouraging drug-like properties, including chemical scalability, metabolic stability and oral bioavailability in mice. Unlike other known inhibitors, Oxy210 antagonizes TGFβ and Hh signaling independently of TGFβ receptor kinase inhibition and downstream of Smoothened, respectively.

Topics: A549 Cells; Animals; Carboplatin; Carcinoma, Non-Small-Cell Lung; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Lung Neoplasms; Mice; Neoplasm Proteins; NIH 3T3 Cells; Oxysterols; Signal Transduction; Transforming Growth Factor beta

Malignant melanoma is the most aggressive form of skin cancer; a substantial percentage of patients present with distant metastases. However, the mechanism of metastasis is not well understood. Here, we demonstrate that the administration of exogenous regulatory T cells (Tregs) into melanoma tumor-bearing mice results in a significant increase in lung metastasis. An increase in the invasive and metastatic phenotype of melanoma was mediated by cell-to-cell contact between melanoma cells and Tregs, which elevated the expression level of transforming growth factor-β (TGF-β) and the subsequent induction of the epithelial-to-mesenchymal transition (EMT).B16-BL6 melanoma tumors co-cultured with Tregs showed a larger population of migrating cells compared to B16-BL6 tumors cultured without Tregs. Additionally, the injection of exogenous Tregs into B16-BL6 melanoma tumors led to the recruitment and infiltration of endogenous Tregs into tumor tissues, thus increasing the overall Treg percentage in the tumor infiltrating lymphocyte population. Collectively, our findings propose novel mechanisms in which exogenous Treg-dependent upregulation of TGF-β and mesenchymal markers is important for augmenting the migration capacity and invasiveness of melanoma, thereby contributing to the metastasis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Melanoma; Melanoma, Cutaneous Malignant; Mice; Mice, Inbred C57BL; Skin Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

The objective of this study is to compare the effects of paravertebral nerve block-propofol intravenous general anesthesia (PPA) and sevoflurane inhalation general anesthesia (SGA) on the expression of serum vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β) in patients undergoing radical resection of lung cancer.Patients undergoing radical resection of lung cancer were divided into PPA group and SGA group. In PPA group, thoracic paraspinal nerve block was performed with 0.5% ropivacaine (2 mg/kg) before general anesthesia. Anesthesia was maintained with 2.5-3.5 μg/mL TCI of propofol. In SGA group, anesthesia was maintained with 1.0-1.5 MAC sevoflurane. The dosage of opioids during and 24 h after operation, the pain score at 2, 8, 24, 48, and 72 h after operation, and the concentrations of serum VEGF and TGF-β before and 24 h after operation were observed in the two groups.The intraoperative dosage of remifentanil in PPA group was significantly less than that in SGA group (P < 0.05). The dosage of sufentanil in SGA group was significantly less than that in SGA group at 24 h after operation (P < 0.05). The VAS score at 2, 8, and 24 h after operation was significantly lower than that in SGA group (P < 0.05). The serum VEGF and TGF-β concentration in PPA group was significantly lower than that in SGA group (P < 0.05).Thoracic paravertebral nerve block-propofol intravenous general anesthesia can reduce the dosage of opioids, improve the effect of postoperative analgesia, and reduce the serum concentration of tumor angiogenesis-related factors in patients undergoing radical resection of lung cancer.

Topics: Anesthesia, General; Anesthesia, Inhalation; Anesthesia, Intravenous; Anesthetics, Inhalation; Anesthetics, Intravenous; Female; Humans; Lung Neoplasms; Male; Middle Aged; Paraspinal Muscles; Pneumonectomy; Propofol; Prospective Studies; Sevoflurane; Thorax; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

Elucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify, through TGFβ-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies.

Topics: Algorithms; Cell Line, Tumor; Computational Biology; Cytophotometry; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Phenotype; Systems Biology; Transforming Growth Factor beta

We investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process.. In this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins.. Here, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-β (TGF-β) derived from TAMs, leading to the invasion and migration of cancer cells.. Our results demonstrated that TAMs contributed the EMT progression through a TGF-β/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-β receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Topics: Animals; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; HCT116 Cells; Humans; Lung Neoplasms; Macrophages; Male; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

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

As one of the leading causes of cancer deaths world-wide, the progression of human non-small cell lung cancer (NSCLC) can be regulated by estrogenic signals. Our present data showed that an industrial endocrine disrupting chemical, bisphenol S (BPS), can promote the in vitro migration of NSCLC cells, which was evidenced by the upregulation of vimentin and matrix metalloproteinase-2 (MMP-2). BPS can increase the mRNA and protein expression of IL-10 and TGF-β. While only targeted inhibition of TGF-β can block BPS induced migration of NSCLC cells. The upregulation of TGF-β can further activate the Smad-2/3 pathways. Further, BPS induced expression of TGF-β was ERα/β or G protein-coupled estrogen receptor (GPER) independent, since targeted inhibition of ERα/β or GPER had no effect on BPS induced transcription of TGF-β. We identified that the inhibitor of ERK1/2 can attenuate BPS induced expression of TGF-β and activation of Smad-2/3 pathways. Collectively, we found that nanomolar BPS can trigger the in vitro migration of NSCLC cells via ERK1/2 mediated activation of TGF-β/Smad-2/3 pathways.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Humans; Lung Neoplasms; Phenols; Sulfones; Transforming Growth Factor beta; Up-Regulation

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.

Topics: A549 Cells; Adenocarcinoma of Lung; Cancer-Associated Fibroblasts; Cellular Reprogramming; Coculture Techniques; Fibrosis; Glycolysis; Humans; Lung; Lung Neoplasms; Neoplasm Staging; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive form of NSCLC. Rarity and poor characterization have limited the development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment.. A training set of 17 biphasic PSCs was selected and tested for the expression of a large panel of 770 genes related to cancer progression using NanoString technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype.. We demonstrated that the transcriptional activation of an epithelial mesenchymal transition (EMT) program drives PSC phylogeny. Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Computational Biology; Dasatinib; Drug Substitution; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Lung Neoplasms; Models, Biological; Phenotype; Protein Kinase Inhibitors; Sarcoma; Transcription, Genetic; Transforming Growth Factor beta

Asbestos exposure increases the risk of asbestosis and malignant mesothelioma (MM). Both fibrosis and cancer have been correlated with the Epithelial to Mesenchymal Transition (EMT)-an event involved in fibrotic development and cancer progression. During EMT, epithelial cells acquire a mesenchymal phenotype by modulating some proteins. Different factors can induce EMT, but Transforming Growth Factor β (TGF-β) plays a crucial role in promoting EMT. In this work, we verified if EMT could be associated with MM development. We explored EMT in human mesothelial cells (MeT-5A) exposed to chrysotile asbestos: we demonstrated that asbestos induces EMT in MeT-5A cells by downregulating epithelial markers E-cadherin, β-catenin, and occludin, and contemporarily, by upregulating mesenchymal markers fibronectin, α-SMA, and vimentin, thus promoting EMT. In these cells, this mechanism is mediated by increased TGF-β secretion, which in turn downregulates E-cadherin and increases fibronectin. These events are reverted in the presence of TGF-β antibody, via a Small Mother Against Decapentaplegic (SMAD)-dependent pathway and its downstream effectors, such as Zinc finger protein SNAI1 (SNAIL-1), Twist-related protein (Twist), and Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), which downregulate the

Topics: Antibodies; Asbestos, Serpentine; beta Catenin; Cadherins; Cell Line; Down-Regulation; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibronectins; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Mesothelioma; Mesothelioma, Malignant; Smad Proteins; Snail Family Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Vimentin; Zinc Finger E-box-Binding Homeobox 1

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

Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-β), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-β-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Collagen; Disease Models, Animal; Extracellular Matrix; Female; Humans; Inflammation; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Mice; Mice, Inbred CBA; Pleural Neoplasms; Thiazolidines; Transforming Growth Factor beta

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

Topics: Humans; Interferon-beta; Lung Neoplasms; Models, Biological; Neoplasm Proteins; Neutrophils; Transforming Growth Factor beta; Tumor Microenvironment

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and has high rates of metastasis. Transforming growth factor beta-inducible protein (TGFBI) is an extracellular matrix component involved in tumour growth and metastasis. However, the exact role of TGFBI in NSCLC remains controversial. Gene silencing via DNA methylation of the promoter region is common in lung tumorigenesis and could thus be used for the development of molecular biomarkers. We analysed the methylation status of the

Topics: Adenocarcinoma of Lung; Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Methylation; Extracellular Matrix Proteins; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Promoter Regions, Genetic; Survival Analysis; Transforming Growth Factor beta; Treatment Outcome

SGA is a sulfated glucan from Antrodia cinnamomea. In this study, we showed that SGA suppressed tumor growth in vitro and in vivo. SGA also potentiated cisplatin-induced cytotoxicity in lung cancer cells. TGFβ signaling and overexpression of Slug are regarded as the critical events in lung tumor malignancy. Functional studies revealed that SGA inhibited the TGFβ/FAK/AKT axis by inducing lipid-raft-mediated lysosome-dependent TGFβ receptor degradation, resulting in suppressing cancer cell viability and migration. Moreover, SGA elimination of TGFβ-mediated intracellular signaling promoted Slug degradation in H1975 cells. Mechanistically, we demonstrated that proteasome-dependent Slug degradation was controlled by TGFβ-mediated downstream signaling pathways; however, inhibitors of AKT and GSK3 abolished Slug degradation. Our findings suggested that SGA targets of the TGFβ/AKT/GSK3β axis played a key role in enhancing Slug degradation and suppressing lung cancer cells. In addition, SGA may be a potential therapeutic supplement for lung cancer.

Topics: Animals; Antrodia; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Survival; Cisplatin; Drug Synergism; Enzyme Activation; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Glucans; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Male; Mice; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Hyper-activation of TGF-β signaling is critically involved in progression of hepatocellular carcinoma (HCC). However, the events that contribute to the dysregulation of TGF-β pathway in HCC, especially at the post-translational level, are not well understood.. Associations of deubiquitinase POH1 with TGF-β signaling activity and the outcomes of HCC patients were examined by data mining of online HCC datasets, immunohistochemistry analyses using human HCC specimens, spearman correlation and survival analyses. The effects of POH1 on the ubiquitination and stability of the TGF-β receptors (TGFBR1 and TGFBR2) and the activation of downstream effectors were tested by western blotting. Primary mouse liver tissues from polyinosinic:polycytidylic acid (poly I:C)- treated Mx-Cre+, poh1. Here we show that POH1 is a critical regulator of TGF-β signaling and promotes tumor metastasis. Integrative analyses of HCC subgroups classified with unsupervised transcriptome clustering of the TGF-β response, metastatic potential and outcomes, reveal that POH1 expression positively correlates with activities of TGF-β signaling in tumors and with malignant disease progression. Functionally, POH1 intensifies TGF-β signaling delivery and, as a consequence, promotes HCC cell metastatic properties both in vitro and in vivo. The expression of the TGF-β receptors was severely downregulated in POH1-deficient mouse hepatocytes. Mechanistically, POH1 deubiquitinates the TGF-β receptors and CAV1, therefore negatively regulates lysosome pathway-mediated turnover of TGF-β receptors.. Our study highlights the pathological significance of aberrantly expressed POH1 in TGF-β signaling hyperactivation and aggressive progression in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Caveolin 1; Cell Line, Tumor; Down-Regulation; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Prognosis; Proteasome Endopeptidase Complex; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transforming Growth Factor beta; Ubiquitination

In this study, we aimed to discuss the clinical significance of CD4. Sixty patients with NSCLC and 35 healthy controls were studied. Peripheral blood CD4. Compared with healthy people, CD4. CD4

Topics: Carcinoma, Non-Small-Cell Lung; Case-Control Studies; CD4-Positive T-Lymphocytes; Female; Flow Cytometry; Humans; Interleukin-10; Lung Neoplasms; Lymphocyte Count; Male; Middle Aged; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Necrosis Factor Receptor Superfamily, Member 7

Multiplex proteome quantification with high accuracy is urgently required to achieve a comprehensive understanding of dynamic cellular and physiological processes. Among the existing quantification strategies, fragment-ion-based methods can provide highly accurate results, but the multiplex capacity is limited to 3-plex. Herein, we developed a multiplex pseudo-isobaric dimethyl labeling (m-pIDL) method to extend the capacity of the fragment-ion-based method to 6-plex by one-step dimethyl labeling with several millidalton and dalton mass differences between precursor ions and enlarging the isolation window of precursor ions to 10 m/ z during data acquisition. m-pIDL showed high quantification accuracy within the 20-fold dynamic range. Notably, the ratio compression was 1.13-fold in a benchmark two-proteome model (5:1 mixed E. coli proteins with HeLa proteins as interference), indicating that by m-pIDL, the ratio distortion of isobaric labeling approaches and the approximate 40% ratio shift of the label-free quantification strategy could be effectively eliminated. Additionally, m-pIDL did not show ratio variation among post-translational modifications (CV = 6.66%), which could benefit the measurement of universal protein properties for proteomic atlases. We further employed m-pIDL to monitor the time-resolved responses of the TGF-β-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma A549 cell lines, which facilitated the finding of new potential regulatory proteins. Therefore, the 6-plex quantification of m-pIDL with the remarkably high accuracy might create new prospects for comprehensive proteome analysis.

Topics: A549 Cells; Adenocarcinoma of Lung; Epithelial-Mesenchymal Transition; Escherichia coli; Escherichia coli Proteins; Humans; Isotope Labeling; Leucine; Lung Neoplasms; Neoplasm Proteins; Peptide Fragments; Proteome; Transforming Growth Factor beta

Because of disease heterogeneity, limited studies on effective chemotherapies and therapeutic agents for advanced gastric cancer are available. Erythrocyte membrane protein band 4.1-like 5 (EPB41L5) has critical roles in renal and breast cancer metastasis. However, its role in metastatic gastric cancer remains unknown.. The specimens of 78 gastric cancer patients were analyzed by oligonucleotide microarray and survival analysis.. Gastric cancer patients with high EPB41L5 levels had poor prognosis and low survival rate. Further, TGFβ1-induced EPB41L5 expression promoted gastric cancer cell migration and invasion by Smad-dependent TGFβ signaling. Phospho-Smad3 recruitment to the. TGFβ/EPB41L5/p120-catenin axis regulates gastric cancer cell metastasis, and EPB41L5 is a promising therapeutic target for advanced gastric cancer.

Topics: Animals; Cell Line, Tumor; Cell Movement; Chick Embryo; Female; Heterografts; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Prognosis; Promoter Regions, Genetic; Signal Transduction; Stomach Neoplasms; Survival Rate; Transforming Growth Factor beta

To detect the protein level of Decorin in non-small cell lung cancer (NSCLC) patients, and to study the mechanism of Decorin inhibiting invasion and metastasis of non-small cell lung cancer from the perspective of in vitro cells, and provide some theoretical support for the treatment of non-small cell lung cancer.. Immunohistochemical staining was used to detect the expression of Decorin protein in 332 cases of stage I-IIIA clinical NSCLC and 70 cases of adjacent tissues. Then, in vitro cell experiments (cell scratch assay and transwell assay) were used to further study the effects of Decorin on migration and invasion of human lung cancer cell line A549; the effect of transforming growth factor-β on the expression of Decorin and Ets-1 protein was verified by Western blotting. The binding sites of Ets-1 and Decorin promoter were analyzed by bioinformatics.. Exogenous Decorin inhibited invasion and metastasis of lung adenocarcinoma cells in vitro. Immunohistochemical staining showed that Decorin was lowly expressed in non-small cell lung cancer and Decorin had a certain effect on lung fibroblast activation. Western blotting results showed that TGF-β affects the expression of Decorin and Ets-1. Bioinformatics results showed that Ets-1 and Decorin gene DNA promoter regions have 18 binding sites.. Decorin inhibits invasion and metastasis of non-small cell lung cancer through the TGF-β signaling pathway.

Topics: Actins; Binding Sites; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Decorin; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-1; Transforming Growth Factor beta

Diacylglycerol (DAG)/phorbol ester-regulated protein kinase C (PKC) isozymes have been widely linked to tumor promotion and the development of a metastatic phenotype. PKCε, an oncogenic member of the PKC family, is abnormally overexpressed in lung cancer and other cancer types. This kinase plays significant roles in proliferation, survival, and migration; however, its role in epithelial-to-mesenchymal transition (EMT) has been scarcely studied. Silencing experiments in non-small lung cancer (NSCLC) cells revealed that PKCε or other DAG-regulated PKCs (PKCα and PKCδ) were dispensable for the acquisition of a mesenchymal phenotype induced by transforming growth factor beta (TGF-β). Unexpectedly, we found a nearly complete down-regulation of PKCε expression in TGF-β-mesenchymally transformed NSCLC cells. PMA and AJH-836 (a DAG-mimetic that preferentially activates PKCε) promote ruffle formation in NSCLC cells via Rac1, however they fail to induce these morphological changes in TGF-β-mesenchymally transformed cells despite their elevated Rac1 activity. Several Rac guanine nucleotide exchange-factors (Rac-GEFs) were also up-regulated in TGF-β-treated NSCLC cells, including Trio and Tiam2, which were required for cell motility. Lastly, we found that silencing or inhibiting PKCε enhances RhoA activity and stress fiber formation, a phenotype also observed in TGF-β-transformed cells. Our studies established a distinctive involvement of PKCε in epithelial and mesenchymal NSCLC cells, and identified a complex interplay between PKCε and small GTPases that contributes to regulation of NSCLC cell morphology and motile activity.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Diglycerides; Down-Regulation; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Protein Kinase C-epsilon; rho GTP-Binding Proteins; Transforming Growth Factor beta; Up-Regulation

Immune dysfunction often occurs in malignant pleural effusion (MPE). In our previous study, TGF-β derived predominantly from macrophages plays an important role in impairing T cell cytotoxicity in MPE. Therefore, we aimed to investigate whether other immunoregulatory cells and factors mediated TGF-β secretion from macrophages, involved in the immunosuppressive microenvironment of MPE, and to provide clues for potential immune therapy for MPE as well. We found that CCL22 level in MPE was significantly higher than that in non-malignant pleural effusion. The high level of CCL22 was closely associated with poor survival in MPE patients with lung cancer. CCL22 was dominantly produced by tumor-associated macrophages (TAMs) in MPE. Meanwhile, TAM-derived TGF-β mediated CCL22 expression in TAMs via c-Fos. CCL22 promoted the recruitment of regulatory T cells (Tregs) in MPE. Lastly, Treg-secreted high level of IL-8 further induced TGF-β production from TAMs, and promoted the immunosuppressive tumor microenvironment in MPE. Our results indicate that macrophage-derived CCL22 plays an important role in the immunosuppressive tumor microenvironment via IL-8 in MPE.

Topics: Cell Line, Tumor; Chemokine CCL22; Humans; Immune Tolerance; Interleukin-8; Lung Neoplasms; Macrophages; Pleural Effusion, Malignant; T-Lymphocytes, Regulatory; THP-1 Cells; Transforming Growth Factor beta; Tumor Microenvironment

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

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

To address the need for improved systemic therapy for non-small-cell lung cancer (NSCLC), we previously demonstrated that mesenchymal NSCLC was sensitive to polo-like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cMet was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal cells. Constitutively active cMet abrogates Plk1 inhibitor-induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor-induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cMet and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cMet phosphorylation via β1-integrin. This research defines a heretofore unknown mechanism of ligand-independent activation of cMet downstream of Plk1 and an effective combination therapy.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Integrin beta1; Lung Neoplasms; Mice, Nude; Phenotype; Phosphorylation; Polo-Like Kinase 1; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Pteridines; Transforming Growth Factor beta; Vimentin; Xenograft Model Antitumor Assays

Importance of growth factor (GF) signaling in cancer progression is widely acknowledged. Transforming growth factor beta (TGFβ) is known to play a key role in epithelial-to-mesenchymal transition (EMT) and metastatic cell transformation that are characterized by alterations in cell mechanical architecture and behavior towards a more robust and motile single cell phenotype. However, mechanisms mediating cancer type specific enhancement of cell mechanical phenotype in response to TGFβ remain poorly understood. Here, we combine high-throughput mechanical cell phenotyping, microarray analysis and gene-silencing to dissect cytoskeletal mediators of TGFβ-induced changes in mechanical properties of on-small-cell lung carcinoma (NSCLC) cells. Our experimental results show that elevation of rigidity and invasiveness of TGFβ-stimulated NSCLC cells correlates with upregulation of several cytoskeletal and motor proteins including vimentin, a canonical marker of EMT, and less-known unconventional myosins. Selective probing of gene-silenced cells lead to identification of unconventional myosin MYH15 as a novel mediator of elevated cell rigidity and invasiveness in TGFβ-stimulated NSCLC cells. Our experimental results provide insights into TGFβ-induced cytoskeletal remodeling of NSCLC cells and suggest that mediators of elevated cell stiffness and migratory activity such as unconventional cytoskeletal and motor proteins may represent promising pharmaceutical targets for restraining invasive spread of lung cancer.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cytoskeleton; Female; Humans; Lung Neoplasms; Male; Mechanical Phenomena; Middle Aged; Myosin Heavy Chains; Neoplasm Invasiveness; Transforming Growth Factor beta; Vimentin

Metastasis is a major cause of death in cancer patients. The extracellular matrix molecule tenascin-C is a known promoter of metastasis, however the underlying mechanisms are not well understood. To further analyze the impact of tenascin-C on cancer progression we generated MMTV-NeuNT mice that develop spontaneous mammary tumors, on a tenascin-C knockout background. We also developed a syngeneic orthotopic model in which tumor cells derived from a MMTV-NeuNT tumor. Tumor cells were transfected with control shRNA or with shRNA to knockdown tenascin-C expression and, were grafted into the mammary gland of immune competent, wildtype or tenascin-C knockout mice. We show that stromal-derived tenascin-C increases metastasis by reducing apoptosis and inducing the cellular plasticity of cancer cells located in pulmonary blood vessels invasions (BVI), before extravasation. We characterized BVI as organized structures of tightly packed aggregates of proliferating tumor cells with epithelial characteristics, surrounded by Fsp1+ cells, internally located platelets and, a luminal monolayer of endothelial cells. We found extracellular matrix, in particular, tenascin-C, between the stromal cells and the tumor cell cluster. In mice lacking stromal-derived tenascin-C, the organization of pulmonary BVI was significantly affected, revealing novel functions of host-derived tenascin-C in supporting the integrity of the endothelial cell coat, increasing platelet abundance, tumor cell survival, epithelial plasticity, thereby promoting overall lung metastasis. Many effects of tenascin-C observed in BVI including enhancement of cellular plasticity, survival and migration, could be explained by activation of TGF-β signaling. Finally, in several human cancers, we also observed BVI to be surrounded by an endothelial monolayer and to express tenascin-C. Expression of tenascin-C is specific to BVI and is not observed in lymphatic vascular invasions frequent in breast cancer, which lack an endothelial lining. Given that BVI have prognostic significance for many tumor types, such as shorter cancer patient survival, increased metastasis, vessel occlusion, and organ failure, our data revealing a novel mechanism by which stromal tenascin-C promotes metastasis in human cancer, may have potential for diagnosis and therapy.

Topics: Animals; Blood Vessels; Cell Line, Tumor; Female; Gene Knockout Techniques; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Rats; Receptor, ErbB-2; Signal Transduction; Stromal Cells; Tenascin; Transforming Growth Factor beta

Long non‑coding RNAs (lncRNAs) can act as carcinogenic or cancer suppressive factors during the pathogenesis, invasion and metastasis of non‑small cell lung cancer (NSCLC). The current study explored the role of long intergenic non‑protein coding RNA 00887 (LINC00887) and competing endogenous RNAs (ceRNAs). It was revealed that LINC00887 interacts with several microRNAs (miRs), which regulates downstream genes such as fibronectin 1, MET proto‑oncogene, receptor tyrosine kinase and mothers against decapentaplegic homolog 4, which are associated with the spread of lung cancer. The experimental results also suggested that LINC00887 can stimulate miR‑613, miR‑206 and miR‑1‑2 to become competing endogenous RNAs, which may regulate the epithelial‑mesenchymal transition of NSCLC cells through the transforming growth factor‑â signal transduction pathway, and therefore promote the migration of cells and the acquisition of stem cell characteristics. Therefore, it can be concluded that high levels of LINC00887 can accelerate the malignant transformation ability of NSCLC cells.

Topics: Adenocarcinoma of Lung; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Approximately 30% of patients with epidermal growth factor receptor (EGFR)-activating mutations have no response to EGFR-tyrosine kinase inhibitors (TKIs) (primary resistance). However, little is known about the molecular mechanism involved in primary resistance to EGFR-TKIs in EGFR-mutant non-small cell lung cancer (NSCLC). Programmed death ligand-1 (PD-L1) plays important regulatory roles in intracellular functions and leads to acquired resistance to EGFR-TKIs in NSCLC. Here, we investigated the mechanistic role of PD-L1 in primary resistance to EGFR-TKIs in EGFR-mutant NSCLC cells.. The expression levels of PD-L1 and the sensitivity to gefitinib in H1975, HCC827 and PC-9 cells were determined by quantitative real-time PCR analysis (qRT-PCR) and Cell Counting Kit-8 (CCK-8) assays, respectively. Molecular manipulations (silencing or overexpression) were performed to assess the effect of PD-L1 on sensitivity to gefitinib, and a mouse xenograft model was used for in vivo confirmation. Western blotting and qRT-PCR were used to analyse the expression of epithelial-mesenchymal transition (EMT) markers. The effect of PD-L1 on migratory and invasive abilities was evaluated using the Transwell assay and mice tail intravenous injection.. Higher expression of PD-L1 was related to less sensitivity to gefitinib in EGFR-mutant NSCLC cell lines. The overexpression or knockdown of PD-L1 presented diametrical sensitivity to gefitinib in vitro and in vivo. Furthermore, the overexpression of PD-L1 led to primary resistance to gefitinib through the induction of EMT, which was dependent on the upregulation of Smad3 phosphorylation. Moreover, in the mouse model, the knockdown of PD-L1 inhibited transforming growth factor (TGF)-β1-induced cell metastasis in vivo.. PD-L1 contributes to primary resistance to EGFR-TKI in EGFR-mutant NSCLC cells, which may be mediated through the induction of EMT via the activation of the TGF-β/Smad canonical signalling pathway.

Topics: Animals; Antineoplastic Agents; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Female; HEK293 Cells; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Protein Kinase Inhibitors; Random Allocation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

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

Late-stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)-dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti-metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti-metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF-κB and TGF-β signalling, including the nuclear translocation of NF-κB subunits and the expression of Smad4, leading to down-regulation of Snail and Slug, which contributed to phenotype epithelial-mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF-κB and TGF-β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Copper; Disulfiram; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; NF-kappa B; Phenotype; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

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

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

Growing evidence shows that lncRNA XIST functions as an oncogene accelerating tumor progression. Transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) plays a key role in tumor metastasis. However, it is still unclear whether lncRNA XIST is implicated in TGF-β-induced EMT and influences cell invasion and metastasis in non-small-cell lung cancer (NSCLC). Here, we observed increased expression of lncRNA XIST and ZEB2 mRNA in metastatic NSCLC tissues. Knockdown of lncRNA XIST inhibited ZEB2 expression, and repressed TGF-β-induced EMT and NSCLC cell migration and invasion. Being in consistent with the in vitro findings, the in vivo experiment of metastasis showed that knockdown of lncRNA XIST inhibited pulmonary metastasis of NSCLC cells in mice. In addition, knockdown of ZEB2 expression can inhibit TGF-β-induced EMT and NSCLC cell migration and invasion. Mechanistically, lncRNA XIST and ZEB2 were targets of miR-367 and miR-141. Furthermore, both miR-367 and miR-141 expression can be upregulated by knockdown of lncRNA XIST. Taken together, our study reveals that lncRNA XIST can promote TGF-β-induced EMT and cell invasion and metastasis by regulating miR-367/miR-141-ZEB2 axis in NSCLC.

Topics: A549 Cells; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Nude; MicroRNAs; RNA Interference; RNA, Long Noncoding; Transforming Growth Factor beta; Transplantation, Heterologous; Zinc Finger E-box Binding Homeobox 2

Cancer cells alter their metabolism to support their malignant properties. In this study, we report that the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (

Topics: A549 Cells; Aldehyde Reductase; Animals; Cell Line, Tumor; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Glucose; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; L-Iditol 2-Dehydrogenase; Lung Neoplasms; MCF-7 Cells; Mice; RNA Interference; RNA, Small Interfering; 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

CD103

Topics: Aged; Antigens, CD; Cadherins; Carcinoma, Non-Small-Cell Lung; CD8-Positive T-Lymphocytes; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha Chains; Kaplan-Meier Estimate; Ki-67 Antigen; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Programmed Cell Death 1 Receptor; Transforming Growth Factor beta

Due to high metastasis and recurrence rate. Recent studies indicated that epithelial-to-mesenchymal transition (EMT) was involved in the progression and metastasis in cancer. Some reports also indicate that HS3ST3B1 played a role in angiogenesis and the proliferation of cancer cells. In this study, we aim to investigate its role in non-small cell lung cancer (NSCLC) .. All cell lines were purchased from ATCC and cultured in our central lab. RT-PCR was performed to study the expession of HS3ST3B1 in tumors and matched normal tissues. Western-blot was used to investigate the expession of HS3ST3B1 in cell lines. We also used luciferase report system to confirm the regulation of HS3ST3B1 by miR-218 in cells.. In this study, we found that HS3ST3B1 was significantly upregulated in NSCLC tissues compared with matched normal tissues (P = 0.02). Its expression was also up-regulated in mesenchymal phenotype of NSCLC cell lines compared with epithelial phenotype (P < 0.05). When TGF-β was applied to induce the epithelial phenotype to mesenchymal phenotype, it was upregulated compared with previous epithelial cell lines. When HS3ST3B1 was knocked down by specific small interfering RNA in the mesenchymal phenotype, mesenchymal phenotype was transformed to epithelial phenotype. Moreover, we also found that it could be targeted by miR-218 in NSCLC.. These findings indicate that HS3ST3B1 is a novel regulator of TGF-beta-mediated EMT and is regulated by miR-218 in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Reporter; Humans; Lung Neoplasms; MicroRNAs; Phenotype; RNA Interference; Sulfotransferases; Transforming Growth Factor beta

Fibroblasts can alter the extracellular matrix (ECM), contributing to cancer progression by providing a scaffold for cancer cells. The influence of lung cancer cells (LCCs) on lung fibroblast-mediated ECM alteration is not well understood.. After incubation in serum-free medium, LCC- or fibroblast-conditioned media were collected. The ECM alteration was assessed by collagen gel contraction assay.. Both LCC-conditioned medium and exogenous transforming growth factor (TGF)-β1 increased collagen gel contraction by lung fibroblasts. TGF-β1 was produced in LCC-conditioned media at approximately 2 ng/ml. SB431542, a specific TGF-β receptor kinase inhibitor, partially inhibited the collagen gel contraction that had been increased by LCC-conditioned media. Lung fibroblast-conditioned medium stimulated TGF-β1 production from LCCs, whereas LCC-conditioned medium decreased fibroblast survival and α-smooth muscle actin expression by fibroblasts.. Interaction between LCCs and lung fibroblasts through TGF-β signaling induces fibroblasts to assume the contractile phenotype and may contribute to cancer progression.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Line, Tumor; Collagen; Fibroblasts; Humans; Lung Neoplasms; Phenotype; Transforming Growth Factor beta; Transforming Growth Factor beta1

Non‑small‑cell lung cancer (NSCLC) is one of the most common malignancies that is responsible for a high level of cancer‑associated mortalities worldwide. Previous evidence has shown that Calotropin is an upstream activator of protein kinase B, which can further inhibit the growth and promote the apoptosis of NSCLC cells. In the present study, the efficacy of Calotropin on growth, aggressiveness and apoptosis of NSCLC cells was investigated, as well as the potential underlying mechanism. The results demonstrated that Calotropin inhibited H358 cell growth, migration and invasion. Flow cytometry assay showed that Calotropin promoted the apoptosis of H358 cells in vitro. Western blot analysis demonstrated that Calotropin inhibited fibronectin (FN), Vimentin (VIM) and E‑cadherin (Eca) protein expression levels in H358 cells in vitro. In addition, Calotropin treatment upregulated pro‑apoptosis gene expression, including caspase‑3, caspase‑8 and apoptotic protease activating factor‑1, and downregulated anti‑apoptosis gene expression, including P53, B‑cell lymphoma (Bcl) 2 and Bcl‑2‑like protein 2 in H358 cells. The results also revealed that the expression levels of cytotoxic T‑lymphocyte associated antigen 4 (CTLA‑4) were decreased by Calotropin treatment in H358 cells. Analyses of the underlying mechanism indicated that Calotropin inhibited transforming growth factor‑β (TGF‑β) and extracellular signal‑regulated kinase (ERK) expression. Overexpression of CTLA‑4 inhibited Calotropin‑mediated downregulation of TGF‑β and ERK expression in H358 cells. In vivo assay revealed that Calotropin administration significantly inhibited tumor growth and prolonged animal survival over the 120‑day observation period. Immunohistochemistry demonstrated that the number of apoptotic cells increased and the expression levels of CTLA‑4 were decreased in the Calotropin‑treated tumor group when compared with control. In addition, the expression levels of TGF‑β and ERK were downregulated in the Calotropin‑treated tumor group compared with control. In conclusion, the results of the present study indicated that Calotropin administration regulated NSCLC apoptosis by downregulating the CTLA‑4‑mediated TGF‑β/ERK signaling pathway, suggesting that Calotropin may be a potential anti‑cancer agent for the treatment of NSCLC.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cardenolides; Cell Line, Tumor; Cell Movement; Cell Proliferation; CTLA-4 Antigen; Disease Models, Animal; Female; Gene Expression; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Tumor-associated macrophages (TAMs) are known to promote tumorigenesis but the mechanism(s) remain elusive. We have developed a mouse model of lung cancer that is initiated through an inducible overexpression of fibroblast growth factor 9 (FGF9) in type-2 pneumocytes. Expression of FGF9 in adult lungs resulted in a rapid development of multiple adenocarcinoma-like tumor nodules, and is associated with an intense immunological reaction. The purpose of this study is to characterize the immune response to the FGF9-induced lung adenocarcinoma and to determine the contribution of TAMs to growth and survival of these tumors.. We used flow cytometry, immunostaining, RT-PCR and in vitro culture system on various cell populations isolated from the FGF9-induced adenocarcinoma mouse lungs.. Immunostaining demonstrated that the majority of the inflammatory cells recruited to FGF9-induced lung tumors were macrophages. These TAMs were enriched for the alternatively activated (M2) macrophage subtype. TAMs performed a significantly high immune suppressive function on T-cells and displayed high levels of arginase-1 expression and activity. The growth and colony forming potential of tumor cells was induced by co-culture with TAMs. Additionally, TAMs were shown to promote fibroblast proliferation and angiogenesis. TAMs had high expression of Tgf-β, Vegf, Fgf2, Fgf10, Fgfr2 and several matrix metalloproteinases; factors that play multiple roles in supporting tumor growth, immune protection, fibroblast activation and angiogenesis.. Our results provide evidence that the Fgf9-induced lung adenocarcinoma is associated with recruitment and activation of M2-biased TAMs, which provided multiple means of support to the tumor. This model represents an excellent means to further study the complex interactions between TAMs, their related chemokines, and progression of lung adenocarcinoma, and adds further evidence to support the importance of TAMs in tumorigenesis.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Disease Progression; Fibroblast Growth Factor 9; Humans; Lung Neoplasms; Macrophages; Mice; Mice, Transgenic; Transforming Growth Factor beta

Myeloid-derived suppressor cells (MDSCs) are known suppressors of antitumor immunity, affecting amino acid metabolism and T cell function in the tumor microenvironment. However, it is unknown whether MDSCs regulate B cell responses during tumor progression. Using a syngeneic mouse model of lung cancer, we show reduction in percentages and absolute numbers of B cell subsets including pro-, pre-, and mature B cells in the bone marrow (BM) of tumor-bearing mice. The kinetics of this impaired B cell response correlated with the progressive infiltration of MDSCs. We identified that IL-7 and downstream STAT5 signaling that play a critical role in B cell development and differentiation were also impaired during tumor progression. Global impairment of B cell function was indicated by reduced serum IgG levels. Importantly, we show that anti-Gr-1 Ab-mediated depletion of MDSCs not only rescued serum IgG and IL-7 levels but also reduced TGF-β1, a known regulator of stromal IL-7, suggesting MDSC-mediated regulation of B cell responses. Furthermore, blockade of IL-7 resulted in reduced phosphorylation of downstream STAT5 and B cell differentiation in tumor-bearing mice and administration of TGF-β-blocking Ab rescued these IL-7-dependent B cell responses. Adoptive transfer of BM-derived MDSCs from tumor-bearing mice into congenic recipients resulted in significant reductions of B cell subsets in the BM and in circulation. MDSCs also suppressed B cell proliferation in vitro in an arginase-dependent manner that required cell-to-cell contact. Our results indicate that tumor-infiltrating MDSCs may suppress humoral immune responses and promote tumor escape from immune surveillance.

Topics: Adoptive Transfer; Animals; B-Lymphocytes; Bone Marrow Cells; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Female; Immunoglobulin G; Interleukin-7; Lung Neoplasms; Mice; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Phosphorylation; Signal Transduction; STAT5 Transcription Factor; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment

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

In lung cancer a deregulation of Transforming Growth Factor-β (TGFβ) signaling has been observed. Yet, the impact of TGFβ in squamous cell carcinoma of the lung (LUSC) remained to be determined. We combined phenotypic and transcriptome-wide studies and showed that the stimulation of the LUSC cell line SK-MES1 with TGFβ results in an increase of migratory invasive properties. The analysis of the dynamics of gene expression by next-generation sequencing revealed that TGFβ stimulation orchestrates the upregulation of numerous motility- and actin cytoskeleton-related genes. Among these the non-muscle myosin 10 (MYO10) showed the highest upregulation in a LUSC patient cohort of the Cancer Genome Atlas (TCGA). Knockdown of MYO10 abrogated TGFβ-induced collagen gel invasion of SK-MES1 cells. The analysis of MYO10 mRNA expression in paired tissues of 151 LUSC patients with corresponding 80-month clinical follow-up data showed that the mRNA expression ratio of MYO10 in tumor and tumor-free tissue is prognostic for overall survival of LUSC patients and predictive for the response of these patients to adjuvant chemotherapy. Thus, MYO10 represents a new clinical biomarker for this aggressive disease and due to its role in cellular motility and invasion could serve as a potential molecular target for therapeutic interventions in patients with LUSC.

Topics: Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Myosins; Neoplasm Invasiveness; Prognosis; RNA, Messenger; Survival Analysis; Transcriptional Activation; Transforming Growth Factor beta

MicroRNA-206 (miR-206) has demonstrated tumor suppressive effects in a variety of cancers. Numerous studies have identified aberrantly expressed targets of miR-206 that contribute to tumor progression and metastasis, however, the broader gene-networks and pathways regulated by miR-206 remain poorly defined. Here, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumors to identify differentially expressed genes, and study the effects on tumor growth and metastasis. In H1299 tumor xenograft assays, stable expression of miR-206 suppressed both tumor growth and metastasis in mice. Profiling of xenograft tumors using small RNA sequencing and a targeted panel of tumor progression and metastasis-related genes revealed a network of genes involved in TGF-β signalling that were regulated by miR-206. Among these were the TGFB1 ligand, as well as direct transcriptional targets of Smad3. Other differentially expressed genes included components of the extracellular matrix involved in TGF-β activation and signalling, including Thrombospondin-1, which is responsible for the activation of latent TGF-β in the stroma. In cultured lung adenocarcinoma cells treated with recombinant TGF-β, ectopic expression of miR-206 impaired canonical signalling, and expression of TGF-β target genes linked to epithelial-mesenchymal transition. This was due at least in part to the suppression of Smad3 protein levels in lung adenocarcinoma cells with ectopic miR-206 expression. Together, these findings indicate that miR-206 can suppress tumor progression and metastasis by limiting autocrine production of TGF-β, and highlight the potential utility of TGF-β inhibitors for the treatment of lung adenocarcinomas.

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; MicroRNAs; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Tumor-derived soluble factors promote the production of Gr-1

Topics: Animals; Antineoplastic Agents; CD11b Antigen; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Drug Evaluation, Preclinical; Female; Gene Expression Regulation, Neoplastic; Humans; Immunity, Innate; Injections, Intravenous; Interferon-gamma; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; MicroRNAs; Myeloid Cells; Oligoribonucleotides; Pyrogallol; Receptor, IGF Type 1; Receptor, Transforming Growth Factor-beta Type II; Receptors, Chemokine; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

Lung cancer is the first leading cause of cancer‑related death in the United States. Non‑small cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with a poor patient prognosis. Identification of promising molecular targets is required for the effective prevention and therapy of NSCLC. Epithelial‑specific ETS‑1 (ESE‑1) belongs to the superfamily of ETS transcription factors. The effect of ESE‑1 on tumorigenesis is controversial in several types of cancer while its role in lung cancer remains unknown. The present study was designed to investigate whether ESE‑1 expression affects tumorigenic activity using human NSCLC cells and a mouse xenograft model. ESE‑1 expression suppressed anchorage‑independent growth in soft agar assay and led to an increase in G1 arrest and apoptosis in human NSCLC cells. ESE‑1 expression suppressed the invasion and migration of human NSCLC cells. Western blot analysis, RT‑PCR and promoter assay indicated that ESE‑1 expression was transcriptionally downregulated by treatment of transforming growth factor (TGF)‑β, an EMT (epithelial‑mesenchymal transition) stimulator. The xenograft study indicated that ESE‑1 expression inhibited tumor formation and development. Our data demonstrated that ESE‑1 plays a key role as a tumor suppressor in human NSCLC.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Proto-Oncogene Proteins c-ets; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Fucosyltransferase 2 (FUT2), the enzyme catalyzing α-1,2-fucosylation in mammals, has been implicated in cancer. The up-regulation of FUT2 has been observed in lung adenocarcinoma (LUAD), and FUT2 can enhance the cell migration and invasion of LUAD cell lines. However, the underlying mechanism of FUT2 in LUAD remains largely unknown. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during lung cancer metastasis and progression. In the present study, we showed that knocking down FUT2 in LUAD cell lines increased the expression of E-cadherin and reduced the expression of Vimentin, N-cadherin, TβRII, p-Smad2, p-Smad3 and Snail, which were the makers of EMT. Meanwhile, the expression of E-cadherin was decreased, and the expression of Vimentin was increased by restoring the expression of FUT2 in RNA interference FUT2 (RNAi-FUT2) cells, suggesting that FUT2 enhanced the EMT process in LUAD. Additionally, silencing FUT2 expression can up-regulate E-cadherin and down-regulate Vimentin, significantly attenuated EMT in vivo. Treated with the SIS3, a new-type inhibitor of p-Smad3 of TGF-β signaling, the expression of E-cadherin, Vimentin and Snail were not affected by RNAi-FUT2 cells, indicating that the effect of FUT2 on EMT depended on TGF-β/Smad signaling. Overall, the current results indicated that FUT2 might promote LUAD metastasis through the EMT initiated by TGF-β/Smad signaling. Therefore, FUT2 might be a prognostic factor and therapeutic target for LUAD.

Topics: Cadherins; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Vimentin

Topics: A549 Cells; Active Transport, Cell Nucleus; Anthracenes; Cell Line, Tumor; Cell Movement; Fatty Acids, Unsaturated; Humans; Lung Neoplasms; MAP Kinase Signaling System; Nuclear Receptor Subfamily 4, Group A, Member 1; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Lung cancer patients with human immunodeficiency virus (HIV) have a poorer prognosis than do patients without HIV infection. HIV1 Tat is a secreted viral protein that penetrates the plasma membrane and interacts with a number of proteins in non-HIV-infected cells. The loss of function of Tat-interacting protein 30 (TIP30) has been linked to metastasis in non-small cell lung cancer (NSCLC). However, it is unknown how the interaction of HIV1 Tat with TIP30 regulates the metastasis of NSCLC cells. In this study, the overexpression of TIP30 decreased tumor growth factor-β-induced epithelial-to-mesenchymal transition (EMT) and invasion of NSCLC cells, whereas the knockdown of TIP30 promoted EMT, invasion and stemness. Exposure to recombinant HIV1 Tat proteins promoted EMT and invasion. A mechanistic study showed that the interaction of HIV1 Tat with TIP30 blocked the binding of TIP30 to importin-β, which is required for the nuclear translocation of Snail. Indeed, the loss of TIP30 promoted the nuclear translocation of Snail. In vivo studies demonstrated that the overexpression of TIP30 inhibited the metastasis of NSCLC cells. In contrast, the coexpression of HIV1 Tat and TIP30 diminished the inhibitory effect of TIP30 on metastasis. Immunohistochemistry confirmed that TIP30 overexpression reduced the nuclear localization of Snail, whereas the coexpression of HIV1 Tat and TIP30 increased nuclear Snail in metastatic tumors. In conclusion, the binding of HIV1 Tat to TIP30 enhanced EMT and metastasis by regulating the nuclear translocation of Snail. Targeting Tat-interacting proteins may be a potential therapeutic strategy to prevent metastasis in NSCLC patients with HIV infection.

Topics: Acetyltransferases; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Nucleus; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; HEK293 Cells; HIV; HIV Infections; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Recombinant Proteins; RNA, Small Interfering; Snail Family Transcription Factors; tat Gene Products, Human Immunodeficiency Virus; Transcription Factors; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The present study aimed to investigate the role of microRNA (miR)‑125a in the development of pneumonitis inpatients with non‑small‑cell lung cancer that received radiotherapy. In addition, the study aimed to determine how the miR‑125a affects its target, transforming growth factor β (TGFβ). Bioinformatics tools were used to identify a potential miR‑125a binding site in the 3'untranslated region of TGFβ, which was subsequently confirmed using a dual‑luciferase reporter system. In addition, tissue samples were collected from patients with lung cancer and genotyped as CC (n=36), CT (n=28) or TT (n=6). The expression levels of miR‑125a and TGFβ in these samples were determined, and CC genotype samples demonstrated upregulated miR‑125a expression, and downregulated TGFβ protein and mRNA expression compared with samples carrying the minor allele, T. To further investigate the association between the rs12976445 polymorphism and the risk of pneumonitis in patients with lung cancer that received radiotherapy, 534 lung cancer patients diagnosed with pneumonitis and 489lung cancer patients without pneumonitis were recruited. rs12976445 was shown to be significantly associated with the risk of pneumonitis. In conclusion, the rs12976445 polymorphism increased expression levels of TGFβ by decreasing the expression of miR‑125a, and therefore may be associated with the development of pneumonitis in patients with lung cancer that receive radiotherapy.

Topics: Base Sequence; Carcinoma, Non-Small-Cell Lung; Demography; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Polymorphism, Single Nucleotide; Radiation Pneumonitis; Risk Factors; RNA Processing, Post-Transcriptional; RNA, Messenger; Transforming Growth Factor beta

Our previous studies have shown that the novel oncogene, cancer upregulated gene 2 (CUG2), activates STAT1, which is linked to anticancer drug resistance, induces epithelial‑mesenchymal transition (EMT) and cancer stem cell‑like phenotypes as determined by MTT, migration and sphere formation assays. We thus aimed to ascertain whether the activation of STAT1 by CUG2 is involved in these malignant phenotypes besides drug resistance. Here, we showed that STAT1 suppression decreased the expression of N‑cadherin and vimentin, biomarkers of EMT, which led to inhibition of the migration and invasion of human lung A549 cancer cells stably expressing CUG2, but did not recover E‑cadherin expression. STAT1 siRNA also diminished CUG2‑induced TGF‑β signaling, which is critical in EMT, and TGF‑β transcriptional activity. Conversely, inhibition of TGF‑β signaling reduced phosphorylation of STAT1, indicating a crosstalk between STAT1 and TGF‑β signaling. Furthermore, STAT1 silencing diminished sphere formation, which was supported by downregulation of stemness‑related factors such as Sox2, Oct4, and Nanog. Constitutive suppression of STAT1 also inhibited cell migration, invasion and sphere formation. As STAT1 acetylation counteracts STAT1 phosphorylation, acetylation of STAT1 by treatment with trichostatin A, an inhibitor of histone deacetylases (HDACs), reduced cell migration, invasion, and sphere formation. As HDAC4 is known to target STAT1, its role was investigated under CUG2 overexpression. HDAC4 suppression resulted in inhibition of cell migration, invasion, and sphere formation as HDAC4 silencing hindered TGF‑β signaling and decreased expression of Sox2 and Nanog. Taken together, we suggest that STAT1‑HDAC4 signaling induces malignant tumor features such as EMT and sphere formation in CUG2‑overexpressing cancer cells.

Topics: A549 Cells; Cell Movement; Chromosomal Proteins, Non-Histone; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Lung Neoplasms; Nanog Homeobox Protein; Repressor Proteins; Signal Transduction; SOXB1 Transcription Factors; Spheroids, Cellular; STAT1 Transcription Factor; Transforming Growth Factor beta

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-β acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-β signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways. Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles. The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21

Topics: Animals; Binding Sites; Cell Line, Tumor; Cell Proliferation; Female; Gene Editing; Gene Knock-In Techniques; Humans; Lung Neoplasms; Mice; Mice, SCID; Phosphorylation; Point Mutation; Signal Transduction; Smad3 Protein; Threonine; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Valine; Xenograft Model Antitumor Assays

Previous studies indicated that transforming growth factor (TGF)-β-mediated exosomal microRNAs (miRNAs) regulate the migration and invasion of lung cancer cells; however, whether and how TGF-β-mediated exosomal long noncoding (lnc) RNAs regulate migration and invasion of lung cancer cells remains unclear. Here, coculture experiments showed that TGF-β pretreatment increased the migration and invasion potential of lung cancer cells and TGF-β pretreated A549 cells increases vascular permeability. Furthermore, we found that TGF-β-mediated exosomes, as carriers of intercellular communication, regulated lung cancer invasion, and vascular permeability. Transcriptional analysis also revealed that lnc-MMP2-2 was highly enriched in TGF-β-mediated exosomes and might function by increasing the expression of matrix metalloproteinase (MMP)2 through its enhancer activity, with ectopic expression and silencing of lnc-MMP2-2 affecting lung cancer invasion and vascular permeability. Additionally, lnc-MMP2-2 and MMP2 expression was assessed semiquantitatively, and tissue-specific correlations between lnc-MMP2-2 and MMP2 expression were evaluated. These results suggested that exosomal lnc-MMP2-2 might regulate the migration and invasion of lung cancer cells into the vasculature by promoting MMP2 expression, suggesting this lncRNA as a novel therapeutic target and predictive marker of tumor metastasis in lung cancer.

Topics: A549 Cells; Cell Line, Tumor; Cell Movement; Coculture Techniques; Exosomes; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Neoplasm Invasiveness; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation

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

Lung cancer is the leading cause of cancer related deaths worldwide. TGF-β-induced epithelial-mesenchymal transition (EMT) is a key cell-intrinsic identity for tumor cell migration, invasion, and stemness acquisition in cancer metastasis. Long noncoding RNAs (lncRNAs) have not been fully investigated for their involvement in regulating TGF-β-induced EMT and metastasis in lung cancer. Here, we demonstrated that the transcription of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) was inhibited by TGF-β1 in a SMAD4-dependent manner. LINP1 suppressed EMT of lung cancer cells, thereby controlling cancer cell migration, invasion, and stemless. Moreover, LINP1 inhibited TGF-β-induced EMT and cell invasion in lung cancer cells. Our study reveals the role of LINP1 in the regulation of TGF-β-induced EMT in human lung cancer.

Topics: A549 Cells; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; RNA, Long Noncoding; Smad4 Protein; 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

The transforming growth factor-β (TGF-β) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-β signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-β-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.

Topics: Cell Cycle; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; Signal Transduction; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta

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

The homeobox transcription factor CUTL1 has been associated with cellular proliferation and cell cycle progression, and CUTL1 functions as an oncogene. The aim of the present study was to investigate whether CUTL1 participates in epithelial-mesenchymal transition (EMT). The expression levels of CUTL1, E-cadherin, N-cadherin and Snail were determined by immunohistochemistry assay, immunofluorescence assay or real-time quantitative reverse transcription PCR. Their roles in non-small cell lung cancer (NSCLC) were assessed by functional analyses. Protein expression was detected by western blot analysis. The CUTL1 expression levels are higher in non-small cell lung cancer (NSCLC) tissues. High CUTL1 expression in NSCLC is associated with the mesenchymal-like phenotype. Mechanistically, CUTL1 upregulates transforming growth factor β receptor I (TβR-I) expression, and the TβR-I inhibitor SB431542 abolishes EMT elicited by ectopic CUTL1 expression. Transforming growth factor β (TGF-β) signaling is essential for CUTL1-induced EMT in NSCLC cells. CUTL1 is downstream of TGF-β signaling and CUTL1 is involved in the expression of the TβR-I. This study indicates that CUTL1 may be a potential target for anti-lung cancer therapy.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Lung Neoplasms; Nuclear Proteins; Repressor Proteins; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

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

A sulfated 1,4-β-d-galactoglucan (B86-III) with 1,6-branches was isolated and identified from Antrodia cinnamomea. The repeating unit of B86-III was proposed based on one-dimensional 1D (

Topics: Antineoplastic Agents; Antrodia; Apoptosis; Cell Line, Tumor; Cell Survival; Focal Adhesion Kinase 1; Galactans; Glucans; Humans; Lung Neoplasms; Polysaccharides, Bacterial; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Non-small cell lung cancer (NSCLC) has the highest mortality rate among all solid tumors with a poor prognosis. The BMP and activin receptor membrane bound inhibitor (BAMBI) has been identified as a hallmark of NSCLC and β-sitosterol possesses antitumor potentiality. This study explores the effect of BAMBI overexpression and β-sitosterol in the context of NSCLC. The results revealed that the transfection of pcDNA‑BAMBI and β-sitosterol treatment significantly reduced the levels of autophagy markers light chain 3 (LC3) II and Beclin 1, whereas the levels of LC3 I and p62 were promoted. The reduced punctate accumulations of GFP-LC3 were detected in pcDNA-BAMBI and β-sitosterol groups, especially in pcDNA-BAMBI + β-sitosterol group. BAMBI overexpression and β-sitosterol induced G0/G1 cell cycle arrest and inhibted cell proliferation in A549 cells. In addition, the levels of transforming growth factor-β (TGF‑β)/p-Smad2/3/c-Myc pathway proteins were decreased. The TGF-β overexpression further confirmed that BAMBI overexpression and β-sitosterol treatment suppre-ssed autohagy and viability of A549 cells was through TGF-β/Smad2/3/c-Myc pathway. Finally, the tumor growth was suppressed in NSCLC xenografts, and the inhibitory effect was stronger under treatment of pcDNA-BAMBI toge-ther with β-sitosterol. These results indicate that BAMBI overexpression and β-sitosterol may serve as novel targets for the treatment of NSCLC.

Topics: A549 Cells; Animals; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Lung Neoplasms; Membrane Proteins; Mice; Signal Transduction; Sitosterols; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Oroxylin A (OA), a naturally occurring monoflavonoid isolated from Scutellariae radix, has previously been reported to inhibit the proliferation of several cancer cell lines. CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an important role in maintenance of immunologic self-tolerance. Tregs also increase in cancer and take part in suppressing antitumor immune responses. Here, we explored how OA affected the Tregs in lung cancer environment and the involved underlying mechanism. It is found that OA reversed the generation of Tregs induced by H460 lung cancer cells co-culture. Furthermore, in vivo, OA reduced tumor formation rate and attenuated Foxp3 expression in tumor-infiltrating lymphocytes. We also found that transforming growth factor-β1 (TGF-β1) neutralizing antibody reversed the enhancement of Treg number and expression of p-Smad3'p-p38'p-JNK'p-ERK1/2 in the co-culture model. Moreover, OA reduced the secretion of TGF-β1 and down-regulated the activation of NF-κB signaling in H460 cells. OA also inhibited Treg activity by a direct inhibition of the T cells' response to TGF-β1. In conclusion, our study demonstrated that OA inhibits the generation of Tregs in lung cancer environment by inhibiting the T cells' response to TGF-β1 and decreasing the secretion of TGF-β1 in lung cancer cells via NF-κB signaling.

Topics: Animals; Biomarkers; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Coculture Techniques; Disease Models, Animal; Flavonoids; Humans; Immunophenotyping; Jurkat Cells; Leukocytes, Mononuclear; Lung Neoplasms; Mice; NF-kappa B; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

C-C chemokine receptor type 4 has been reported to correlate with lung cancer. However, the role of CCR4 in human non-small cell lung cancer patients is not well defined. Here, we demonstrated that increased expression of CCR4 was associated with clinical stage and CCR4 was an independent risk factor for overall survival in NSCLC patients. Moreover, tumor-infiltrating Treg cells were higher expression than matched adjacent tissues in CCR4+ NSCLC. Higher expression of chemokine CCL17 and CCL22 could recruit Treg cells to tumor sites in NSCLC. Treg in TIL exhibit a higher level of suppressive activity on effector T cells than matched adjacent tissues in NSCLC patients. Significant NK cell reduction was observed in tumor regions compared to non-tumor regions. NK cells demonstrated that reduced the killing capacity against target cells and the expression of CD69 

Topics: Carcinoma, Non-Small-Cell Lung; Chemotaxis; Female; Humans; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Receptors, CCR4; T-Lymphocytes; Transforming Growth Factor beta

A key feature of cancer cachexia is the loss of adipose tissue, mainly due to increased lipolysis and an impairment of adipogenesis. Recent findings have shown that cancer exosomes promoted lipolysis in adipose tissue. However, effects of cancer exosomes on adipogenesis were not reported. In this study, we found that lung cancer exosomes could be internalized by human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and significantly inhibited hAD-MSC adipogenesis as demonstrated by Oil Red O staining and decreased expression of adipogenic-specific genes. Specifically, TGFβ signaling pathway was demonstrated to be involved in the inhibitive effects of lung cancer exosomes on hAD-MSC adipogenesis. Additionally, TGFβ was detected in A549 exosomes. Herein, this study reports that the effect of lung cancer cell exosomes on hAD-MSC adipogenic differentiation was mediated by TGFβ signaling pathway and suggests the involvement of cancer exosomes in weight loss of cancer cachexia.

Topics: A549 Cells; Adipogenesis; Exosomes; Humans; Lung Neoplasms; Mesenchymal Stem Cells; Neoplasm Proteins; Signal Transduction; Transforming Growth Factor beta

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules.. We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets.. We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules.. Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Pharmacological; Carcinoma, Non-Small-Cell Lung; Deoxycytidine; DNA Repair; Down-Regulation; Drug Resistance, Neoplasm; Etoposide; Female; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Retrospective Studies; Signal Transduction; Smad4 Protein; Topoisomerase I Inhibitors; Transforming Growth Factor beta

Cancer chemoresistance and metastasis are tightly associated features. However, whether they share common molecular mechanisms and thus can be targeted with one common strategy remain unclear in non-small cell lung cancer (NSCLC). Here, we report that high levels of microRNA-128-3p (miR-128-3p) is key to concomitant development of chemoresistance and metastasis in residual NSCLC cells having survived repeated chemotherapy and correlates with chemoresistance, aggressiveness and poor prognosis in NSCLC patients. Mechanistically, miR-128-3p induces mesenchymal and stemness-like properties through downregulating multiple inhibitors of Wnt/β-catenin and TGF-β pathways, leading to their overactivation. Importantly, antagonism of miR-128-3p potently reverses metastasis and chemoresistance of highly malignant NSCLC cells, which could be completely reversed by restoring Wnt/β-catenin and TGF-β activities. Notably, correlations among miR-128-3p levels, activated β-catenin and TGF-β signalling, and pro-epithelial-to-mesenchymal transition/pro-metastatic protein levels are validated in NSCLC patient specimens. These findings suggest that miR-128-3p might be a potential target against both metastasis and chemoresistance in NSCLC.

Topics: Animals; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Our previous work identified a 13-gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non-Small Cell Lung Cancer cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signalling. We hypothesized that TGFβ signalling controls expression of the miRNA genes comprising an erlotinib response signature in NSCLC. Western analysis revealed that TGFβ signalling via Smad2/3/4 occurred differently between erlotinib-resistant A549 and erlotinib- sensitive PC9 cells. We showed that TGFβ induced an interaction between Smad4 and putative Smad Binding Elements in PC9. However, qRT-PCR analysis showed that endogenous miR-140/141/200c expression changes resulted from time in treatments, not the treatments themselves. Moreover, flow cytometry indicated that cells exited the cell cycle in the same manner. Taken together these data indicated that the miRNA comprising the signature are likely regulated by the cell cycle rather than by TGFβ. Importantly, this work revealed that TGFβ did not induce EMT in PC9 cells, but rather TGFβ-inhibition induced an EMT-intermediate. These data also show that growth/proliferation signals by constitutively-activated EGFR may rely on TGFβ and a possible relationship between TGFβ and EGFR signalling may prevent EMT progression in this context rather than promote it.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial-Mesenchymal Transition; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Phenotype; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Smad Proteins; Time Factors; Transforming Growth Factor beta

Nonsense-mediated mRNA decay (NMD) is a highly conserved pathway that selectively degrades aberrant RNA transcripts. In this study, we proved that NMD regulates the epithelial-mesenchymal transition (EMT) of lung adenocarcinoma (ADC). Moreover, we found that NMD core factor UP-frameshift 1 tends to be expressed at lower levels in human ADC tissues than in normal lung tissues, thereby raising the possibility that NMD may be downregulated to permit ADC oncogenesis. Our experiments in human ADC cell lines showed that downregulating NMD can promote EMT. Moreover, EMT can be inhibited by upregulating NMD. We tested the role of TGF-ß signaling and found that NMD influences EMT by targeting the TGF-ß signaling pathway. Our findings reveal that NMD is a potential tumor regulatory mechanism and may be a potential therapeutic target for ADC.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Disease-Free Survival; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice, Nude; Middle Aged; Nonsense Mediated mRNA Decay; Pneumonectomy; RNA Helicases; RNA Interference; RNA Stability; RNA, Messenger; Signal Transduction; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta; Treatment Outcome

Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) has been reported to interact with many cancer-related proteins and plays an important role in cancer progression. However, little is known about the biological functions of NHERF1 in lung cancer cells. The aim of the current study was to explore whether NHERF1 is involved in transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer cells.. The expression of NHERF1 and EMT-associated markers including E-cadherin, N-cadherin, snail family transcriptional repressor 1 (SNAI1) and snail family transcriptional repressor 2 (SLUG) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The migratory properties of cells were assessed using a wound-healing assay.. TGF-β1-induced a pro-migratory response in the A549 lung cancer cell line, that was consistently associated with corresponding changes in the expression levels of EMT-related genes. The expression of NHERF1 significantly decreased in the TGF-β1-induced A549 cells. Overexpression of NHERF1 significantly inhibited the migratory ability of cells and reversed the TGF-β1-induced mesenchymal phenotype of A549 cells.. These data showed an important role of NHERF1 in the EMT of non-small-cell lung cancer cells, as well as migration.

Topics: A549 Cells; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphoproteins; Sodium-Hydrogen Exchangers; Transforming Growth Factor beta

Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B).. To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM.. In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-β signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA2, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model.. Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

Topics: A549 Cells; Animals; Arachidonic Acid; Carcinoma, Non-Small-Cell Lung; Culture Media, Conditioned; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Phospholipases A2, Secretory; Protein Kinase C-delta; Pulmonary Surfactant-Associated Protein B; Radiation Tolerance; Radiation, Ionizing; Signal Transduction; 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

The epithelial-to-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal features and contributes to metastasis and resistance to treatment. This process involves epigenetic reprogramming for gene expression. We explored global histone modifications during TGF-β1-induced EMT in two non-small cell lung cancer (NSCLC) cell lines and tested different epigenetic treatment to modulate or partially reverse EMT.. Loss of classical epithelial markers and gain of mesenchymal markers were verified in A549 and H358 cell lines during TGF-β1-induced EMT. In addition, we noticed increased expression of the axonal guidance protein semaphorin 3C (SEMA3C) and PD-L1 (programmed death-ligand 1) involved in the inhibition of the immune system, suggesting that both SEMA3C and PD-L1 could be the new markers of TGF-β1-induced EMT. H3K79me3 and H2BK120me1 were decreased in A549 and H358 cell lines after a 48-h TGF-β1 treatment, as well as H2BK120ac in A549 cells. However, decreased H3K79me3 was not associated with expression of the histone methyltransferase DOT1L. Furthermore, H3K79me3 was decreased in tumors compared in normal tissues and not associated with cell proliferation. Associations of histone deacetylase inhibitor (SAHA) with DOT1L inhibitors (EPZ5676 or SGC0946) or BET bromodomain inhibitor (PFI-1) were efficient to partially reverse TGF-β1 effects by decreasing expression of PD-L1, SEMA3C, and its receptor neuropilin-2 (NRP2) and by increasing epithelial markers such as E-cadherin.. Histone methylation was modified during EMT, and combination of epigenetic compounds with conventional or targeted chemotherapy might contribute to reduce metastasis and to enhance clinical responses.

Topics: A549 Cells; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Regulatory Networks; Histone Deacetylase Inhibitors; Histones; Humans; Lung Neoplasms; Methylation; Transforming Growth Factor beta

To investigate the role of TGF-β and IL-6 in myofibroblasts (MFs) - lung cancer cell interactions, lung cancer cells (Lewis and CTM-167 cell lines) were stimulated by IL-6, MF-conditioned medium (MF-CM) or MFs, with or without TGF-β signaling inhibitor - SB431542 and/or JAK2/STAT3 inhibitor - JSI-124. MFs were stimulated by TGF-β, cancer cell-CM or cancer cells, with or without SB431542 and JSI-124. Cell proliferation, the levels of cytokines, expression of mRNA and protein were determined. Mice bearing xenograft tumors were intraperitoneally treated with SB431542 or JSI-124 and monitored for up to 45 days. In co-culture systems, MFs secreted high levels of IL-6, while cancer cells produced high levels of TGF-β. Recombinant IL-6 and MF-CM activated STAT3 and upregulated TGF-β in cancer cells. In contrast, cancer cell-CM or TGF-β stimulated MFs to produce IL-6. Blockade of JAK2/STAT3 and TGF-β signaling by specific inhibitors significantly inhibited cell proliferation in vitro and tumor growth in vivo of lung cancer cells. Our study demontrated that the TGF-β and IL-6/JAK2/STAT3 signaling pathways form a positive feedback signaling loop that mediated the interactions between MFs and lung cancer cells. Targeted inhibiton of this signaling loop could be a new approach for lung cancer prevention and therapy.

Topics: Animals; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cytokines; Disease Models, Animal; Gene Expression; Humans; Interleukin-6; Janus Kinase 2; Lung Neoplasms; Male; Mice; Molecular Targeted Therapy; Myofibroblasts; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

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

Lung cancer is one of the major causes of cancer morbidity and mortality around the world, and the resistance to cisplatin is a critical issue to chemotherapy in lung cancer patients. Transforming growth factor β (TGF-β) signal pathway abnormality is widely observed in drug resistance during lung cancer chemotherapy. Here, we investigated the effects of heat-shock protein 27 (HSP27) in the TGF-β-induced cisplatin resistance in lung cancer cell. In this study, our results indicated that the mRNA and protein expression of HSP27 were significantly increased in human lung cancer tissues. TGF-β induced the mRNA and protein expression of HSP27 in human lung cancer cell (A549). Treatment of TGF-β-induced cisplatin resistance in A549 cell through blocking the cisplatin-induced apoptosis and cell death, which characterized as the increasing of cell viability and decreasing of PARP and caspase3 cleavage in the cisplatin-treated cell. Knockdown of SMAD3 attenuated the TGF-β-induced HSP27 expression and restored the TGF-β-induced cisplatin resistance in A549 cell. Additionally, the knockdown of HSP27 blocked TGF-β-induced cisplatin resistance via decreasing cell viability and increasing cell apoptosis in A549 cell. These data therefore suggested that HSP27 is critical to lung cancer progression and TGF-β-induced cisplatin resistance in human lung cancer cell, and may provide an effective clinical strategy in lung cancer patients with resistance to chemotherapy.

Topics: A549 Cells; Apoptosis; Cell Line, Tumor; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Lung Neoplasms; Molecular Chaperones; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Pericytes represent important support cells surrounding microvessels found in solid organs. Emerging evidence points to their involvement in tumor progression and metastasis. Although reported to be present in the human lung, their specific presence and functional orientation within the tumor microenvironment in non-small cell lung cancer (NSCLC) has not yet been adequately studied. Using a multiparameter approach, we prospectively identified, sorted and expanded mesenchymal cells from human primary NSCLC samples based on co-expression of CD73 and CD90 while lacking hematopoietic and endothelial lineage markers (CD45, CD31, CD14 and Gly-A) and the epithelial marker EpCAM. Compared to their normal counterpart, tumor-derived Lineage-EpCAM-CD73+CD90+ cells showed enhanced expression of the immunosuppressive ligand PD-L1, a higher constitutive secretion of IL-6 and increased basal αSMA levels. In an in vitro model of 3D microvessels, both tumor-derived and matched normal Lineage-EpCAM-CD73+CD90+ cells supported the assembly of perfusable vessels. However, tumor-derived Lineage-EpCAM-CD73+CD90+ cells led to the formation of vessels with significantly increased permeability. Together, our data show that perivascular-like cells present in NSCLC retain functional abnormalities in vitro. Perivascular-like cells as an eventual target in NSCLC warrants further investigation.

Topics: 5'-Nucleotidase; B7-H1 Antigen; Biomarkers; Capillary Permeability; Carcinoma, Non-Small-Cell Lung; Epithelial Cell Adhesion Molecule; Humans; Interleukin-6; Lung Neoplasms; Mesenchymal Stem Cells; Microvessels; Neovascularization, Pathologic; Pericytes; Stromal Cells; Thy-1 Antigens; Transforming Growth Factor beta

Arctigenin (ARC) is a lignan that is abundant in Asteraceae plants, which show anti-inflammatory and anti-cancer activities. The current study investigated whether ARC affects cancer progression and metastasis, focusing on EMT using invasive human non-small cell lung cancer (NSCLC) cells. No toxicity was observed in the cells treated with different doses of ARC (12-100 μM). The treatment of ARC repressed TGF-β-stimulated changes of metastatic morphology and cell invasion and migration. ARC inhibited TGF-β-induced phosphorylation and transcriptional activity of smad2/3, and expression of snail. ARC also decreased expression of N-cadherin and increased expression of E-cadherin in dose-dependent and time-dependent manners. These changes were accompanied by decreased amount of phospho-smad2/3 in nucleus and nuclear translocation of smad2/3. Moreover, ARC repressed TGF-β-induced phosphorylation of ERK and transcriptional activity of β-catenin. Our data demonstrate anti-metastatic activity of ARC in lung cancer model.

Topics: Antineoplastic Agents, Phytogenic; Asteraceae; beta Catenin; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Survival; Epithelial-Mesenchymal Transition; Furans; Humans; Lignans; Lung; Lung Neoplasms; Neoplasm Invasiveness; Phosphorylation; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Radiotherapy is used to treat tumors of different origins and nature, but often lead to development of radioresistance and metastasis of cells. Interestingly, radiation induces epithelial-mesenchymal transition (EMT), a process by which epithelial cells undergo mesenchymal phenotype and stimulates tumor progression capability. Our study investigated the effect of Trichostatin A (TSA), a natural derivate isolated from Streptomyces, upon radiation-induced lung EMT and we tried to understand the role of signaling molecules in irradiated lung cancer cells (A549). The cells were categorized into four groups: untreated control, radiation alone (R; 8Gy, X-ray), radiation combined with TSA (R + T) and TSA (100nM). Radiation-induced lung EMT were evidenced by decreased expression of epithelial marker like E-cadherin, Zona occluden1 (ZO-1) and increased expression of N-cadherin and Vimentin. The Snail protein, a master regulator of EMT, was observed to be elevated after radiation treatment. In addition, TGF-β1 signaling (smad2, 3, and 4) proteins were activated upon irradiation. Western blot data were supported by the altered m-RNA expression of E-cadherin, TGF-β and Snail genes and this effect were reversed by TSA treatment. In addition to this, as supportive evidence, we performed docking studies between snail protein and TSA using Auto docking software and results suggested that less binding energy was needed for the putative binding of TSA on C-terminal domain of Snail protein. Based on our report, we suggest that TSA can effectively inhibit radiation-induced EMT (i) by altering epithelial and mesenchymal markers (ii) by modulating signaling molecules of TGFβ1 pathway (iii) by inhibiting cancer cell migratory potential in A549 cells (iv)by effectively binding to Snail which is an enhancer of EMT.

Topics: A549 Cells; Cell Movement; Epithelial-Mesenchymal Transition; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Smad Proteins; Snail Family Transcription Factors; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFβ-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFβ. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. TRAF3 autophagy is driven by RAS and results in activation of the NF-κB family member RELB. We show that RELB represses TGFβ target promoters independently of DNA binding at NF-κB recognition sequences, instead binding with SMAD family member(s) at SMAD-response elements. Thus, autophagy antagonises TGFβ gene expression. Finally, autophagy-deficient A549 cells regain tumorigenicity upon SMAD4 knockdown. Thus, at least in this setting, a physiologic function for autophagic regulation of gene expression is tumour growth.

Topics: A549 Cells; Animals; Autophagy; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice; Smad Proteins; TNF Receptor-Associated Factor 3; Transcription Factor RelB; Transforming Growth Factor beta; Transplantation, Heterologous

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

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

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

A recent multicenter study led by our institution demonstrated that local recurrence of non-small cell lung cancer (NSCLC) was significantly more frequent in patients with diabetes, raising the possibility of different tumor biology in diabetics. Epithelial-to-mesenchymal transition (EMT) plays a key role in local tumor recurrence and metastasis. In the present study, we investigated differences of tumor microenvironment between patients with and without diabetes by examining expression of EMT markers. Seventy-nine NSCLC patients were selected from the cohort of our early multicenter study. These patients were classified into 4 groups: 39 with adenocarcinoma with (n = 19) and without (n = 20) diabetes, and 40 with squamous cell carcinoma with (n = 20) and without (n = 20) diabetes. Immunohistochemical expression of eight EMT markers was analyzed, including transforming growth factor-beta (TGF-β), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), vimentin, E-cadherin, N-cadherin, HtrA1, and beta-catenin. Five markers (E-cadherin, HtrA1, TGF-β, IGF-1R and vimentin) demonstrated significantly higher expression in diabetics than in non-diabetics in both histology types. N-cadherin had higher expression in diabetics, though the difference did not reach statistical significance. EGFR showed a higher expression in diabetics in squamous cell carcinoma only. Beta-catenin was the only marker with no difference in expression between diabetics versus non-diabetics. Our findings suggest that diabetes is associated with enhanced EMT in NSCLC, which may contribute to growth and invasiveness of NSCLC.

Topics: Adenocarcinoma; Aged; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Diabetes Mellitus; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Neoplasm Recurrence, Local; Transforming Growth Factor beta; Vimentin

Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-β (TGF-β)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-β-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-β-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-β in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells.

Topics: Antigens, CD; Apoptosis; Blotting, Western; Cadherins; Cell Proliferation; Chromatin; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Histones; Humans; Immunoprecipitation; Lung Neoplasms; MicroRNAs; Polycomb Repressive Complex 2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Long Noncoding; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-β, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg.

Topics: CD4-Positive T-Lymphocytes; Cell Cycle; Cyclin D1; Forkhead Box Protein O1; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Killer Cells, Natural; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Natural Cytotoxicity Triggering Receptor 1; Receptors, CXCR3; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Cancer upregulated gene 2 (CUG2) enhances cell migration and invasion, but the underlying mechanism has not been revealed. Herein, CUG2 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin, characteristics of the epithelial-mesenchymal transition (EMT). A CUG2 deletion mutant, lacking interaction with nucleophosmin 1 (NPM1), or suppression of NPM1 reduced wound healing and cell invasion, indicating that CUG2-mediated EMT requires NPM1. CUG2 enhanced activation of Smad2/3 and expression of Snail and Twist, while the CUG2 silence decreased these TGF-β signaling pathways, leading to suppression of EMT. NPM silence also inhibited the CUG2-induced TGF-β signaling. These results suggest that TGF-β signaling is involved in CUG2-induced EMT. Treatment with EW-7197, a novel inhibitor of TGF-β signaling, diminished CUG2-mediated EMT and inhibition of Akt, ERK, JNK, and p38 MAPK, non-canonical TGF-β signaling molecules, also decreased expression of Smad2/3, Snail and Twist, leading to inhibition of EMT. The results confirm that TGF-β signaling is essential for CUG2-mediated EMT. Interestingly, TGF-β enhanced CUG2 expression. We further found that both CUG2-induced TGF-β production and TGF-β-induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF-β promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-β. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF-β-induced EMT, indicating that the effect of TGF-β on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF-β signaling.

Topics: A549 Cells; Cadherins; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Nucleophosmin; Signal Transduction; Transforming Growth Factor beta; Vimentin

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

Topics: Animals; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Extracellular Vesicles; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; Male; Mesenchymal Stem Cells; Mice; Osteosarcoma; Signal Transduction; STAT3 Transcription Factor; Tissue Array Analysis; Transforming Growth Factor beta

Mesenchymal stem cells (MSCs) are considered for potential use as an ideal vehicle to efficiently deliver therapeutic agents in treatment against cancers including melanoma. However, emerging evidence indicates that MSCs promote tumor growth and progression. Therefore, a comprehensive understanding of the role of MSCs is very important to evaluate the MSCs-based therapy in melanoma. B16 melanoma cells treated by MSC conditioned medium (CM), showed significantly enhanced migration and invasion, which was also confirmed in a lung metastasis mice model in vivo. Later, it was found that MSC CM induced an epithelial mesenchymal transition (EMT) in B16 cells. The ELISA assay showed that transforming growth factor-β (TGF-β) was secreted by MSCs and EMT was also induced by recombinant TGF-β in B16 melanoma cells, which suggests the important role of TGF-β in mediating EMT caused by MSC CM. When TGF-β signaling was inhibited by SB431542, the EMT process was significantly eliminated in vitro and in xenograft tumors in vivo. Snail, as a downstream target of TGF-β signaling and an EMT regulator, was upregulated by MSC CM and inhibited by SB431542, which confirms the key role of TGF-β signaling in EMT induced by MSC CM in B16 cells. Taken together, this study shows that MSC induces EMT in melanoma cells in a paracrine manner, which might be mediated by the TGF-β/Snail signaling pathway. Thus, caution should be exercised when considering MSCs-based therapy in melanoma and also in other cancers. Targeting TGF-β signaling and Snail could be further investigated as potential therapeutic approaches for melanoma.

Topics: Animals; Benzamides; Cell Movement; Culture Media, Conditioned; Dioxoles; Epithelial-Mesenchymal Transition; Lung Neoplasms; Melanoma, Experimental; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Paracrine Communication; Recombinant Proteins; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Compared with CD4

Topics: Animals; B7-1 Antigen; B7-2 Antigen; Cell Proliferation; Clonal Anergy; CTLA-4 Antigen; Cytotoxicity, Immunologic; Dendritic Cells; Early Growth Response Protein 2; Exosomes; Gene Expression Regulation; Histocompatibility Antigens Class I; Interleukin-10; Interleukin-2; Lung Neoplasms; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Transplantation; Ovalbumin; Primary Cell Culture; Signal Transduction; Survival Analysis; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Neuropilins (NRP1 and NRP2) are co-receptors for heparin-binding growth factors and class 3 semaphorins. Different isoforms of NRP1 and NRP2 are produced by alternative splicing. We found that in non-small cell lung cancer (NSCLC) cell lines, transforming growth factor-β (TGFβ) signaling preferentially increased the abundance of NRP2b. NRP2b and NRP2a differ only in their carboxyl-terminal regions. Although the presence of NRP2b inhibited cultured cell proliferation and primary tumor growth, NRP2b enhanced cellular migration, invasion into Matrigel, and tumorsphere formation in cultured cells in response to TGFβ signaling and promoted metastasis in xenograft mouse models. These effects of overexpressed NRP2b contrast with the effects of overexpressed NRP2a. Hepatocyte growth factor (HGF)-induced phosphorylation of the kinase AKT was specifically promoted by NRP2b, whereas inhibiting the HGF receptor MET attenuated NRP2b-dependent cell migration. Unlike NRP2a, NRP2b did not bind the PDZ domain scaffolding protein GAIP carboxyl terminus-interacting protein (GIPC1) and only weakly recruited phosphatase and tensin homolog (PTEN), potentially explaining the difference between NRP2b-mediated and NRP2a-mediated effects. Analysis of NSCLC patient tumors showed that NRP2b abundance correlated with that of the immune cell checkpoint receptor ligand PD-L1 as well as with epithelial-to-mesenchymal transition (EMT) phenotypes in the tumors, acquired resistance to epidermal growth factor receptor (EGFR) inhibitors, disease progression, and poor survival in patients. NRP2b knockdown attenuated the acquisition of resistance to the EGFR inhibitor gefitinib in cultured NSCLC cells. Thus, in NSCLC, NRP2b contributed to the oncogenic response to TGFβ and correlated with tumor progression in patients.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Neuropilin-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sequence Homology, Amino Acid; Survival Analysis; Transforming Growth Factor beta; Transplantation, Heterologous

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

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

Accumulating evidence suggests that long noncoding RNAs (lncRNAs) are crucial regulators of the Epithelial-Mesenchymal-Transition (EMT). TGF-β signaling is a major inducer of EMT and can facilitate lung cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here, we have identified 291 lncRNAs which were differentially expressed in lung cancer tissues compared with adjacent normal tissues. Of these, the gene body or vicinity of 19 transcripts were also bound by SMAD3. The expression of LINC01186 was significantly decreased in A549 cells treated with TGF-β1. Furthermore, LINC01186 was stably down-regulated in lung cancer tissues compared with normal tissues in TCGA data sets and another published lung cancer data sets. The bioinformatics analysis suggested that LINC01186 was associated with TGF-β and might participate in EMT process. Moreover, knocking-down LINC01186 promoted cell migration and invasion, whereas, LINC01186 overexpression prevented cell metastasis. Importantly, LINC01186 expression was regulated by SMAD3. And LINC01186 affected several EMT markers expression. These findings suggest that LINC01186, a mediator of TGF-β signaling, can play a significant role in the regulation of EMT and lung cancer cell migration and invasion.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Movement; Cells, Cultured; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Microarray Analysis; Neoplasm Invasiveness; RNA, Long Noncoding; Smad3 Protein; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT), the transdifferentiation of epithelial cells into mesenchymal cells, has been implicated in the metastasis and provides novel strategies for cancer therapy. Osthole (OST), a dominant active constituent of Chinese herb Cnidium monnieri, has been reported to inhibit cancer metastasis while the mechanisms remains unclear. Here, we studied the inhibitory effect and mechanisms of OST on TGF-β1-induced EMT in A549 cells. Cells were treated with TGF-β1 in the absence and presence of OST. The morphological alterations were observed with a microscopy. The protein and mRNA expressions were determined by Western blotting and real-time PCR. The protein localization was detected with immunofluorescence. The adhesion, migration, and invasion were determined by Matrigel, wound-healing, and Transwell assays. TGF-β1 treatment induced spindle-shaped alterations of cells, upregulation of N-cadherin, Vimentin, NF-κB p65, and downregulation of E-cadherin. Dysregulated membrane expression and mRNA expression of E-cadherin and N-cadherin were observed after TGF-β1 treatment. TGF-β1 increased abilities of migration and invasion and triggered the nuclear translocation of NF-κB p65. These alterations were dramatically inhibited by OST. Furthermore, PDTC, a NF-κB inhibitor, showed similar effects. In addition, TGF-β1-induced expression of Snail was significantly inhibited by OST and silenced Snail partially reversed TGF-β1-induced EMT biomarkers without affecting NF-κB p-65. In conclusion, OST inhibited TGF-β1-induced EMT, adhesion, migration, and invasion through inactivation of NF-κB-Snail pathways in A549 cells. This study provides novel molecular mechanisms for the anti-metastatic effect of OST.

Topics: A549 Cells; Cadherins; Coumarins; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; NF-kappa B; Proline; Snail Family Transcription Factors; Thiocarbamates; Transcription Factor RelA; Transforming Growth Factor beta

The tumour microenvironment is critical for various characteristics of tumour malignancies. Platelets, as part of the tumour microenvironment, are associated with metastasis formation via increasing the rate of tumour embolus formation in microvasculature. However, the mechanisms underlying the ability of tumour cells to acquire invasiveness and extravasate into target organs at the site of embolization remain unclear. In this study, we reported that platelet aggregation-inducing factor podoplanin expressed on tumour cell surfaces were found to not only promote the formation of tumour-platelet aggregates via interaction with platelets, but also induced the epithelial-mesenchymal transition (EMT) of tumour cells by enhancing transforming growth factor-β (TGF-β) release from platelets. In vitro and in vivo analyses revealed that podoplanin-mediated EMT resulted in increased invasiveness and extravasation of tumour cells. Treatment of mice with a TGF-β-neutralizing antibody statistically suppressed podoplanin-mediated distant metastasis in vivo, suggesting that podoplanin promoted haematogenous metastasis in part by releasing TGF-β from platelets that was essential for EMT of tumour cells. Therefore, our findings suggested that blocking the TGF-β signalling pathway might be a promising strategy for suppressing podoplanin-mediated haematogenous metastasis in vivo.

Topics: A549 Cells; Animals; Antibodies, Neutralizing; Blood Platelets; Carcinoma, Small Cell; Cell Movement; Diffusion Chambers, Culture; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Membrane Glycoproteins; Mice; Mice, Inbred ICR; Neoplasm Invasiveness; Platelet Aggregation; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

MicroRNA-381 (miR-381) has been reported to play suppressive or promoting roles in different malignancies. However, the expression level, biological function, and underlying mechanisms of miR-381 in gastric cancer remain poorly understood. Our previous study indicated that transmembrane protein 16A (TMEM16A) contributed to migration and invasion of gastric cancer and predicted poor prognosis. In this study, we found that miR-381 inhibited the metastasis of gastric cancer through targeting TMEM16A expression.. MiR-381 expression was analyzed using bioinformatic software on open microarray datasets from the Gene Expression Omnibus (GEO) and confirmed by quantitative RT-PCR (qRT-PCR) in human gastric cancer tissues and cell lines. Cell proliferation was investigated using MTT and cell count assays, and cell migration and invasion abilities were evaluated by transwell assay. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Luciferase reporter assay, western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were employed to explore the mechanisms of the effect of miR-381 on gastric cancer cells.. MiR-381 was significantly down-regulated in gastric cancer tissues and cell lines. Low expression of miR-381 was negatively related to lymph node metastasis, advanced tumor stage and poor prognosis. MiR-381 decreased gastric cancer cell proliferation, migration and invasion in vitro and in vivo. TMEM16A was identified as a direct target of miR-381 and the expression of miR-381 was inversely correlated with TMEM16A expression in gastric cancer tissues. Combination analysis of miR-381 and TMEM16A revealed the improved prognostic accuracy for gastric cancer patients. Moreover, miR-381 inhibited TGF-β signaling pathway and down-regulated epithelial-mesenchymal transition (EMT) phenotype partially by mediating TMEM16A.. MiR-381 may function as a tumor suppressor by directly targeting TMEM16A and regulating TGF-β pathway and EMT process in the development of progression of gastric cancer. MiR-381/TMEM16A may be a novel therapeutic candidate target in gastric cancer treatment.

Topics: 3' Untranslated Regions; Animals; Anoctamin-1; Cell Line, Tumor; Cell Movement; Cell Proliferation; Computational Biology; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; MicroRNAs; Neoplasm Proteins; Neoplasm Staging; Neoplasm Transplantation; Signal Transduction; Stomach Neoplasms; Transforming Growth Factor beta

This study aimed to investigate the gene expression changes associated with carcinoma-associated fibroblasts (CAFs) involving in non-small cell lung carcinoma (NSCLC).. We downloaded the GEO series GSE22862, which contained matched gene expression values for 15 CAF and normal fibroblasts samples, and series GSE27289 containing SNP genotyping for four matched NSCLC samples. The differentially expressed genes in CAF samples were identified using the limma package in R. Then we performed gene ontology (GO) and pathway enrichment analysis and protein-protein interaction (PPI) network construction using the identified DEGs. Moreover, aberrant cell fraction, ploidy, allele-specific copy number, and loss of heterozygosity (LOH) within CAF cells were analyzed using the allele-specific copy number analysis.. We obtained 545 differentially expressed genes between CAF and normal fibroblasts samples. The up-regulated genes are mainly involved in GO terms such as positive regulation of cell migration and extracellular region, while the down-regulated genes participate in the lung development and extracellular region. Multiple genes including bone morphogenetic protein 4 (BMP4) and transforming growth factor, beta 3 (TGFB3) are involved in the TGF-β signaling pathway. Genes including BMP4, TGFBI and matrix Gla protein (MGP) were hub genes. Moreover, no LOH event for BMP4 and MGP was found, that for sphingosine kinase 1 (SPHK1) was 70%, and for TGFBI was 40%.. Our data suggested that BMP4, MGP, TGFBI, and SPHK1 may be important in CAFs-associated NSCLC, and the abnormal expression and high LOH frequency of them may be used as the diagnosis targets of CAFs in NSCLC.

Topics: Alleles; Cancer-Associated Fibroblasts; Carcinoma; Carcinoma, Non-Small-Cell Lung; Down-Regulation; Gene Dosage; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Genetic Association Studies; Humans; Loss of Heterozygosity; Lung Neoplasms; Protein Interaction Maps; Tissue Array Analysis; Transforming Growth Factor beta; Up-Regulation

TGFβ-SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Early Growth Response Transcription Factors; Epithelial-Mesenchymal Transition; Feedback, Physiological; Humans; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice, Knockout; Patients; Promoter Regions, Genetic; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

TIF1γ is a novel regulator of transforming growth factor (TGF)-β/Smad signaling. Our previous studies show that dysregulated expression of transcriptional intermediary factor 1 γ (TIF1γ) and abnormal TGF-β/Smad signaling are implicated in non-small-cell lung cancer (NSCLC) separately. However, how TIF1γ contributes to NSCLC by controlling TGF-β/Smad signaling is poorly understood. Here, we investigated the mechanistic role of TIF1γ in TGF-β-induced epithelial-mesenchymal transition (EMT), as well as a link between TIF1γ and SOX2 in NSCLC. We show that TIF1γ is a downstream target of SOX2 in NSCLC cells. SOX2 overexpression negatively regulated TIF1γ promoter activity and thereby attenuated TIF1γ mRNA and protein expression levels; SOX2 knockdown significantly enhanced TIF1γ promoter activity and augmented TIF1γ expression. Moreover, TIF1γ mRNA expression was downregulated in human NSCLC tissues and negatively correlated with SOX2 protein, which was upregulated in NSCLC tissues. Importantly, knockdown of TIF1γ or SOX2 overexpression augmented SMAD4 (human Mad (mothers against decapentaplegic)-related homologous protein 4)-dependent transcriptional responses, and enhanced TGF-β-induced EMT and human NSCLC cell invasion; knockdown of SOX2 impaired TGF-β-induced EMT and NSCLC cell invasion. In an in vivo model of metastasis, knockdown of TIF1γ promotes NSCLC cell metastasis. In addition, our data suggested that TIF1γ inhibited TGF-β-induced EMT through competing with SMAD4 in NSCLC cells. Taken together, our findings reveal a new mechanism by which SOX2-mediated transcription repression of TIF1γ promotes TGF-β-induced EMT in NSCLC.

Topics: Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Nude; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; SOXB1 Transcription Factors; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta

The process of epithelial-mesenchymal transition (EMT), in addition to being an initiating event for tumor metastasis, is implicated in conferring several clinically relevant properties to disseminating cancer cells. These include stem cell-like properties, resistance to targeted therapies and ability to evade immune surveillance. Enrichment analysis of gene expression changes during transforming growth factor-β (TGF-β)-induced EMT in lung cancer cells identified complement cascade as one of the significantly enriched pathway. Further analysis of the genes in the complement pathway revealed an increase in the expression of complement inhibitors and a decrease in the expression of proteins essential for complement activity. In this study, we tested whether EMT confers resistance to complement-dependent cytotoxicity (CDC) in lung cancer cells and promotes tumor progression. CD59 is a potent inhibitor of membrane attack complex that mediates complement-dependent cell lysis. We observed a significant increase in the CD59 expression on the surface of cells after TGF-β-induced EMT. Furthermore, CD59 knockdown restored susceptibility of cells undergoing EMT to cetuximab-mediated CDC. TGF-β-induced CD59 expression during EMT is dependent on Smad3 but not on Smad2. Chromatin immunoprecipitation analysis confirmed that Smad3 directly binds to the CD59 promoter. Stable knockdown of CD59 in A549 cells inhibited experimental metastasis. These results demonstrate that TGF-β-induced EMT and CD59 expression confers an immune-evasive mechanism to disseminating tumor cells facilitating tumor progression. Together, our data demonstrates that CD59 inhibition may serve as an adjuvant to enhance the efficacy of antibody-mediated therapies, as well as to inhibit metastasis in lung cancer.

Topics: Adenocarcinoma; Animals; CD59 Antigens; Cell Line, Tumor; Cetuximab; Complement Activation; Complement Membrane Attack Complex; Cytotoxicity, Immunologic; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Mice; Mice, Mutant Strains; Mice, SCID; Neoplasm Proteins; Promoter Regions, Genetic; Proteins; RNA Interference; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Smad3 Protein; Transcriptome; Transforming Growth Factor beta; Tumor Escape; Vesicular Transport Proteins; Xenograft Model Antitumor Assays

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

A multilayered paper-based platform is used to investigate the interactions between human lung tumor cells and fibroblasts that are isolated from primary patient tumor samples.

Topics: Cell Line, Tumor; Cell Movement; Coculture Techniques; Enzyme-Linked Immunosorbent Assay; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Immunohistochemistry; Lung Neoplasms; Microscopy, Fluorescence; Paper; Transforming Growth Factor beta

Nuclear Factor (NF)-κB is positioned to provide the interface between COPD and carcinogenesis through regulation of chronic inflammation in the lungs. Using a tetracycline-inducible transgenic mouse model that conditionally expresses activated IκB kinase β (IKKβ) in airway epithelium (IKTA), we found that sustained NF-κB signaling results in chronic inflammation and emphysema by 4 months. By 11 months of transgene activation, IKTA mice develop lung adenomas. Investigation of lung inflammation in IKTA mice revealed a substantial increase in M2-polarized macrophages and CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Depletion of alveolar macrophages in IKTA mice reduced Tregs, increased lung CD8+ lymphocytes, and reduced tumor numbers following treatment with the carcinogen urethane. Alveolar macrophages from IKTA mice supported increased generation of inducible Foxp3+ Tregs ex vivo through expression of TGFβ and IL-10. Targeting of TGFβ and IL-10 reduced the ability of alveolar macrophages from IKTA mice to induce Foxp3 expression on T cells. These studies indicate that sustained activation of NF-κB pathway links COPD and lung cancer through generation and maintenance of a pro-tumorigenic inflammatory environment consisting of alternatively activated macrophages and regulatory T cells.

Topics: Animals; Blotting, Western; CD8-Positive T-Lymphocytes; Cells, Cultured; Epithelium; Female; Flow Cytometry; Humans; I-kappa B Kinase; Immunosuppressive Agents; Inflammation; Interleukin-10; Lung; Lung Neoplasms; Macrophages, Alveolar; Male; Mice; Mice, Transgenic; NF-kappa B; Pulmonary Disease, Chronic Obstructive; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Increasing evidence shows that micro RNAs (miRNAs) play a critical role in tumor development. However, the role of miRNAs in non-small cell lung cancer (NSCLC) metastasis remains largely unknown. Here, we found that miR-124 expression was significantly impaired in NSCLC tissues and associated with its metastasis. In vitro and in vivo experiments indicate that restoring miR-124 expression in NSCLC cells had a marked effect on reducing cell migration, invasion and metastasis. Mechanistic analyses show that Smad4, a cobinding protein in transforming growth factor-β (TGF-β) pathway, was identified as a new target gene of miR-124. Restoring Smad4 expression in miR-124-infected cells could partially rescue miR-124-induced abolition of cell migration and invasion. Notably, upon TGF-β stimulation, phosphorylation of Smad2/3 was modulated by alteration of miR-124 or Smad4 expression, followed by inducing some special transcription of downstream genes including Snail, Slug and ZEB2, all of which may trigger epithelial-mesenchymal transition and be associated with NSCLC metastasis. Moreover, activation of TGF-β pathway may enhance expression of DNMT3a, leading to hypermethylation on miR-124 promoter. Therefore, heavily loss of miR-124 expression further enhances Smad4 level by this feedback loop. Taken together, our data show for the first time that the feedback loop between miR-124 and TGF-β pathway may play a significant role in NSCLC metastasis. Targeting the loop may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for NSCLC.

Topics: Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Movement; Chromatin Immunoprecipitation; Feedback, Physiological; Heterografts; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, Inbred BALB C; MicroRNAs; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

MicroRNA (miR) was implicated in the tumorigenesis of many types of cancer, but no study was conducted on the exact role of miR-133 in lung cancer.. We have identified miR-133 as a putative regulator of FOXQ1 expression, and investigated the potential involvement of miR-133 in the migration and invasion of lung cancer cells, as well as the underlying molecular mechanism.. MiR-133 directly targeted and down-regulated FOXQ1 expression, which in turn reduced TGF-β level. MiR-133 was down-regulated in lung cancer cell lines A549 and HCC827, and its re-expression significantly inhibited the migration and invasion of the lung cancer cells. Further investigation revealed that this inhibition was caused by reversing the epithelial-mesenchymal transition, evidenced by miR-133 induced elevation of epithelial marker E-cadherin, and reduction of mesenchymal marker Vimentin.. Our study is the first to identify miR-133 as a biomarker for lung cancer. It functions to down-regulate FOXQ1, and inhibit epithelial-mesenchymal transition, which antagonizes lung cancer tumorigenesis. Therefore our data support the role of miR-133 as a potential molecular therapeutic tool in treating lung cancer.

Topics: 3' Untranslated Regions; Antigens, CD; Cadherins; Cell Adhesion; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; Neoplasm Proteins; RNA, Neoplasm; Transforming Growth Factor beta; Vimentin

Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF-) β separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-β on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-β under hypoxia/reoxygenation conditions. Combined treatment with TGF-β and hypoxia activated epidermal growth factor receptor (EGFR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-β, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-β and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS), while treatment with N-acetyl-l-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-β under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR), and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-β and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; ErbB Receptors; Humans; Ligands; Lung Neoplasms; Models, Biological; NF-E2-Related Factor 2; Oxygen; Phosphorylation; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Epithelial to mesenchymal transition (EMT) plays a key role in tumor progression and metastasis as a crucial event for cancer cells to trigger the metastatic niche. Transforming growth factor-β (TGF-β) has been shown to play an important role as an EMT inducer in various stages of carcinogenesis. Previous reports had shown that antitumor vanadium inhibits the metastatic potential of tumor cells by reducing MMP-2 expression and inducing ROS-dependent apoptosis. However, the role of vanadium in (TGF-β)-induced EMT remains unclear. In the present study, we report for the first time on the inhibitory effects of vanadium on (TGF-β)-mediated EMT followed by down-regulation of ex vivo cancer stem cell markers. The results demonstrate blockage of (TGF-β)-mediated EMT by vanadium and reduction in the mitochondrial potential of tumor cells linked to EMT and cancer metabolism. Furthermore, combination of vanadium and carboplatin (a) resulted in synergistic antitumor activity in ex vivo cell cultures, and (b) prompted G0/G1 cell cycle arrest and sensitization of tumor cells to carboplatin-induced apoptosis. Overall, the findings highlight the multifaceted antitumor action of vanadium and its synergistic antitumor efficacy with current chemotherapy drugs, knowledge that could be valuable for targeting cancer cell metabolism and cancer stem cell-mediated metastasis in aggressive chemoresistant tumors.

Topics: A549 Cells; Antineoplastic Agents; Carboplatin; Cell Line, Tumor; Drug Synergism; Epithelial-Mesenchymal Transition; Flow Cytometry; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Molecular Structure; Signal Transduction; Transforming Growth Factor beta; Vanadium

Homing of CD8(+) T lymphocytes to the tumor microenvironment is an important step for mounting a robust antitumor immune response. TGFβ is responsible for CD103 (αEβ7) integrin induction in activated intraepithelial CD8(+) T lymphocytes. However, the interplay between TGFβ and CD103 and their contribution to T-cell infiltration and antitumor activity remain unknown. Here, we used viable human lung tumor slices and autologous tumor antigen-specific T-lymphocyte clones to provide evidence that CD103 is directly involved in T-lymphocyte recruitment within epithelial tumor islets and intratumoral early T-cell signaling. Moreover, TGFβ enhanced CD103-dependent T-cell adhesion and signaling, whereas it inhibited leukocyte function-associated antigen (LFA)-1 (αLβ2) integrin expression and LFA-1-mediated T-lymphocyte functions. Mechanistic investigations revealed that TGFβ bound to its receptors (TGFBR), which promoted the recruitment and phosphorylation of integrin-linked kinase (ILK) by TGFBR1. We further show that ILK interacted with the CD103 intracellular domain, resulting in protein kinase B (PKB)/AKT activation, thereby initiating integrin inside-out signaling. Collectively, our findings suggest that the abundance of TGFβ in the tumor microenvironment may in fact engage with integrin signaling pathways to promote T-lymphocyte antitumor functions, with potential implications for T-cell-based immunotherapies for cancer. Cancer Res; 76(7); 1757-69. ©2016 AACR.

Topics: Antigens, CD; CD3 Complex; Humans; Integrin alpha Chains; Lung Neoplasms; Microscopy, Confocal; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment

RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-β in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-β repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-β. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Cullin Proteins; Cyclin-Dependent Kinase Inhibitor p21; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; MafK Transcription Factor; Mice; Mitochondria; NF-E2-Related Factor 2; RNA-Binding Proteins; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) is a complex multistep process in which phenotype switches are mediated by a network of transcription factors (TFs). Systematic characterization of all dynamic TFs controlling EMT state transitions, especially for the intermediate partial-EMT state, represents a highly relevant yet largely unexplored task. Here, we performed a computational analysis that integrated time-course EMT transcriptomic data with public cistromic data and identified three synergistic master TFs (ETS2, HNF4A and JUNB) that regulate the transition through the partial-EMT state. Overexpression of these regulators predicted a poor clinical outcome, and their elimination readily abolished TGF-β-induced EMT. Importantly, these factors utilized a clique motif, physically interact and their cumulative binding generally characterized EMT-associated genes. Furthermore, analyses of H3K27ac ChIP-seq data revealed that ETS2, HNF4A and JUNB are associated with super-enhancers and the administration of BRD4 inhibitor readily abolished TGF-β-induced EMT. These findings have implications for systematic discovery of master EMT regulators and super-enhancers as novel targets for controlling metastasis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Azepines; Cell Cycle Proteins; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 4; Histones; Humans; Lung Neoplasms; Nuclear Proteins; Phenotype; Proto-Oncogene Protein c-ets-2; RNA, Small Interfering; Sequence Analysis, RNA; Signal Transduction; Smad3 Protein; Survival Analysis; Transcription Factors; Transcriptome; Transforming Growth Factor beta; Triazoles

Epithelial-to-mesenchymal transition (EMT) is a malignant cancer phenotype characterized by augmented invasion and metastasis, chemoresistance, and escape from host-immunity. This study sought to identify efficient methods for inducing EMT reversion, to evaluate alterations in chemosensitivity and immune-protectiveness, and to elucidate the underlying mechanisms. In this study, the human lung adenocarcinoma cell lines PC-9 and HCC-827, harboring an EGFR mutation, were treated with TGF-β and FGF-2 to induce EMT. The phenotypic alterations were evaluated by RT-PCR, fluorescent immunohistochemistry, cell-mobility, and flow cytometry. Chemosensitivity to gefitinib and cisplatin was evaluated using an MTT assay and apoptosis. Immune-protectiveness was evaluated by PD-L1 expression. A combination of TGF-β and FGF-2 efficiently induced EMT in both cell lines: through Smad3 pathway in PC-9, and through Smad3, MEK/Erk, and mTOR pathways in HCC-827. The mTOR inhibitor PP242, metformin, and DMSO reverted EMT to different extent and through different pathways, depending on the cell lines. EMT induction reduced the sensitivity to gefitinib in both cell lines and to cisplatin in HCC-827, and it increased PD-L1 expression in both cell lines. EMT reversion using each of the 3 agents partly restored chemosensitivity and suppressed PD-L1 expression. Thus, chemoresistance and increased PD-L1 expression caused by EMT can be successfully reverted by EMT-reverting agents.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; B7-H1 Antigen; Cell Line, Tumor; Cell Survival; Cisplatin; Dimethyl Sulfoxide; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Fibroblast Growth Factor 2; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Indoles; Lung Neoplasms; Metformin; Mutation; Purines; Quinazolines; Transforming Growth Factor beta

GLI pathogenesis-related 1 (GLIPR1) was originally identified in glioblastomas and its expression was also found to be down-regulated in prostate cancer. Functional studies revealed both growth suppression and proapoptotic activities for GLIPR1 in multiple cancer cell lines. GLIPR1's role in lung cancer has not been investigated. Protein arginine methyltransferase 5 (PRMT5) is a protein arginine methyltransferase and forms a stoichiometric complex with the WD repeat domain 77 (WDR77) protein. Both PRMT5 and WDR77 are essential for growth of lung epithelial and cancer cells. But additional gene products that interact genetically or biochemichally with PRMT5 and WDR77 in the control of lung cancer cell growth are not characterized.. DNA microarray and immunostaining were used to detect GLIPR1 expression during lung development and lung tumorigenesis. GLIPR1 expression was also analyzed in the TCGA lung cancer cohort. The consequence of GLIPR1 on growth of lung cancer cells in the tissue culture and lung tumor xenografts in the nude mice was observed.. We found that GLIPR1 expression is negatively associated with PRMT5/WDR77. GLIPR1 is absent in growing epithelial cells at the early stages of mouse lung development and highly expressed in the adult lung. Expression of GLIPR1 was down-regulated during lung tumorigenesis and its expression suppressed growth of lung cancer cells in the tissue culture and lung tumor xenografts in mice. GLIPR1 regulates lung cancer growth through the V-Erb-B avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3).. This study reveals a novel pathway that PRMT5/WDR77 regulates GLIPR1 expression to control lung cancer cell growth and GLIPR1 as a potential therapeutic agent for lung cancer.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung; Lung Neoplasms; Membrane Proteins; Mice, Nude; Neoplasm Proteins; Nerve Tissue Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Xenograft Model Antitumor Assays

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFβ-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFβ effectors, SMAD4 and TβRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFβ-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

Topics: Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Movement; Cell Proliferation; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Mice; MicroRNAs; Osteosarcoma; Protein Isoforms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wound Healing; Xenograft Model Antitumor Assays

Chronic exposure to TGFβ, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFβ. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFβ, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFβ stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3β, resulting from the high Akt activity. Notably, integrin β3, whose expression was markedly increased upon sustained exposure to TGFβ, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin β3 mRNA levels. Therefore, we suggest that the integrin β3-Akt-GSK3β signaling axis plays an important role in non-canonical TGFβ signaling, determining the invasive properties of tumor cells chronically exposed to TGFβ.

Topics: A549 Cells; Adenocarcinoma; Glycogen Synthase Kinase 3 beta; Humans; Integrin beta3; Kaplan-Meier Estimate; Lung Neoplasms; Neoplasm Invasiveness; Prognosis; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. Cancer cells with mesenchymal features produced TGF-β and also increased the levels of TGF-β receptor. TGF-β increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-β receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-β and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment.

Topics: A549 Cells; Animals; Base Sequence; Cell Line, Tumor; Cells, Cultured; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Proto-Oncogene Proteins c-crk; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

Little is known about Yu-Ping-Feng (YPF), a typical Chinese herbal decoction, for its antitumor efficacy in non-small-cell lung cancer (NSCLC). Here, we found that YPF significantly inhibited the growth of Lewis lung cancer, prolonged the survival of tumor-bearing mice, promoted NK cell tumor infiltration, increased the population of NK cells in spleen, and enhanced NK cell-mediated killing activity. The growth suppression of tumors by YPF was significantly reversed by the depletion of NK cells. Furthermore, we found that YPF significantly downregulated the expression of TGF-β, indoleamine 2,3-dioxygenase, and IL-10 in tumor microenvironment. These results demonstrated that YPF has a NK cell-dependent inhibitory effect on Lewis lung cancer.

Topics: Animals; Carcinoma, Lewis Lung; Drugs, Chinese Herbal; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Killer Cells, Natural; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Transforming Growth Factor beta

Confluence-dependent resistance (CDR) is a phenomenon in which the efficacy of anti-cancer agents decreases when cell density increases. CDR in lung cancer has never been reported. The purpose of this study is to investigate if CDR can occur in NSCLC cells and to find a role for transforming growth factor (TGF)-β as a mechanism of CDR.. Non-small cell lung cancer (NSCLC) cell lines A549 and H2228 were exposed to cisplatin in a variety of cell density conditions. RNA interference targeting TGF-β receptor I was performed to silence the TGF-β pathway.. CDR to cisplatin was induced in NSCLC cells, whereas CDR to crizotinib, an inhibitor of activin receptor-like kinase, was not observed. During confluent conditions, the TGF-β1 concentration in the culture medium was the highest. Exogenous TGF-β1 inhibited cell proliferation and reduced sensitivity to cisplatin. Inhibition of the TGF-β pathway increased in terms of sensitivity to cisplatin at confluency.. CDR to cisplatin can occur in NSCLC cells, and the TGF-β pathway is associated with the regulation of CDR.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Count; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Metabolic Networks and Pathways; Transforming Growth Factor beta; Transforming Growth Factor beta1

For clinical application, there is a great need for small-molecule inhibitors (SMIs) that could control pathogenic effects of transforming growth factor (TGF-β) and/or modulate effects of TGF-β in normal responses. Selective SMIs of the TGF-β signaling pathway developed for therapeutics will also be powerful tools in experimentally dissecting this complex pathway, especially its cross-talk with other signaling pathways. In this study, we characterized (1'R,5'S,6'S)-2-(3',5'-dibromo-1',6'-dihydroxy-4'-oxocyclohex-2'-enyl) acetonitrile (DT), a member of a new class of small-molecule inhibitors related to bromotyrosine derivate from Pseudoceratina sp., which inhibits the TGF-β type I receptor serine/threonine kinase known as activin receptor-like kinase (ALK) 5. The inhibitory effects of DT on TGF-β-induced Smad signaling and epithelial-to-mesenchymal transition (EMT) were investigated in epithelial cells using in vitro kinase assay, luciferase reporter assays, immunoblotting, confocal microscopy, and wound healing assays. The novel ALK5 inhibitor, DT, inhibited the TGF-β-stimulated transcriptional activations of 3TP-Lux. In addition, DT decreased phosphorylated Smad2/3 levels and the nuclear translocation of Smad2/3 increased by TGF-β. In addition, DT inhibited TGF-β-induced EMT and wound healing of A549 cells. Our results suggest that DT is a potential therapeutic agent for fibrotic disease and cancer treatment. J. Cell. Biochem. 117: 2800-2814, 2016. © 2016 Wiley Periodicals, Inc.

Topics: Acetonitriles; Animals; Apoptosis; Blotting, Western; Cell Proliferation; Cyclohexanones; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Phosphorylation; Porifera; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tyrosine

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.

Topics: A549 Cells; Aminopyridines; Antigens, CD; Antineoplastic Agents; Benzamides; Cadherins; DNA Methylation; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Lung Neoplasms; Phosphorylation; Promoter Regions, Genetic; Protein Processing, Post-Translational; Transforming Growth Factor beta

Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFβ elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFβ signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFβ pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFβ-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFβ signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR.

Topics: Adenocarcinoma; Aged; Animals; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Case-Control Studies; Cell Movement; Cell Proliferation; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Follow-Up Studies; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Membrane Proteins; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

Topics: Animals; Bone Neoplasms; Brain Neoplasms; Cancer-Associated Fibroblasts; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Chemokine CCL2; Chemokine CCL5; Chemokine CCL7; Chemokine CXCL16; Chemokines; Chemokines, CXC; Hedgehog Proteins; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Invasiveness; Paracrine Communication; Receptors, Scavenger; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

MicroRNAs (miRNAs) have been proved to play important role in development of various cancers, including non-small cell lung cancer (NSCLC). Our previous studies have shown that miR-203 and miR-145 are associated with cellular invasion in NSCLC and nasopharyngeal cancer, respectively. However, the mechanistic role of miR-203 and miR-145 in TGF-β-induced epithelial-mesenchymal transition (EMT) has not yet been elucidated in human cancers, including NSCLC.. Real-time quantitative reverse transcriptase PCR (qRT-PCR), western blot analysis, luciferase reporter gene assays, small RNA interference and transwell migration and invasion assays were carried on human NSCLC cell lines A549 and 95C. Thirty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected.. Both miR-145 and miR-203 can directly target the 3'-untranslated region (3'-UTR) of SMAD3, and overexpression of the two miRNAs in NSCLC cells inhibited the expression of SMAD3 mRNA and protein, whereas inhibition of endogenous miR-145 or miR-203 caused an increased expression of SMAD3. Moreover, miR-145 and/or miR-203 repressed TGF-β-induced EMT and attenuated cell migration and invasion in A549 and 95C cells. siRNA-mediated knockdown of SMAD3 copied the phenotype of miR-145 and miR-203 overexpression in A549 and 95C cells.. MiR-145 and miR-203 inhibited TGF-β-induced EMT and invasion through repression of SMAD3 in NSCLC cells. Our findings provided insights into the miRNA-based mechanism for controlling TGF-β-induced EMT of NSCLC cells and a strategy for targeted therapy of NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Molecular Targeted Therapy; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

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

The tumor microenvironment is composed of different types of stromal cells that represent a key component of tumor progression. Cancer-associated fibroblasts (CAFs) secrete several factors that promote tumorigenesis. The purpose of this study was to clarify the role of the interleukin-6 (IL-6) secreted from CAFs in the communication between CAFs and NSCLC cells that modulates chemoresistance.. We used standard NSCLC cell lines as well as NSCLC cells, lung normal fibroblasts, and CAFs obtained from specimens from patients with NSCLC to evaluate phenotypic changes. Immunohistochemical analysis was also utilized to examine the stromal changes in tumor specimens obtained from patients with NSCLC who had undergone chemotherapy.. IL-6 significantly increased transforming growth factor-β1-induced epithelial-to-mesenchymal transition (EMT) changes in cancer cells. Cisplatin treatment increased expression of transforming growth factor-β in cancer cells, and the conditioned media from cancer cells activated fibroblasts and increased their IL-6 production. Expression of IL-6 was increased in CAFs compared with in lung normal fibroblasts. The conditioned media from CAFs induced EMT and resistance to cisplatin in NSCLC cells through IL-6 signaling. Immunohistochemical analysis showed that stromal IL-6 expression was correlated with EMT changes in cancer cells as well as with a diffuse distribution of smooth muscle actin-stained fibroblasts. Univariate and multivariate analyses indicated that stromal IL-6 expression was an independent prognostic factor in patients with NSCLC.. IL-6 from CAFs enhanced EMT in NSCLC cells. IL-6 may contribute to maintenance of a paracrine loop that functions as part of the communication between CAFs and NSCLC cells, resulting in chemoresistance.

Topics: Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Interleukin-6; Lung Neoplasms; Transforming Growth Factor beta

Dysregulated epithelial to mesenchymal transition (EMT) in cancer cells endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs and therefore play a critical role in transforming early-stage carcinomas into invasive malignancies. EMT has also been associated with tumor recurrence and drug resistance and cancer stem cell initiation. Therefore, better understanding of the mechanisms behind EMT could ultimately contribute to the development of novel prognostic approaches and individualized therapies that specifically target EMT processes. As an effort to characterize the central transcriptome changes during EMT, we have developed a Transforming growth factor (TGF)-beta-based in vitro EMT model and used it to profile EMT-related gene transcriptional changes in two different cell lines, a non-small cell lung cancer cell line H358, and a breast cell line MCF10a. After 7 days of TGF-beta/Oncostatin M (OSM) treatment, changes in cell morphology to a mesenchymal phenotype were observed as well as concordant EMT-associated changes in mRNA and protein expression. Further, increased motility was noted and flow cytometry confirmed enrichment in cancer stem cell-like populations. Microarray-based differential expression analysis identified an EMT-associated gene expression signature which was confirmed by RT-qPCR and which significantly overlapped with a previously published EMT core signature. Finally, two novel EMT-regulating transcription factors, IRF5 and LMCD1, were identified and independently validated.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Co-Repressor Proteins; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Interferon Regulatory Factors; LIM Domain Proteins; Lung Neoplasms; Reproducibility of Results; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transcriptome; Transforming Growth Factor beta

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

Topics: Animals; Anti-Mullerian Hormone; Bone Morphogenetic Protein Receptors, Type II; Carcinoma, Non-Small-Cell Lung; Cell Plasticity; Drug Resistance, Neoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Heat-Shock Proteins; Lung Neoplasms; Mesenchymal Stem Cells; Mice; Mice, SCID; NF-kappa B; Receptors, Peptide; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-β on Claudin-4 expression. We have observed that TGF-β induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-β-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-β-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-β-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-β-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-β regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells.

Topics: Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Claudin-4; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-β (TGF-β) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-β1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-β1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-β-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antigens, Nuclear; Cadherins; Carcinogenesis; Cell Line, Tumor; Claudin-1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Nerve Tissue Proteins; Respiratory Mucosa; RNA Splicing; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Malignant pleural effusion (MPE) is an indication of advanced cancer. Immune dysfunction often occurs in MPE. We aimed to identify the reason for impaired T cell activity in MPE from lung cancer patients and to provide clues toward potential immune therapies for MPE. The surface inhibitory molecules and cytotoxic activity of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. Levels of inflammatory cytokines in MPE and PB were tested using ELISA. TGF-β expression in tumor-associated macrophages (TAMs) was also analyzed. The effect of TAMs on T cells was verified in vitro. Lastly, changes in T cells were evaluated following treatment with anti-TGF-β antibody. We found that expression levels of Tim-3, PD-1 and CTLA-4 in T cells from MPE were upregulated compared with those from PB, but levels of IFN-γ and Granzyme B were downregulated (p < 0.05). The amount of TGF-β was significantly higher in MPE than in PB (p < 0.05). TGF-β was mainly produced by TAMs in MPE. When T cells were co-cultured with TAMs, expression levels of Tim-3, PD-1 and CTLA-4 were significantly higher than controls, whereas levels of IFN-γ and Granzyme B were significantly decreased, in a dose-dependent manner (p < 0.05). In vitro treatment with anti-TGF-β antibody restored the impaired T cell cytotoxic activity in MPE. Our results indicate that macrophage-derived TGF-β plays an important role in impaired T cell cytotoxicity. It will therefore be valuable to develop therapeutic strategies against TGF-β pathway for MPE therapy of lung cancer.

Topics: CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Humans; Lung Neoplasms; Macrophages; Pleural Effusion, Malignant; Transforming Growth Factor beta

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

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer.

Topics: A549 Cells; Adenocarcinoma; Blood Coagulation; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha5; Integrin beta Chains; Lung Neoplasms; MAP Kinase Signaling System; Protein Kinases; Receptor, PAR-1; Smad3 Protein; Thrombin; Transforming Growth Factor beta; Up-Regulation

In advanced stages of cancers, TGF-β promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, mechanisms that underpin the signaling cooperation and convert TGF-β from a potent growth inhibitor to a tumor promoter are not fully understood. We report here that TGF-β directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF-β and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35-positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF-β and has a global influence on protein products of genes instrumental to epithelial-to-mesenchymal transition and metastasis.

Topics: Alternative Splicing; Animals; Cell Line, Tumor; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Exons; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Hyaluronan Receptors; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphorylation; Promoter Regions, Genetic; Protein Isoforms; RNA Precursors; RNA-Binding Proteins; Signal Transduction; Smad3 Protein; Threonine; Transforming Growth Factor beta

Increased collagen expression in tumors is associated with increased risk of metastasis, and triple-negative breast cancer (TNBC) has the highest propensity to develop distant metastases when there is evidence of central fibrosis. Transforming growth factor-β (TGF-β) ligands regulated by cancer-associated fibroblasts (CAFs) promote accumulation of fibrosis and cancer progression. In the present study, we have evaluated TNBC tumors with enhanced collagen to determine whether we can reduce metastasis by targeting the CAFs with Pirfenidone (PFD), an anti-fibrotic agent as well as a TGF-β antagonist. In patient-derived xenograft models, TNBC tumors exhibited accumulated collagen and activated TGF-β signaling, and developed lung metastasis. Next, primary CAFs were established from 4T1 TNBC homograft tumors, TNBC xenograft tumors and tumor specimens of breast cancer patients. CAFs promoted primary tumor growth with more fibrosis and TGF-β activation and lung metastasis in 4T1 mouse model. We then examined the effects of PFD in vitro and in vivo. We found that PFD had inhibitory effects on cell viability and collagen production of CAFs in 2D culture. Furthermore, CAFs enhanced tumor growth and PFD inhibited the tumor growth induced by CAFs by causing apoptosis in the 3D co-culture assay of 4T1 tumor cells and CAFs. In vivo, PFD alone inhibited tumor fibrosis and TGF-β signaling but did not inhibit tumor growth and lung metastasis. However, PFD inhibited tumor growth and lung metastasis synergistically in combination with doxorubicin. Thus, PFD has great potential for a novel clinically applicable TNBC therapy that targets tumor-stromal interaction.

Topics: Animals; Antineoplastic Agents; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Proliferation; Collagen; Dose-Response Relationship, Drug; Female; Humans; Lung Neoplasms; Mice, Inbred BALB C; Molecular Targeted Therapy; Pyridones; Signal Transduction; Time Factors; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Tumor Burden; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Multidrug resistance (MDR) is one of the most important contributors to the high mortality of cancer and remains a major concern. We previously found that zinc finger protein 32 (ZNF32), an important transcription factor associated with cancer in Homo sapiens, protects tumor cells against cell death induced by oxidative stress and other stimuli. We thus hypothesized that ZNF32 might enable the tolerance of cancer cells to anti-tumor drugs because higher ZNF32 expression has been found in cancer tissues and in drug-resistant lung adenocarcinoma (AC) cells. In this study, we found that ZNF32 is upregulated by Sp1 (specificity protein 1) in response to drug treatment and that ZNF32 promotes drug resistance and protects AC cells against cisplatin or gefitinib treatment. ZNF32 overexpression in AC cells conferred resistance to EGFR (epidermal growth factor receptor) inhibitors by enhancing MEK/ERK activation. Moreover, ZNF32 was found to directly bind to the TGF-βR2 (transforming growth factor-beta receptor 2) promoter to promote its expression, and ZNF32-induced resistance was mediated by enhancing TGF-βR2 expression and activating the TGF-βR2/SMAD2 pathway. In both a mouse model and ex vivo cultured patient samples, a high level of ZNF32 expression was closely associated with worse overall survival and cisplatin resistance. ZNF32 appears to be a potential inducer of drug resistance that could increase the expression of the drug resistance-associated gene TGF-βR2 and subsequently facilitate the induction of drug resistance during both conventional chemotherapy and novel target therapy. Thus, ZNF32-associated target therapy is a potential novel adjuvant therapy that might effectively prevent the occurrence of multidrug resistance (MDR) during chemotherapy and improve the survival of patients with AC.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; HEK293 Cells; Humans; Kruppel-Like Transcription Factors; Lung Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Sp1 Transcription Factor; Survival Analysis; Transforming Growth Factor beta; Up-Regulation

Previously, we showed that levels of sphingosine-1 phosphate receptor 3 (S1PR3) are increased in a panel of cultured human lung adenocarcinoma cell lines, and that S1PR3-mediated signaling pathways regulate proliferation, soft agar growth, and invasion of human lung adenocarcinoma cells in vitro In the present study, we examine S1PR3 levels in human lung adenocarcinoma specimens. cDNA array and tumor microarray analysis shows that mRNA and protein levels of S1PR3 are significantly increased in human lung adenocarcinomas when compared with normal lung epithelial cells. Promoter analysis shows 16 candidate SMAD3 binding sites in the promoter region of S1PR3. ChIP indicates that TGF-β treatment stimulates the binding of SMAD3 to the promoter region of S1PR3. Luciferase reporter assay demonstrates that SMAD3 transactivates S1PR3 promoter. TGF-β stimulation or ectopic expression of TGF-β up-regulates S1PR3 levels in vitro and ex vivo Pharmacologic inhibition of TGF-β receptor or SMAD3 abrogates the TGF-β-stimulated S1PR3 up-regulation. Moreover, S1PR3 knockdown dramatically inhibits tumor growth and lung metastasis, whereas ectopic expression of S1PR3 promotes the growth of human lung adenocarcinoma cells in animals. Pharmacological inhibition of S1PR3 profoundly inhibits the growth of lung carcinoma in mice. Our studies suggest that levels of S1PR3 are up-regulated in human lung adenocarcinomas, at least in part due to the TGF-β/SMAD3 signaling axis. Furthermore, S1PR3 activity promotes the progression of human lung adenocarcinomas. Therefore, S1PR3 may represent a novel therapeutic target for the treatment of deadly lung adenocarcinomas.

Topics: Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Cell Proliferation; Cells, Cultured; Female; Humans; Lung; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad3 Protein; Sphingosine-1-Phosphate Receptors; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

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

The role of the different circulating regulatory T-cells (Treg) subsets, as well as their correlation with clinical outcome of non-small cell lung cancer (NSCLC) patients is poorly understood. Peripheral blood from 156 stage III/IV chemotherapy-naive NSCLC patients and 31 healthy donors (HD) was analyzed with flow cytometry for the presence and functionality of CD4

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; CD4 Antigens; Disease-Free Survival; Female; Humans; Interleukin-10; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFβ) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFβ requires translational activation via the non-canonical TGFβ signaling branch acting through eIF4E phosphorylation.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-4E; Female; Lung Neoplasms; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 3; Mice; Phosphorylation; Protein Biosynthesis; RNA, Messenger; Snail Family Transcription Factors; Transcription Factors; 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

Accumulating evidence shows that an imbalance in regulatory T cells (Tregs)/T helper IL-17-producing cells (Th17) exists in malignant pleural effusion (MPE). However, the cause of this phenomenon in MPE and the underlying mechanism remain uncertain. The percentages of Tregs and Th17 cells in MPE and parapneumonic effusion (PPE) were determined by flow cytometry. Their specific transcription factors, forkhead box P3 (FoxP3) and retinoic acid-related orphan receptor γt (RORγt); related cytokines, interleukin-6 (IL-6), IL-17, IL-10, and transforming growth factor-β1 (TGF‑β1); and chemokines, C-C motif ligand 17 (CCL17) and CCL20, were analyzed by real-time PCR and ELISA, respectively. Compared to patients with PPE, patients with MPE presented a higher percentage of Tregs but a lower frequency of Th17 cells. Foxp3 mRNA expression level in the cells in the pleural effusion was significantly increased in patients with MPE compared to the levels in patients with PPE (MPE vs. PPE: 3.05±0.62 vs. 0.52±0.11, p=0.0012). It was also noted that high levels of IL-10, TGF-β1 and CCL17 were observed in MPE when compared to PPE (MPE vs. PPE: IL-10, 166.3±39.53 vs. 40.38±10.92 pg/ml, p=0.0307; TGF-β1, 10,720±1,274 vs. 1,747±293.2 pg/ml, p<0.0001; CCL17, 341.1±88.22 vs. 119.2±19.80 pg/ml, p=0.0427). Furthermore, a high ratio of Tregs/Th17 cells in MPE was highly correlated to poor survival. An alteration in CCL17 and CCL20 might contribute to the Treg/Th17 imbalance in MPE, which partially predicts a poor prognosis in patients with lung cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Chemokine CCL17; Chemokine CCL20; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Lung Neoplasms; Male; Middle Aged; Nuclear Receptor Subfamily 1, Group F, Member 3; Pleural Effusion; Pleural Effusion, Malignant; Pneumonia; Prognosis; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

PIK3R3, an isoform of class IA phosphoinositide 3-kinase (PI3K), specifically interacts with cell proliferation regulators, such as retinoblastoma and proliferation cell nuclear antigen, to promote cell proliferation. However, the mechanisms behind the upstream signaling pathway of PIK3R3 remain unclear to date. This study showed that PIK3R3 expression was regulated by transforming growth factor-β (TGF-β) signaling and that PIK3R3 mediated the TGF-β-induced inhibition of lung adenocarcinoma cell proliferation. TGF-β down-regulated PIK3R3 expression in lung adenocarcinoma cells. However, this TGF-β-induced inhibition of cell proliferation can be attenuated by PIK3R3 overexpression. In addition, TGF-β can attenuate the transcriptional activity of NKX2.1, a transcription factor that binds to the promoter of PIK3R3. This result indicated that TGF-β regulated PIK3R3 expression by targeting NKX2.1. We confirmed the correlation between NKX2.1 and PIK3R3 in clinical samples. Therefore, the TGF-β/NKX2.1/PIK3R3 axis is crucial in the TGF-β-induced inhibition of cell proliferation, and the NKX2.1/PIK3R3 axis might become a target in TGF-β receptor-repressed lung adenocarcinoma.

Topics: Adenocarcinoma; Base Sequence; Cell Line, Tumor; DNA Primers; Humans; Lung Neoplasms; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Thyroid Nuclear Factor 1; Transcription Factors; Transforming Growth Factor beta

The cytotoxic activity and count of natural killer (NK) cells appear to be reduced after surgery; however, it is unknown whether the magnitude of this immune suppression is similar among different types of oncological surgery. In this study, we compared the innate immune function of patients undergoing three different oncological surgeries.. We compared the number and function of NK cells obtained from patients who had undergone mastectomies (n = 17), thoracotomies (n = 21), or liver resections for cancer (n = 22). Cytotoxicity assays were performed to measure the function of NK cells. We also determined the plasma concentrations of interleukins (IL) 2 and 4, interferon-γ, granzyme B, perforin, soluble major histocompatibility complex class I-related chain A, and epinephrine, both before and 24 h after surgery. Differences in immunologic parameters were compared preoperatively and postoperatively and by type of surgery. P values <0.05 were considered statistically significant.. The preoperative NK cell count differed statistically (P < 0.006) among all three types of surgeries; however, within surgery postoperative counts and changes compared with baseline did not. The postoperative function of NK cells was similar among types of surgeries, but was significantly reduced compared with preoperative levels (mastectomy P < 0.0001, thoracotomy P = 0.001, and liver resections P = 0.002). We observed a significant increase in the postoperative plasma concentrations of epinephrine, whereas the concentrations of major histocompatibility class I polypeptide-related sequence A and the IL-2 and/or IL-4 ratio remained unchanged before and after surgery.. The magnitude of innate immune suppression is similar among different oncological procedures. More studies are needed to better understand this complex phenomenon.

Topics: Adult; Aged; Breast Neoplasms; Colorectal Neoplasms; Cytokines; Epinephrine; Female; Humans; Immunity, Innate; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

Transforming growth factor b (TGFb) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g., Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFb receptor trafficking and signaling. Here, we report that downstream TGFb-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFb pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb dependent apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Lung Neoplasms; MAP Kinase Signaling System; Multiprotein Complexes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Serine Endopeptidases; Smad2 Protein; Smad4 Protein; TNF Receptor-Associated Factor 6; Transforming Growth Factor beta

The present study aimed to investigate the biofunctions of microRNA (miR)‑125b on lung cancer cells. A miR genechip array was used to examine the differential expression of miRs between 95D lung cancer cells and 16 human bronchial epithelial (HBE) cells. Overexpression of miR‑125b was observed in the cell lines and in the lung carcinoma tissues compared with the adjacent tissues, confirmed using reverse transcription quantitative polymerase chain reaction. Bioinformatic analysis of miR‑125b was also performed, including target prediction, gene ontology and pathway analysis. MTT, flow cytometry and Transwell assays were also used to examine the effect of downregulated miR‑125b on the proliferation, apoptosis, invasive ability and cell cycle of 95D cells. Significant differences were observed in the expression of 45 miRs in the 95D cells compared with those in 16HBE cells and the expression of miR‑125b was significantly higher in 95D cells compared with that in 16HBE cells as well as in lung tumor tissues compared with that in adjacent tissues. In addition, inhibition of the expression of miR‑125b in 95D cells induced apoptosis, G1/S phase arrest and reduction of their invasive ability. In addition, bioinformatics software predicted that miR‑125b was involved in the regulation of several pathways associated with cancer, including the transforming growth factor‑β, Wnt and mitogen‑activated protein kinase signaling pathways. These data indicated for the first time, to the best of our knowledge, that miR‑125b may function as an oncogene in lung cancer.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Computational Biology; Gene Expression; Gene Expression Profiling; Humans; Lung Neoplasms; MicroRNAs; Oncogenes; Transforming Growth Factor beta; Wnt Signaling Pathway

Lung cancer is one of the primary causes of mortality worldwide and drug resistance is the key contributing factor which results in the failure of lung cancer chemotherapy. Previous studies have shown that microRNA (miR)‑10a was involved in the reversal of cisplatin (DDP) resistance in numerous types of tumors; however, the underlying mechanism of action of this remains to be fully elucidated. In the present study, miR‑10a silencing in human DDP‑resistant lung cancer A549/DDP cells was demonstrated to improve DDP sensitivity, apoptosis, intracellular rhodamine‑123 content as well as the expression and activity of caspase‑3/8. In addition, miR‑10a suppressed the cellular expression of P‑glycoprotein, multi‑drug resistance protein (MDR) 1, MDR‑associated protein 1, RhoE, B cell lymphoma‑2 and survivin in A549/DDP cells. Furthermore, miR‑10a silencing inhibited the secretion of transforming growth factor (TGF)‑β, phosphorylation of Sma‑ and Mad‑related protein (Smad)2, signal transducer and activator of transcription (STAT)3 and STAT5, the transcriptional activity of hypoxia‑inducible factor and eukaryotic translation initiation factor 4E in human lung cancer A549/DDP cell line. These results therefore indicated that miR‑10a may be a potential target for improving the effectiveness of lung cancer chemotherapy via regulation of the TGF‑β/Smad2/STAT3/STAT5 pathway.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Gene Silencing; Humans; Lung Neoplasms; MicroRNAs; Signal Transduction; Smad2 Protein; STAT3 Transcription Factor; STAT5 Transcription Factor; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation.. Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected.. Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively.. We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

Topics: Antigens, CD; Base Sequence; Binding Sites; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Nucleotide Motifs; Promoter Regions, Genetic; Protein Binding; Smad3 Protein; Smad4 Protein; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Topics: Animals; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Mice; Neoplasm Invasiveness; RNA, Long Noncoding; Transcriptome; Transforming Growth Factor beta

The translation initiation factor eIF4E is an oncogene that is commonly overexpressed in primary breast cancers and metastases. In this article, we report that a pharmacologic inhibitor of eIF4E function, ribavirin, safely and potently suppresses breast tumor formation. Ribavirin administration blocked the growth of primary breast tumors in several murine models and reduced the development of lung metastases in an invasive model. Mechanistically, eIF4E silencing or blockade reduced the invasiveness and metastatic capability of breast cancer cells in a manner associated with decreased activity of matrix metalloproteinase (MMP)-3 and MMP-9. Furthermore, eIF4E silencing or ribavirin treatment suppressed features of epithelial-to-mesenchymal transition, a process crucial for metastasis. Our findings offer a preclinical rationale to explore broadening the clinical evaluation of ribavirin, currently being tested in patients with eIF4E-overexpressing leukemia, as a strategy to treat solid tumors such as metastatic breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-4E; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Ribavirin; Transforming Growth Factor beta

Cathepsin L, a lysosomal acid cysteine protease, was found to be overexpressed in several types of human carcinomas. However, its functional roles in tumor progression and the underlying mechanisms remain largely unclear. In the present study, we investigated a novel functional aspect of cathepsin L in regulating transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT) in A549 and MCF‑7 cells and examined its possible mechanisms. We found that TGF‑β‑induced cell morphologic changes of EMT were associated with the increased protein level of cathepsin L in A549 and MCF‑7 cells, suggesting that cathepsin L may be involved in the regulation of EMT. Furthermore, we showed that silencing of cathepsin L blocked TGF‑β‑induced cell migration, invasion and actin remodeling and inhibited TGF‑β‑mediated EMT. We also demonstrated that the mechanism of how cathepsin L knockdown regulates EMT may be explained by the suppression of EMT‑inducing molecules, such as Snail, which is associated with the phosphatidylinositol 3‑kinase (PI3K)‑AKT and Wnt signaling pathways. Moreover, we proved that cathepsin L knockdown in A549 cells significantly inhibited xenograft tumor growth and EMT in vivo. The results showed a new mechanism to determine cathepsin L involvement in the regulation of cancer invasion and migration. These results showed that cathepsin L knockdown is important in regulating EMT and suggest that cathepsin L may be utilized as a new target for enhancing the efficacy of chemotherapeutics against epithelial cancer.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cathepsin L; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Heterografts; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Transplantation; RNA, Bacterial; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

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

Non-small cell lung cancer (NSCLC) is one of the leading causes of death in all lung cancer patients due to its metastatic spread. Even though cisplatin treatment after surgical resection of the primary tumor has been established as a standard chemotherapy for residual disease including metastatic spread, NSCLC often acquires a resistance against chemotherapy, and metastatic disease is often observed. Amongst many potential mechanisms, epithelial-to-mesenchymal transition (EMT) has been considered as an important process in acquiring both metastatic spread and chemo-resistance of NSCLC. In this study, we identified MCL-1 as a critical molecule for chemo-resistance in A549 cells associated with TGF-β-induced EMT. Importantly, downregulation of MCL-1 by siRNA or inhibition of MCL-1 with pan-BCL2 inhibitor to inhibit MCL-1 was able to overcome the EMT-associated chemo-resistance in A549 cells. Collectively, MCL-1 can be a new therapeutic target for overcoming EMT-associated chemo-resistance in NSCLC patients in the context of post-operative chemotherapies.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Minor Histocompatibility Antigens; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta

Overexpression and mutational activation of the epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC). EGFR tyrosine-kinase inhibitors (TKIs) are given as a primary therapy for advanced patients with EGFR-activating mutations; however, the majority of these tumors relapse and patients eventually develop resistance to TKIs. To address a potential role of protein kinase C (PKC) isozymes in the resistance to TKIs, we used the isogenic NSCLC H1650 cell line and its erlotinib-resistant derivative H1650-M3, a cell line that displays a mesenchymal-like morphology driven by transforming growth factor-β signaling. We found that H1650-M3 cells display remarkable PKCα upregulation and PKCδ downregulation. Notably, silencing PKCα from H1650-M3 cells using RNA interference caused a significant reduction in the expression of epithelial-to-mesenchymal transition (EMT) markers vimentin, Zeb2, Snail, and Twist. Moreover, pharmacological inhibition or PKCα RNA interference depletion and PKCδ restoring sensitized H1650-M3 cells to erlotinib. Whereas ectopic overexpression of PKCα in parental H1650 cells was not sufficient to alter the expression of EMT genes or to confer resistance to erlotinib, it caused downregulation of PKCδ expression, suggesting a unidirectional crosstalk. Finally, mechanistic studies revealed that PKCα upregulation in H1650-M3 cells is driven by transforming growth factor-β. Our results identified important roles for specific PKC isozymes in erlotinib resistance and EMT in lung cancer cells, and highlight PKCα as a potential target for lung cancer treatment.

Topics: Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Mutation; Protein Kinase C-alpha; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

During the last decades, changes have been observed in the frequency of different histologic subtypes of lung cancer, one of the most common causes of morbidity and mortality, with a declining proportion of squamous cell carcinomas and an increasing proportion of adenocarcinomas, particularly in developed countries. This suggests the emergence of new etiologic factors and mechanisms, including those defining the lung microenvironment, promoting tumor growth. Assuming that the lung is the main portal of entry for broadly used nanomaterials and their established proinflammatory propensities, we hypothesized that nanomaterials may contribute to changes facilitating tumor growth. Here, we report that an acute exposure to single-walled carbon nanotubes (SWCNT) induces recruitment and accumulation of lung-associated myeloid-derived suppressor cells (MDSC) and MDSC-derived production of TGFβ, resulting in upregulated tumor burden in the lung. The production of TGFβ by MDSC requires their interaction with both SWCNT and tumor cells. We conclude that pulmonary exposure to SWCNT favors the formation of a niche that supports ingrowth of lung carcinoma in vivo via activation of TGFβ production by SWCNT-attracted and -presensitized MDSC.

Topics: Adenocarcinoma; Animals; Cell Proliferation; Cells, Cultured; Immune Tolerance; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Nanotubes, Carbon; Transforming Growth Factor beta; Tumor Burden; Tumor Escape

Epithelial-to-mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, miRNA, DNA methylation, and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma. The resulting data were integrated with functional profiles consisting of cell invasiveness, adhesion, and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. Other cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin-binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFβ. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Cytoskeleton; DNA Methylation; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Microfilament Proteins; MicroRNAs; Proteomics; Transforming Growth Factor beta; Up-Regulation; Vimentin

Malignant tumors are exemplified by excessive proliferation and aggressive migration/invasion contributing to increased mortality of cancer patients. Matrix metalloproteinase 9 (MMP9) expression is positively correlated with lung cancer malignancy. The mechanism underlying an elevated MMP9 expression is not clearly defined. We demonstrate here that the transcriptional modulator megakaryocytic leukemia 1 (MKL1) was activated by hypoxia and transforming growth factor (TGF-β), two prominent pro-malignancy factors, in cultured lung cancer cells. MKL1 levels were also increased in more invasive types of lung cancer in humans. Depletion of MKL1 in lung cancer cells attenuated migration and invasion both in vitro and in vivo. Overexpression of MKL1 potentiated the induction of MMP9 transcription by hypoxia and TGF-β, whereas MKL1 silencing diminished MMP9 expression. Of interest, MKL1 knockdown eliminated histone H3K4 methylation surrounding the MMP9 promoter. Further analyses revealed that MKL1 recruited ASH2, a component of the H3K4 methyltransferase complex, to activate MMP9 transcription. Depletion of ASH2 ameliorated cancer cell migration and invasion in an MMP9-dependent manner. Together our data indicate that MKL1 potentiates lung cancer cell migration and invasion by epigenetically activating MMP9 transcription.

Topics: Animals; Cell Line, Tumor; Cell Movement; DNA Methylation; Epigenesis, Genetic; Epigenomics; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Methyltransferases; Mice; Neoplasm Invasiveness; Promoter Regions, Genetic; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

Lung cancer is associated with poor prognosis. The aim of this study was to evaluate the clinical usefulness of HMGB-1 (high-mobility group protein B1) and TGF-β (transforming growth factor beta) in patients with advanced non-small cell lung cancer (NSCLC). We studied 45 patients with NSCLC prior to chemotherapy, 23 patients with Besnier-Boeck-Schaumann (BBS) disease (sarcoidosis), and 15 healthy volunteers. HMGB-1 and TGF-β levels were measured in serum and BALF samples using ELISA method. A higher serum HMGB-1 and TGF-β levels were in NSCLC patients compared with the other groups. TGF-β concentration in BALF was significantly higher in NSCLC than in healthy controls (p=0.047) but lower than in BBS (p=0.016). Serum HMGB-1 in NSCLC correlated with age and gender while its level in BALF was associated with distant metastasis. A higher levels of HMGB-1 in the serum of NSCLC patients with progressive disease was linked with shorter overall survival and disease-free survival. We found a positive correlation between HMGB-1 and TGF-β in BALF of IIIB NSCLC group and overall survival (p=0.04; p=0.003). Our findings confirmed that the measurement of HMGB-1 and TGF-β levels in serum and BALF of patients with NSCLC prior to treatment may have clinical usefulness and predict poor prognosis.

Topics: Aged; Biomarkers, Tumor; Bronchoalveolar Lavage Fluid; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; HMGB1 Protein; Humans; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Prognosis; Transforming Growth Factor beta

Specific p53 mutations abrogate tumor-suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort TGF-β signaling, which paradoxically displays both tumor-suppressive and pro-oncogenic functions. The molecular mechanisms of how mutant p53 simultaneously antagonizes the tumor-suppressive and synergizes the tumor-promoting function of the TGF-β pathway remain elusive. Here we demonstrate that mutant p53 differentially regulates subsets of TGF-β target genes by enhanced binding to the MH2 domain in Smad3 upon the integration of ERK signaling, therefore disrupting Smad3/Smad4 complex formation. Silencing Smad2, inhibition of ERK, or introducing a phosphorylation-defective mutation at Ser-392 in p53 abrogates the R175H mutant p53-dependent regulation of these TGF-β target genes. Our study shows a mechanism to reconcile the seemingly contradictory observations that mutant p53 can both attenuate and cooperate with the TGF-β pathway to promote cancer cell malignancy in the same cell type.

Topics: Animals; Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mutant Proteins; Mutation; Signal Transduction; Smad2 Protein; Smad3 Protein; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Protein p53

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Kaplan-Meier Estimate; Lung Neoplasms; Mice, Inbred C57BL; Neoplastic Stem Cells; Paracrine Communication; Proto-Oncogene Proteins; Smad2 Protein; Transcriptome; Transforming Growth Factor beta; Wnt Proteins; Wnt-5a Protein

Lung cancer is the leading cause of cancer-related death in the world. Previous report has identified ribosomal protein s15a (RPS15A) as a TGF-β-responsible gene in the lung adenocarcinoma cell line A549. In this study, we used specific si-RNA to downregulate RPS15A expression in A549 cells and found that decreased RPS15A expression significantly inhibited cell proliferation and survival, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Moreover, A549 cells were obviously accumulated in the G0/G1 phase in response to RPS15A knockdown, suggesting that RPS15A inhibition could induce a diminution of proliferation through cell cycle arrest. In addition, immunohistochemistry analysis further revealed that RPS15A was overexpressed in surgically resected lung cancer tissues. In conclusion, we identify RPS15A as a novel potential oncogenic gene involved in lung carcinogenesis. This study may provide a preliminary experimental basis for a gene therapy approach for treating lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Ribosomal Proteins; Transforming Growth Factor beta

To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo.. TGF-β-mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody.. The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation.. Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chemotherapy, Adjuvant; Comet Assay; DNA Damage; Histones; Humans; In Vitro Techniques; Lung Neoplasms; Mice; Phosphorylation; Protein Serine-Threonine Kinases; Radiation Tolerance; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Xanthenes

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.. iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.. High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.. Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Topics: Activating Transcription Factor 3; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Female; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Mice; Molecular Targeted Therapy; Neoplasm Invasiveness; Nitric Oxide Synthase Type II; Prognosis; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Tumor Burden; Xenograft Model Antitumor Assays

Tumors are complex ecosystems composed of networks of interacting 'normal' and malignant cells. It is well recognized that cytokine-mediated cross-talk between normal stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and cancer cells, influences all aspects of tumor biology. Here we demonstrate that the cross-talk between CAFs and cancer cells leads to enhanced growth of oncolytic virus (OV)-based therapeutics. Transforming growth factor-β (TGF-β) produced by tumor cells reprogrammed CAFs, dampened their steady-state level of antiviral transcripts and rendered them sensitive to virus infection. In turn, CAFs produced high levels of fibroblast growth factor 2 (FGF2), initiating a signaling cascade in cancer cells that reduced retinoic acid-inducible gene I (RIG-I) expression and impeded the ability of malignant cells to detect and respond to virus. In xenografts derived from individuals with pancreatic cancer, the expression of FGF2 correlated with the susceptibility of the cancer cells to OV infection, and local application of FGF2 to resistant tumor samples sensitized them to virotherapy both in vitro and in vivo. An OV engineered to express FGF2 was safe in tumor-bearing mice, showed improved therapeutic efficacy compared to parental virus and merits consideration for clinical testing.

Topics: Aged; Animals; Antiviral Agents; Cell Line, Tumor; Chlorocebus aethiops; Coculture Techniques; Female; Fibroblast Growth Factor 2; Fibroblasts; Green Fluorescent Proteins; Humans; Lung Neoplasms; Male; Mice; Microscopy, Fluorescence; Middle Aged; Neoplasm Transplantation; Oncolytic Virotherapy; Oncolytic Viruses; Ovarian Neoplasms; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment; Vero Cells

In contrast to tumor-associated macrophages, myeloid-derived suppressor cells, or inflammatory monocytes, functions of tissue resident macrophages, including alveolar macrophages (AM), in cancer were not well studied. Using a mouse model of breast cancer, we show that AM promote cancer metastasis to the lungs by suppressing antitumor T cells in this organ. AM accumulated in the premetastatic lungs through complement C5a receptor-mediated proliferation but not through recruitment from the circulation. AM preconditioned by breast tumors inhibited Th1 and favored generation of Th2 cells that had lower tumoricidal activity than Th1 cells. In addition, AM reduced the number and maturation of lung dendritic cells by regulating TGF-β in the lung environment. Depletion of AM reversed immunosuppression imposed by these cells and strengthened local Th1 responses, which significantly reduced lung metastatic burden. C5a receptor deficiency, which also lessens myeloid-derived suppressor cells in the premetastatic niche, synergized with the depletion of AM in preventing metastasis, leading to protection of mice from lung metastases. This study identifies AM as a new component of the premetastatic niche, which is harnessed by tumors to impose immunosuppression, and as a new target for cancer immunotherapies to eliminate or reduce metastasis. Because the lungs are the most common target for hematogenous metastasis, this research offers a plausible explanation for susceptibility of the lungs to cancer metastasis.

Topics: Animals; Cell Line; Cell Proliferation; Dendritic Cells; Female; Humans; Lung; Lung Neoplasms; Macrophages, Alveolar; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Receptor, Anaphylatoxin C5a; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Microenvironment

We previously demonstrated that haemoptysis as a prognostic factor in lung adenocarcinoma and haemoptysis was associated with severe vascular invasion and high circulating white blood cell count. Epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion. We hypothesized there was some relationship between tumor-associated inflammatory cells, tumor invasion, EMT, and haemoptysis. Immunohistochemistry (IHC) was used to detect CD66b and E-cadherin expression in tumor tissue. By co-culture tumor cells with polymorphonuclear neutrophils (PMNs), the expressions of EMT markers were assessed by western blotting. TGF-β1 concentrations in the supernatant and the migration activities of tumor cells were performed by ELISA and migration assays. Intratumoral CD66b(+) PMN expression was negatively associated with E-cadherin expression. Haemoptysis was significantly associated with neutrophil infiltration (OR = 4.25, 95 % CI 1.246-14.502). Neutrophils promoted EMT of tumor cells in vitro and enhanced the migration activity of tumor cells. In addition, TGF-β1 was up-regulated and Smad4 translocated into nucleus, indicating that TGF-β/Smad signaling pathway was initiated during the process. We indicated that lung adenocarcinoma with haemoptysis was associated with more PMN infiltration and PMNs promoted EMT, partly via TGF-β/Smad signal pathway. This may provide mechanistic reasons for why haemoptysis was associated with poor outcome in lung adenocarcinoma.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Cadherins; Cell Movement; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Epithelial-Mesenchymal Transition; Granulocytes; Humans; Immunoenzyme Techniques; Lung Neoplasms; Neoplasm Invasiveness; Neutrophils; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin

Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype associated with a poor prognosis. The mechanism involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumor subtype. In this study, by performing quantitative proteomic analyses in highly metastatic and parental breast cancer cell line, we found that RAB1B, a member of the RAS oncogene family, was significantly down-regulated in highly metastatic breast cancer cells. Moreover, down-regulation of RAB1B was also found to promote the proliferation and migration of TNBC cells in vitro and in vivo. Mechanistically, loss of RAB1B resulted in elevated expression of TGF-β receptor 1 (TβR1) through decreased degradation of ubiquitin, increased levels of phosphorylated SMAD3 and TGF-β-induced epithelial-mesenchymal transition (EMT). Furthermore, low RAB1B expression correlated with poor prognosis in breast cancer patients. Taken together, our findings reveal that RAB1B acts as a metastasis suppressor in TNBC by regulating the TGF-β/SMAD signaling pathway and RAB1B may serve as a novel biomarker of prognosis and the response to anti-tumor therapeutics for patients with TNBC.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Enzyme Activation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Prognosis; rab1 GTP-Binding Proteins; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad3 Protein; Tissue Array Analysis; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Ubiquitin

Transforming growth factor-β (TGF-β) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-β antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-β mediates metastasis-promoting effects is poorly understood. Establishment of TGF-β-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-β oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-β receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-β pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-β tumor-promoting activity.

Topics: Animals; Benzamides; Cell Line, Tumor; Dioxoles; Eukaryotic Initiation Factors; Female; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Proteome; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The eIF3e protein is a component of the multisubunit eIF3 complex, which is essential for cap-dependent translation initiation. Decreased eIF3e expression is often observed in breast and lung cancer and has been shown to induce epithelial-to-mesenchymal transition (EMT) in breast epithelial cells by an unknown mechanism. Here, we study the effect of decreased eIF3e expression in lung epithelial cells by creating stable clones of lung epithelial cells (A549) that express an eIF3e-targeting shRNA. Our data indicate that decreased eIF3e expression in lung epithelial cells leads to EMT, as it does in breast epithelial cells. Importantly, we show that decreased eIF3e expression in both lung and breast epithelial cells leads to the overproduction of the TGFβ cytokine and that inhibition of TGFβ signaling can reverse eIF3e-regulated EMT in lung epithelial cells. In addition, we discovered that several mRNAs that encode important EMT regulators are translated by a cap-independent mechanism when eIF3e levels are reduced. These findings indicate that EMT mediated by a decrease in eIF3e expression may be a general phenomenon in epithelial cells and that it requires activation and maintenance of the TGFβ signaling pathway.. These results indicate that inhibition of TGFβ signaling could be an efficient way to prevent metastasis in patients with NSCLC that display reduced eIF3e expression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Eukaryotic Initiation Factor-3; Humans; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

MAP3K3 is involved in both the immune response and in tumor progression. Its potential biological role in vitro in lung cancer cell lines and the association of mRNA/protein expression patterns with clinical outcome of primary lung tumors were investigated in this study. Silencing MAP3K3 using siRNA in lung cancer cell lines resulted in decreased cell proliferation, migration and invasion. These effects were associated with down-regulation of the JNK, p38, AKT, and GSK3β pathways as determined using phospho-protein and gene expression array analyses. However, MAP3K3 mRNA and protein overexpression in primary lung tumors correlated significantly with favorable patient survival. Gene cluster and pathway analyses of primary tumor datasets indicated that genes positively-correlated with MAP3K3 are significantly involved in immune response rather than the cell cycle regulators observed using in vitro analyses. These results indicate that although MAP3K3 overexpression has an oncogenic role in vitro, in primary lung adenocarcinomas it correlates with an active immune response in the tumor environment that correlates with improved patient survival. MAP3K3 may potentially not only serve as diagnostic/prognostic markers for patients with lung cancer but also provide an indicator for future investigations into immunomodulatory therapies for lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; cdc25 Phosphatases; Cell Movement; Cell Proliferation; Cyclin E; Epithelial-Mesenchymal Transition; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; MAP Kinase Kinase Kinase 3; Oncogene Proteins; Prognosis; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 (Iβ3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype.

Topics: Adenocarcinoma; Animals; Bone Neoplasms; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Elastic Modulus; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin beta3; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Nuclear Proteins; Osteolysis; Pliability; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Tumor Microenvironment; Xenograft Model Antitumor Assays; Zinc Finger Protein Gli2

The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor‑β (TGF‑β)‑induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF‑β‑induced EMT and migration was investigated in human lung cancer A549 cells. TGF‑β induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF‑β‑induced upregulation of Twist2 and zinc finger E‑box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad‑independent signaling pathways were not affected. Further analysis showed that the TGF‑β receptor I (TβRI) and TGF‑β receptor II (TβRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TβRI, significantly attenuated TGF‑β‑induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-β-induced EMT and migration by downregulating TβRI and TβRII in A549 cells.

Topics: Antineoplastic Agents; Bufanolides; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Lung; Lung Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-β (TGFβ) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it.. Generalized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-κB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-κB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFβ stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFβ-induced the NF-κB/RelA pathway by observing a 2-fold change in NF-κB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFβ-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression.. These data indicate that NF-κB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Topics: Epithelial Cells; Epithelial-Mesenchymal Transition; High-Throughput Nucleotide Sequencing; Humans; Lung Neoplasms; NF-kappa B; Signal Transduction; Stem Cells; Transcription Factor RelA; Transforming Growth Factor beta

The formation of tumor-promoting premetastatic microenvironment plays a pivotal role on metastatic progression. Understanding how the primary tumor can promote the formation of premetastatic microenvironment in the lung will aid discovery of a final cure for metastatic breast cancer. The murine 4T1 mammary carcinoma cells were injected into the mammary fat pads of the BALB/c mice. Days 0-14 were considered the premetastatic phase. Lung tissues were examined using hematoxylin-eosin staining and transmission electron microscopy. After intravenous injection of TGFβ1 pretreated 4T1 cells, the relative pulmonary vascular permeability was quantified, the extravasation, survival, and proliferation of tumor cells in premetastatic lungs were evaluated, and the levels of S100A8, S100A9, VEGF, and Angpt2 were detected in tumor-bearing mice. The results showed that during the premetastatic phase, an inflammatory response and inflammation-induced vascular hyperpermeability were established, leading to an abnormal pulmonary microenvironment, which facilitated extravasation of circulating tumor cells, and subsequent survival and proliferation of metastatic tumor cells in a TGFβ-dependent manner. Moreover, the expressions of S100A8, S100A9, VEGF, and Angpt2 were increased, and an induction of these genes by TGFβ was further observed in premetastatic lungs. Thus, this study demonstrated that TGFβ promoted the creation of premetastatic microenvironment by modulating certain crucial inflammatory cytokines and growth factors, and finally enhanced the ability of circulating cells to seed the lung.

Topics: Angiopoietin-2; Animals; Calgranulin A; Calgranulin B; Capillary Permeability; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytokines; Disease Models, Animal; Female; Humans; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

To study the effect of Fuzheng Sanjie recipe in regulating tumor-associated macrophages (TAMs) in Lewis lung cancer mice.. Efforts were made to establish the Lewis lung cancer mouse model, weigh tumors and calculate the anti-tumor rate. The immunohistochemical method was used to examine the infiltration degree of CD68 + in tumor tissues in each group. ELISA was used to examine the content of IFN-γ, TGF-β, IL-4, IL-13, IL-6, IL-10, IL-12, TNF-α in mice serum.. Compared with the tumor-bearing model group, all of the other groups showed higher tumor inhibition rates, i. e. 50.28% for the DDP group, 34.37% for the TCM-preventing group and 66.76% for the Chinese and western medicine group, with statistical difference (P < 0.05), but without statistical difference in the infiltration degree of CD68+. The expressions of the IFN-γ, IL-6, IL-12 in tumor-bearing groups were lower than that in the blank control group, but with higher contents of IL-4, IL-13, TGF-β. Intervened with different drugs, there were significant differences in content among some relevant cytokines (P < 0.05), as well as statistical differences among the TCM prevention group, the Chinese and western medicine group and the tumor-bearing control group (P <0. 05) , but without statistical difference in TNF-α and IL-10 content from the tumor-bearing control group (P < 0.05).. Fuzheng Sanjie recipe could reverse the immune remodeling effect and control the tumor growth by down-regulating the expressions of IL-4, IL-13, TGF-α in lung cancer immune microenvironment and up-regulating the expression of IFN-γ.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Interleukin-10; Interleukin-12; Interleukin-13; Lung Neoplasms; Macrophages; Male; Mice; Mice, Inbred C57BL; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

RNF111/Arkadia is a critical regulator of TGF-β signaling, being required for SMAD3-mediated responses such as TGF-β-induced repression of E-cadherin. Previous studies show that mutations in RNF111 in human cancers are rare and RNF111 promotes lung tumor metastasis. However, the epigenetic mechanisms underlying the role of RNF111 in non-small cell lung cancer (NSCLC) metastasis remain unknown. Here, we mainly focused on low- (95C) and high-metastatic (95D) NSCLC cell lines, which share a similar genetic background, and investigated the methylation-based regulation of RNF111 expression.. Clonal bisulfite sequencing, real-time qRT-PCR, western blot analysis, luciferase reporter assays, RNA interference, chromatin immunoprecipitation (ChIP) assay and transwell migration and invasion assays were performed on human NSCLC cell lines 95C and 95D.. RNF111 was high-expressed in 95D cells, which showed low-level methylation at -459CpG site in RNF111 promoter. The opposite results were obtained in 95C cells. Cell-based and biochemical assays revealed that -459CpG methylation can inhibit RNF111 transcriptional expression by interfering with the recruitment of Sp1 to RNF111 promoter. On TGF-β stimulation, siRNA-mediated RNF111 knockdown inhibited TGF-β/Smad signaling activity and Snail (an inducer of metastasis) expression, and enhanced E-cadherin (an epithelial-to-mesenchymal transition marker) expression in 95C and 95D cells. Furthermore, demethylation-induced upregulation of RNF111 enhanced phosphorylation of SMAD3 and Snail expression, and repressed E-cadherin expression in 95C cells expressing low RNF111.. Our results suggest that -459CpG methylation in Sp1-binding site of RNF111 promoter transcriptionally decreases RNF111 expression, which inhibits TGF-β/Smad signaling associated invasion in NSCLC cells.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Methylation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Mutation; Neoplasm Invasiveness; Nuclear Proteins; Phosphorylation; Promoter Regions, Genetic; RNA Interference; RNA, Messenger; Smad3 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Up-Regulation

TRIM66 belongs to the family of tripartite motif (TRIM)-containing proteins. Alterations in TRIM proteins have been implicated in several malignancies. This study was aimed at elucidating the expression and biological function of TRIM66 in osteosarcoma. Here, TRIM66 expression level was higher in osteosarcoma tissues than in normal tissues. High TRIM66 expression was correlated with high rate of local recurrence and lung metastasis, and short survival time. Then, we found that knockdown of TRIM66 in two osteosarcoma cell lines, MG63 and HOS, significantly inhibited cell proliferation and induced G1-phase arrest. Moreover, inhibition of TRIM66 in osteosarcoma cells significantly induced cell apoptosis, while remarkably inhibited cell migration, invasion as well as tumorigenicity in nude mice. Gene set enrichment analysis in Gene Expression Omnibus dataset revealed that apoptosis, epithelial-mesenchymal transition (EMT) and transforming growth factor-β (TGF-β) signaling pathway-related genes were enriched in TRIM66 higher expression patients, which was confirmed by western blot analysis in osteosarcoma cells with TRIM66 silenced. In conclusion, TRIM66 may act as an oncogene through suppressing apoptosis pathway and promoting TGF-β signaling in osteosarcoma carcinogenesis. TRIM66 may be a prognostic factor and potential therapeutic target in osteosarcoma.

Topics: Animals; Apoptosis; Carcinogenesis; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Oncogenes; Osteosarcoma; Retrospective Studies; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

The present study aimed to investigate the effects of the tumor suppressor gene differentially expressed in adenocarcinoma of the lung 1 (DAL‑1)/4.1B on early‑stage adenocarcinoma of the lung. The role of DAL‑1/4.1B in the epithelial‑mesenchymal transition (EMT), which is implicated in cancer metastasis, was examined using DAL‑1 knockdown and overexpression, followed by polymerase chain reaction and western blot analysis of EMT markers, as well as cell counting and cell migration/invasion assays. The results showed that DAL‑1/4.1B has a role in transforming growth factor (TGF)‑β‑induced EMT in non‑small cell lung cancer cells. Silencing of DAL‑1/4.1B with inhibitory RNAs altered the expression of numerous EMT markers, including E‑cadherin and β‑catenin, whereas overexpression of DAL‑1/4.1B had the opposite effect. In addition, DAL‑1/4.1B expression was induced following TGF‑β treatment at the protein and mRNA level. DAL‑1/4.1B deficiency impaired TGF‑β‑induced EMT and increased cell migration and invasion. These results suggested that DAL‑1/4.1B contributed to the EMT and may be important for tumor metastasis in lung cancer. Together with the results of a previous study by our group, the present study suggested that DAL‑1/4.1B acts as a tumor suppressor in the early transformation process in lung cancer, while in later stages, it functions as an oncogene affecting the biological features of human lung carcinoma cells. The results of the present study provided evidence for the feasibility of utilizing DAL‑1/4.1B as a target for lung cancer gene therapy.

Topics: Antigens, CD; beta Catenin; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Microfilament Proteins; RNA, Messenger; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.

Topics: Adult; Animals; Antigens, CD; Cadherins; Carcinoma, Adenoid Cystic; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice, Inbred NOD; Mice, SCID; Middle Aged; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; Salivary Gland Neoplasms; Signal Transduction; Snail Family Transcription Factors; Thioredoxin Reductase 1; Thioredoxins; Time Factors; Transcription Factors; Transfection; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFβ-induced EMT. Nevertheless, whether or not the blockade of TGFβ-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFβ-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFβ-induced EMT in lung cancer cells.

Topics: Blotting, Western; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Microscopy, Confocal; Phosphoprotein Phosphatases; Phosphorylation; PTEN Phosphohydrolase; Transfection; Transforming Growth Factor beta

Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.

Topics: Absorptiometry, Photon; Animals; Bone Neoplasms; Breast Neoplasms; Calcium; Calcium Signaling; Camurati-Engelmann Syndrome; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Nude; Mice, SCID; Multiple Myeloma; Muscle Contraction; Muscle Proteins; Muscle Strength; Muscle Weakness; Muscle, Skeletal; NADPH Oxidase 4; NADPH Oxidases; Neoplasms; Osteolysis; Oxidation-Reduction; Prostatic Neoplasms; Ryanodine Receptor Calcium Release Channel; Transforming Growth Factor beta; Up-Regulation; X-Ray Microtomography

Distant relapse after chemotherapy is an important clinical issue for treating breast cancer patients and results from the development of cancer stem-like cells (CSCs) during chemotherapy. Here we report that blocking epithelial-to-mesenchymal transition (EMT) suppresses paclitaxel-induced CSCs properties by using a MDA-MB-231-xenografted mice model (in vivo), and breast cancer cell lines (in vitro). Paclitaxel, one of the cytotoxic taxane-drugs such as docetaxel, increases mesenchymal markers (Vimentin and Fibronectin) and decreases an epithelial marker (Zo-1). Blocking TGF-β signaling with the TGF-β type I receptor kinase (ALK5) inhibitor, EW-7197, suppresses paclitaxel-induced EMT and CSC properties such as mammosphere-forming efficiency (MSFE), aldehyde dehydrogenase (ALDH) activity, CD44+/CD24- ratio, and pluripotency regulators (Oct4, Nanog, Klf4, Myc, and Sox2). The combinatorial treatment of EW-7197 improves the therapeutic effect of paclitaxel by decreasing the lung metastasis and increasing the survival time in vivo. We confirmed that Snail is increased by paclitaxel-induced intracellular reactive oxygen species (ROS) and EW-7197 suppresses the paclitaxel-induced Snail and EMT by attenuating paclitaxel-induced intracellular ROS. Knock-down of SNAI1 suppresses paclitaxel-induced EMT and CSC properties. These data together suggest that blocking the Snail-induced EMT with the ALK5 inhibitor attenuates metastasis after paclitaxel-therapy and that this combinatorial approach could prove useful in treating breast cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Combined Modality Therapy; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Kruppel-Like Factor 4; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Paclitaxel; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

Cullin 4A (Cul4A) promotes oncogenesis through overexpression and then ubiquitination‑mediated proteolysis of tumor suppressors in various types of cancers. Transforming growth factor β‑induced (TGFBI) has been implicated as a tumor suppressor, which enhances gemcitabine chemosensitivity in lung cancer cells. The present study aimed to investigate the association of TGFBI and Cul4A and the mechanism by which Cul4A regulates TGFBI. In addition, we also evaluated the therapeutic value of Cul4A RNAi using adenoviral transfection of Cul4A RNAi in nude mouse xenograft models. We observed that knockdown of Cul4A was associated with increased sensitivity to gemcitabine through upregulation of TGFBI in lung cancer cells. Cul4A regulated TGFBI through direct interaction and then ubiquitin‑mediated protein degradation. In the nude mouse xenograft models, adenoviral transfection of Cul4A RNAi in combination with gemcitabine chemotherapy inhibited lung cancer tumor growth. As the result, combination of Cul4A RNAi with chemotherapy may provide a new approach to lung cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Cullin Proteins; Deoxycytidine; Drug Resistance, Neoplasm; Extracellular Matrix Proteins; Gemcitabine; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Mice; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

To investigate the effect of crude antigens of Ascaris lumbricoides on the secretion of IL-6 and TGF-β of human lung adenocarcinoma cells (A549 cells), and the apoptosis of A549 cells.. Crude antigens of A. lumbricoides were prepared. A549 cells were co-cultured with 25, 125, and 500 µg/ml crude antigens of A. lumbricoides for 1, 18, and 24 h, named as low concentration group, medium concentration group, and high concentration group, respectively. Meanwhile, A549 cells were co-cultured with culture medium (negative control) and 12.5 µg/ml adriamycin (positive control). The apoptosis rate was detected by using Annexin V-FITC apoptosis detection kit. The cell changes were determined by flow cytometry. The levels of mRNA expression of IL-6 and TGF-β were detected by ELISA and real time PCR, respectively.. The apoptosis rate of A549 cells induced by crude antigens for 1, 18, and 24 h was significantly higher than that of negative control (P < 0.01). The apoptosis rate in medium concentration group (treated for 18 h) was highest [(47.10 ± 3.68)%]. After co-culture with 125 µg/ml crude antigens for 18 h, the proportion of G0/G1 phase cells increased and that of S phase cells decreased, and there was a significant difference between medium concentration group and negative control group. At the same time, the level of IL-6 increased with the increasing concentration of crude antigens. However, no significant difference was found in the level of TGF-β among the groups. In the medium concentration group, mRNA expression levels of IL-6 (5.95 ± 0.31) and TGF-β (3.43 ± 0.35) of A549 cells reached peak on the 18th hour, and were significantly higher than that of the control (P < 0.01).. The cell cycle of A549 cells is blocked in G0/G1 phase induced by crude antigens of A. lumbricoides. And the apoptosis may be related to the changes in the level of TGF-β and IL-6.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; Ascaris lumbricoides; Cell Cycle; Cell Line, Tumor; Humans; Interleukin-6; Lung Neoplasms; Transforming Growth Factor beta

Tumours frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer-testis antigens (CTAs). The extent to which CTA expression represents epiphenomena or confers tumorigenic traits is unknown. In this study, to address this, we implemented a multidimensional functional genomics approach that incorporates 7 different phenotypic assays in 11 distinct disease settings. We identify 26 CTAs that are essential for tumor cell viability and/or are pathological drivers of HIF, WNT or TGFβ signalling. In particular, we discover that Foetal and Adult Testis Expressed 1 (FATE1) is a key survival factor in multiple oncogenic backgrounds. FATE1 prevents the accumulation of the stress-sensing BH3-only protein, BCL-2-Interacting Killer (BIK), thereby permitting viability in the presence of toxic stimuli. Furthermore, ZNF165 promotes TGFβ signalling by directly suppressing the expression of negative feedback regulatory pathways. This action is essential for the survival of triple negative breast cancer cells in vitro and in vivo. Thus, CTAs make significant direct contributions to tumour biology.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antigens, Neoplasm; Apoptosis Regulatory Proteins; Carcinogenesis; Cell Line; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; DNA-Binding Proteins; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Immunoblotting; In Vitro Techniques; Lung Neoplasms; Membrane Proteins; Mice, Inbred NOD; Mitochondrial Proteins; Neoplasm Transplantation; Neoplasms; Prognosis; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad7 Protein; Transcription Factors; Transforming Growth Factor beta; Triple Negative Breast Neoplasms; Ubiquitin-Protein Ligases; Wnt Signaling Pathway

Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation on the airways, which might be mediated by Th17 cells. This inflammatory environment may contribute to tumor development. In contrast, some reports indicate that tumors may induce immunosuppressive Treg cells to dampen immune reactivity, supporting tumor growth and progression. Thus, we aimed to analyze whether chronic inflammation or immunosuppression predominates at the systemic level in lung adenocarcinoma patients, and several cytokines and Th17 and Treg cells were studied. Higher proportions of IL-17-producing CD4(+) T-cells were found in smoking control subjects and in lung adenocarcinoma patients compared to nonsmoking control subjects. In addition, lung adenocarcinoma patients increased both plasma concentrations of IL-2, IL-4, IL-6, and IL-10, and proportions of Latency Associated Peptide (LAP) TGF-β subset of CD4(+)CD25(+)CD127(-) Treg cells, which overexpressed LAP TGF-β. This knowledge may lead to the development of immunotherapies that could inhibit the suppressor activity mediated by the LAP TGF-β subset of CD4(+)CD25(+)CD127(-) Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; CD4-Positive T-Lymphocytes; Female; Humans; Immune Tolerance; Inflammation; Interleukin-10; Interleukin-17; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Interleukin-6; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Peptides; Protein Precursors; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

Topics: Animals; Axl Receptor Tyrosine Kinase; Benzocycloheptenes; Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Lung Neoplasms; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Metastasis; Neoplastic Stem Cells; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; RNA Interference; Signal Transduction; Smad Proteins; Thrombospondins; Transforming Growth Factor beta; Transplantation, Heterologous; Triazoles

Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFβ1 mRNA, and addition of exogenous TGFβ1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFβR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFβ signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.

Topics: Animals; Bronchioles; Cells, Cultured; Disease Models, Animal; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Inhibitor of Differentiation Proteins; Lung Neoplasms; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Protein Kinase C-alpha; Proto-Oncogene Proteins p21(ras); Pulmonary Alveoli; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stem Cells; Transforming Growth Factor beta; WT1 Proteins

Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intraepithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF)β pathway in prostate epithelial cells. TGFβ signaling is known to have a tumor suppressive role in many cancer types, and reduced expression of TGFβ receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten or expression of constitutively active AKT1, inactivation of TGFβ signaling by deletion of the TGFβ type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFβ signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFβ signaling by Pten loss or AKT activation suggests that the same signaling events that have key roles in tumor initiation also induce the activity of a pathway that restrains disease progression.

Topics: Adenocarcinoma; Animals; Disease Progression; Epithelial Cells; Gene Deletion; Homozygote; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Neoplasm Invasiveness; Prostate; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

S100 family of calcium-binding proteins is commonly upregulated in a variety of tumor types and is often associated with tumor progression. Among several S100 members, altered expression of S100A2 is a potential diagnostic and prognostic marker in cancer. Several reports suggest a role for S100A2 in metastasis. Earlier, our studies established regulation of S100A2 by transforming growth factor-β (TGF-β) and its involvement in TGF-β-mediated cancer cell invasion and migration. However, the molecular mechanisms of S100A2 protumorigenic actions remain unexplored. In the present study, we demonstrate that overexpression of S100A2 in A549 lung cancer cells induced epithelial-mesenchymal transition (EMT) followed by increased invasion, loose colony morphology in soft agar and enhanced Akt phosphorylation (Ser-473). Furthermore, overexpression of S100A2 led to increased tumor growth in immunocompromised mice. In agreement, immunohistochemical examination of resected xenograft tumors established inverse correlation between S100A2 and E-cadherin expression together with activated Akt signaling. Interestingly, our study demonstrates a strong dependence of S100A2 and Smad3 in TGF-β-induced Hep3B cell EMT and invasion. Most importantly, we demonstrate that these effects of S100A2 are manifested through functional interaction with Smad3, which is enhanced in the presence of high calcium and TGF-β. S100A2 stabilizes Smad3 and binds to its C-terminal MH2 domain. Additionally, loss of S100A2 attenuates the transcription of TGF-β/Smad3 target genes involved in tumor promotion, such as PA1-1 and vimentin. Collectively, our findings present the first mechanistic details of S100A2 protumorigenic actions and its involvement in TGF-β-mediated cancer cell invasion and EMT.

Topics: Animals; Calcium; Cell Line, Tumor; Cell Proliferation; Chemotactic Factors; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Nude; Phosphatidylinositol 3-Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; S100 Proteins; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

Overcoming cellular mechanisms of de novo and acquired resistance to drug therapy remains a central challenge in the clinical management of many cancers, including non-small cell lung cancer (NSCLC). Although much work has linked the epithelial-mesenchymal transition (EMT) in cancer cells to the emergence of drug resistance, it is less clear where tractable routes may exist to reverse or inhibit EMT as a strategy for drug sensitization. Here, we demonstrate that extracellular signal-regulated kinase (ERK) 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) signaling plays a key role in directing the mesenchymal character of NSCLC cells and that blocking ERK signaling is sufficient to heighten therapeutic responses to EGF receptor (EGFR) inhibitors. MEK1/2 (MAPKK1/2) inhibition promoted an epithelial phenotype in NSCLC cells, preventing induction of EMT by exogenous TGF-β. Moreover, in cells exhibiting de novo or acquired resistance to the EGFR inhibitor gefitinib, MEK inhibition enhanced the sensitivity to gefitinib and slowed cell migration. These effects only occurred, however, if MEK was inhibited for a period sufficient to trigger changes in EMT marker expression. Consistent with these findings, changes in EMT phenotypes and markers were also induced by the expression of mutant KRAS in a MEK-dependent manner. Our results suggest that prolonged exposure to MEK or ERK inhibitors may not only restrain EMT but also overcome naïve or acquired resistance of NSCLC to EGFR-targeted therapy in the clinic.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Transforming Growth Factor beta

Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to the upregulation of secreted protein, acidic and rich in cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates transforming growth factor beta (TGF-β), extracellular signal-regulated kinase (ERK)1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-β receptor 2 (TGF-βR2) are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulates SPARC expression. Inhibition of MAP-ERK kinase (MEK) phosphorylation downregulates SPARC. MiR-29b is downregulated in Snail-overexpressing cell lines, whereas overexpression of miR-29b inhibits SPARC expression. In addition, miR-29b is upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.

Topics: Bronchi; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Progression; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; Osteonectin; Phosphorylation; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

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

Umbelliprenin is a member of the 7-prenyloxycoumarins with potential therapeutic properties such as cytotoxic effects on various cancer cells. The present study investigates the effect of umbelliprenin on predominance of Th1 and Th2 responses in Lewis lung cancer (LLC) mouse model. The cytotoxic effect of umbelliprenin was explored on LLC cells and mouse splenocytes by MTT assay. Mice into which LLC had been transplanted were treated with umbelliprenin on alternate days, at 2.5 mg/200 µl intraperitoneally. Foxp3, TNF-α and TGF-β mRNA expressions were assessed in tumor and lung tissues of LLC mice. In addition, IL-10, IFN-γ and IL-4 levels were determined in sera and also in splenocyte culture supernatants at the presence of tumor cell lysate (10 µg/ml) and Con A (3 µg/ml) after 72 h. Results showed the cytotoxic effects of umbelliprenin on LLC cells (IC₅₀ = 51.6 ± 5.4 µM) while no adverse effect was seen at this concentration on normal splenocytes. TNF-α mRNA expression in both lung and tumor tissues was increased. However, Foxp3 and TGF-β expressions were decreased in tumor tissues. Serum level of IFN-γ was elevated in the umbelliprenin treated cancerous mice compared to the control group while IL-10 and IL-4 secretions were reduced. Tumor size was also decreased in umbelliprenin treated group. In summary, umbelliprenin has shown a partially Th1 bias with a reduction of regulatory immune response. Although the mechanism behind this action is not known, it is speculated that upon changing the Th1/Th2 balance in favour of Th1, umbelliprenin induces its antitumor activity.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Female; Forkhead Transcription Factors; Interferon-gamma; Interleukin-10; Lung Neoplasms; Mice; Neoplasm Proteins; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbelliferones

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

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

TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as a direct target gene of transforming growth factor-β (TGF-β)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF-β/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI-H23, and RERF-LC-KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF-β receptor kinase antagonist, SB208, and by TGF-β neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF-β stimulation. Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-β/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI-H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD-SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Cell Line, Tumor; Female; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; Smad Proteins; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Burden

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

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

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

Epithelial-mesenchymal transition (EMT), a key step in the early stages of cancer metastasis, is orchestrated by several signaling pathways, including IL-6/JAK/STAT3 and TGF-β/Smad signaling. However, an association between the two signaling pathways during the EMT process is largely unknown. Here, we focused on lung cancer and demonstrated that TGF-β1 induced the phosphorylation of Smad3 (p-Smad3), upregulation of Snail, a fibroblast-like morphology, and downregulation of E-cadherin as well as upregulation of vimentin in lung cancer cell lines. SIS3 (an inhibitor of Smad3) suppressed TGF-β1-induced activation of Smad3, upregulation of Snail and the EMT process. Importantly, the JAK2/STAT3-specific inhibitor AG490 blocked Stat3 phosphorylation, resulting in attenuated levels of TGF-β1-induced p-Smad3, Snail, MMP2, and Smad-mediated PAI-1 promoter reporter gene activity in A549 and H1650 cells. Subsequently, AG490 inhibited TGF-β-induced cell migration and invasion. Moreover, exogenous IL-6 treatment stimulated Stat3 activation, enhanced TGF-β-induced expression of p-Smad3 and Snail, aggravated the EMT process, and increased lung cancer cell migration and invasion induced by TGF-β1. Our findings show that the JAK/STAT3 pathway is required for TGF-β-induced EMT and cancer cell migration and invasion via upregulation of the expression of p-Smad3 and Snail, and the IL-6/JAK/STAT3 and TGF-β/Smad signaling synergistically enhance EMT in lung carcinomas. The present study suggests a novel rationale for inhibiting cancer metastasis using anti-IL-6/JAK/STAT3 and anti-TGF-β/Smad therapeutic strategies.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Janus Kinases; Lung Neoplasms; Neoplasm Invasiveness; Phosphorylation; Signal Transduction; Smad3 Protein; Snail Family Transcription Factors; STAT3 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Tyrphostins

Metastatic lung cancer is one of the leading causes of cancer death. In recent years, epithelial-to-mesenchymal transition (EMT) has been found to contribute to metastasis, as it enables migratory and invasive properties in cancer cells. Previous genome-wide studies found that DNA methylation was unchanged during EMT induced by TGF-β in AML12 cells. In this study, we aimed to discover EMT-related changes in DNA methylation in cancer cells, which are poorly understood.. We employed a next-generation sequencing-based method, MSCC (methyl-sensitive cut counting), to investigate DNA methylation during EMT in the A549 lung cancer cell line.. We found that methylation levels were highly correlated to gene expression, histone modifications and small RNA expression. However, no differentially methylated regions (DMRs) were found in A549 cells treated with TGF-β for 4 h, 12 h, 24 h and 96 h. Additionally, CpG islands (CGIs) showed no overall change in methylation levels, and at the single-base level, almost all of the CpGs showed conservation of DNA methylation levels. Furthermore, we found that the expression of DNA methyltransferase 1, 3a, 3b (DNMT1, DNMT3a, DNMT3b) and ten-eleven translocation 1 (TET1) was altered after EMT. The level of several histone methylations was also changed.. DNA methylation-related enzymes and histone methylation might have a role in TGF-β-induced EMT without affecting the whole DNA methylome in cancer cells. Our data provide new insights into the global methylation signature of lung cancer cells and the role of DNA methylation in EMT.. The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1112892497119603.

Topics: Cell Line, Tumor; CpG Islands; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histones; Humans; Lung Neoplasms; Methylation; Mixed Function Oxygenases; Neoplasm Invasiveness; Proto-Oncogene Proteins; RNA, Neoplasm; Time Factors; Transforming Growth Factor beta

Identification of target cells in lung tumorigenesis and characterization of the signals that control their behavior is an important step toward improving early cancer diagnosis and predicting tumor behavior. We identified a population of cells in the adult lung that bear the EpCAM+CD104+CD49f+CD44+CD24loSCA1+ phenotype and can be clonally expanded in culture, consistent with the properties of early progenitor cells. We show that these cells, rather than being restricted to one tumor type, can give rise to several different types of cancer, including adenocarcinoma and squamous cell carcinoma. We further demonstrate that these cells can be converted from one cancer type to the other, and this plasticity is determined by their responsiveness to transforming growth factor (TGF)-beta signaling. Our data establish a mechanistic link between TGF-beta signaling and SOX2 expression, and identify the TGF-beta/SMAD/SOX2 signaling network as a key regulator of lineage commitment and differentiation of lung cancer cells.

Topics: Animals; Cell Differentiation; Cell Lineage; Humans; Lung Neoplasms; Mice; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

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

Cancer tissues are comprised of various components including tumor cells and the surrounding tumor stroma, which consists of the extracellular matrix and inflammatory cells. Since the tumor stroma plays critical roles in tumor development, investigation of the tumor stroma in addition to tumor cells is important to identify useful tumor-associated markers. To discover novel and useful sero-diagnostic markers, a comparative study of tumor-associated autoantibodies (AAbs) in sera from lung adenocarcinoma (AC) patients was investigated by two-dimensional immunoblotting with AC cell lines or each autologous AC tissues. Autoantigens identified from tissue and cell line samples comprised 58 (45 antigens) and 53 spots (41 antigens), respectively. Thirty-six proteins including Transforming growth factor-beta-induced protein ig-h3 (BIGH3) and Hyaluronan and proteoglycan link protein 1 (HAPLN1) were detected only from tissues, 32 proteins only from cell lines, and 9 proteins from both. BIGH3 and HAPLN1 expressions were confirmed in the tumor stroma, but not in AC cell lines by immunostaining and immunoblotting. These data suggest that autologous tumor tissue and serum are important to coincidently detect AAbs derived from the tumor stroma in addition to tumor cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Annexin A2; Antigens, Neoplasm; Autoantibodies; Autoantigens; Biomarkers, Tumor; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Extracellular Matrix Proteins; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Microfilament Proteins; Middle Aged; Neoplasms; Nuclear Proteins; Proteoglycans; Transforming Growth Factor beta

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 migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non-small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non-small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration.

Topics: Actins; Adenocarcinoma; Apoptosis; Blotting, Western; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cells, Cultured; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Immunoprecipitation; Lung Neoplasms; Microscopy, Fluorescence; Phenanthrenes; Stress Fibers; Transforming Growth Factor beta

A post-transcriptional pathway by which TGF-β modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-β for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-β induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-β treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-β-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma.

Topics: Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Focal Adhesion Protein-Tyrosine Kinases; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Lung Neoplasms; Phosphorylation; Protein Isoforms; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Transforming Growth Factor beta

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

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor β (TGFβ)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFβ-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFβ-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; MicroRNAs; Organ Specificity; Pancreatic Neoplasms; Phenotype; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

TGF-β signaling plays an important role in breast cancer progression and metastasis. Epithelial-mesenchymal transition (EMT) is an important step in the progression of solid tumors to metastatic disease. We previously reported that IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppressed renal fibrosis in obstructive nephropathy (Moon et al., 2006). Here, we show that IN-1130 suppressed EMT and the lung metastasis of mammary tumors in mouse models. Treating human and mouse cell lines with IN-1130 inhibited TGF-β-mediated transcriptional activation, the phosphorylation and nuclear translocation of Smad2, and TGF-β-induced-EMT, which induces morphological changes in epithelial cells. Additionally, we demonstrated that IN-1130 blocked TGF-β-induced 4T1 mammary cancer cell migration and invasion. The TGF-β-mediated increase in matrix metalloproteinase (MMP)-2 and MMP-9 expression was restored by IN-1130 co-treatment with TGF-β in human epithelial cells and in 4T1 cells. Furthermore, we found that lung metastasis from primary breast cancer was inhibited by IN-1130 in both 4T1-xenografted BALB/c mice and MMTV/c-Neu transgenic mice without any change in primary tumor volume. IN-1130 prolonged the life span of tumor-bearing mice. In summary, this study indicated that IN-1130 has therapeutic potential for preventing breast cancer metastasis to the lung.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Nucleus; Drug Screening Assays, Antitumor; Epithelial-Mesenchymal Transition; Female; Humans; Imidazoles; Lung; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Transplantation; Protein Serine-Threonine Kinases; Protein Transport; Quinoxalines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Burden

The change of oligosaccharide structure has been revealed to be crucial for glycoproteins' biological functions and cell biological characteristics. N-acetylglucosaminy transferase V (GnT-V), a key enzyme catalysing the reaction of adding β1, 6-N-acetylglucosamine (GlcNAc) on asparagine-linked oligosaccharides of cell proteins, has been implicated to a metastastic-promoting oncoprotein in some carcinomas. However, this correlation might not be subjected to all types of cancers, for example, in non-small cell lung cancers, low level of GnT-V expression is associated with relatively short survival time and poor prognosis. To explain the role of GnT-V in lung cancer progression, we studied the association of GnT-V expression with lung cancer EMT behaviour. We found that GnT-V expression was correlated with epithelial marker positively and mesenchymal marker negatively. GnT-V levels, as well as β1,6-GlcNAc branched N-glycans, were strongly reduced in TGF-β1-induced EMT of human lung adenocarcinoma A549 cells. Further studies showed that suppression of β1,6-GlcNAc branched N-glycans by inhibitor or GnT-V silencing in A549 cells could promote TGF-β1-induced EMT-like changes, cell migration and invasion. Meanwhile, overexpression of GnT-V impaired TGF-β1-induced EMT, migration and invasion. It suggests that GnT-V suppresses the EMT process of lung cancer cells through inhibiting the TGF-β/Smad signalling and its downstream transcription factors in a GnT-V catalytic activity-dependent manner. Taken together, the present study reveals a novel mechanism of GnT-V as a suppressor of both EMT and invasion in human lung cancer cells, which may be useful for fully understanding N-glycan's biological roles in lung cancer progression.

Topics: Acetylglucosamine; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cells, Cultured; Epithelial-Mesenchymal Transition; Flow Cytometry; Fluorescent Antibody Technique; HEK293 Cells; Humans; Immunoenzyme Techniques; Luciferases; Lung Neoplasms; Mesoderm; N-Acetylglucosaminyltransferases; Polysaccharides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signaling plays important roles in embryogenesis and tumorigenesis by controlling cell growth, differentiation and migration. The transmembrane prostate androgen-induced protein (TMEPAI) is elevated in several cancers. TMEPAI expression is induced by TGF-β signaling, and in turn, expression of TMEPAI negatively regulates TGF-β signaling, but the molecular mechanisms of TMEPAI induced TGF-β signaling inhibition are not well understood. Here we report that TMEPAI is localized to the lysosome and late endosome, and that association of TMEPAI with the E3 ubiquitin ligase Nedd4 is required for its transport to the lysosome. TMEPAI associates with the TGF-β type I receptor (TβRI) and promotes its degradation in the lysosome. Depletion of TMEPAI in A549 lung cancer cells inhibits cell proliferation, migration and invasion, while TMEPAI expression in nude mice promotes tumorigenesis. These results reveal a novel function for TMEPAI in regulating TGF-β signaling through the modulation of TβRI levels, which has important implications for cancer development in vivo.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Endosomal Sorting Complexes Required for Transport; Endosomes; HEK293 Cells; Humans; Lung Neoplasms; Lysosomes; Membrane Proteins; Mice; Mice, Nude; Nedd4 Ubiquitin Protein Ligases; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous; Ubiquitin-Protein Ligases

In previous work, we demonstrated that transcription factor Trim28 (Tripartite motif containing 28) plays a tumor-suppressor role in early-staged adenocarcinoma of the lung due to its ability to restrain transcription of cell cycle-regulating genes. Herein we examine Trim28's role in the epithelial-to-mesenchymal transition (EMT) which is strongly implicated in cancer metastasis. We found that Trim28 plays a role in TGF-β-induced EMT in non-small cell lung cancer cells. Silencing Trim28 with inhibitory RNAs alters the expression of numerous EMT markers, such as E-cadherin and N-cadherin, whereas overexpression of Trim28 has an opposite effect. Trim28 expression is induced following TGF-β treatment at both protein and mRNA levels. Trim28 deficiency impairs TGF-β-induced EMT and decreases cell migration and invasion. Finally, we demonstrate that the expression of Trim28 affects the acetylation and methylation of histones on E-cadherin and N-cadherin promoters. These results suggest that Trim28 contributes to EMT and might be important for tumor metastasis in lung cancer. Taken together with our previous work these results suggest a model in which Trim28 is a tumor suppressor early in the transformation process in lung cancer, but in later stages it functions as an oncogene.

Topics: Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Repressor Proteins; Transforming Growth Factor beta; Tripartite Motif-Containing Protein 28

Most lung cancer (LC) patients have metastatic disease at time of diagnosis, which influence the treatment regimen and is the most important prognostic factor. The main purpose of our study was to evaluate the relationship between cell proliferation (Ki-67 label index), p53, transforming growth factor-β (TGF-β) and lysyl oxidase (LOX), and the metastatic stages of different lung cancers. The secondary aim was to correlate these parameters with the standardized uptake value (SUVmax) of the primary lesion during positron emission tomography-computed tomography (PET-CT).. Eighty-five treatment-naive patients with LC were enrolled. All patients were examined with PET-CT. Ki-67, p53, TGF-β and LOX were evaluated histopathologically.. Small cell lung cancer (SCLC) showed the most intense staining in all parameters. A well-differentiated adenocarcinoma (AC) demonstrated a more diffuse and intense staining than squamous cell carcinoma (SCC). There was no statistically significant relationship between the four parameters and metastases of SCLC and SCC. However, a significant relationship between TGF-β, LOX and metastatic AC was demonstrated with regards to diffusivity and intensity. p53 and Ki-67 did not show a significant relationship. No correlation between SCLC and SCC and SUVmax was found. However, in AC, the diffusivity and intensity of the LOX and p53 staining showed a statistically significant relationship to the SUVmax.. LOX and TGF-β may play roles in metastatic AC. LOX and TGF-β may become markers of metastatic disease and inhibition could be explored for treatment.

Topics: Carcinoma, Non-Small-Cell Lung; Cohort Studies; Female; Fluorodeoxyglucose F18; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Multimodal Imaging; Positron-Emission Tomography; Protein-Lysine 6-Oxidase; Radiopharmaceuticals; Small Cell Lung Carcinoma; Tomography, X-Ray Computed; Transforming Growth Factor beta; Tumor Suppressor Protein p53

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

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-β and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.

Topics: Animals; Breast Neoplasms; Chemokine CXCL12; DNA-Binding Proteins; Fibroblasts; Heat Shock Transcription Factors; Heterografts; Humans; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Transcription Factors; Transforming Growth Factor beta

Phenotype reprogramming during transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) is an extensive and dynamic process, orchestrated by the integration of biological signaling across multiple time scales. As part of the numerous transcriptional changes necessary for EMT, TGFβ-initiated Smad3 signaling results in remodeling of the redox environment and decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated activity through antioxidants, the possibility of a positive feedback loop exists, albeit the time scales on which these mechanisms operate are quite different. We hypothesized that the decreased nucleophilic tone acquired during EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Previous findings supporting such a mechanism were characterized independent of each other; we sought to investigate these relationships within a singular experimental context. In this study, we characterized multivariate representations of phenotype as they evolved over time, specifically measuring expression of epithelial/mesenchymal differentiation, redox regulators, and Smad transcription factors. In-cell Western (ICW) assays were developed to evaluate multivariate phenotype states as they developed during EMT. Principal component analysis (PCA) extracted anticorrelations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected a compensatory up-regulation of Smad2 and Smad4 following cessation of TGFβ signaling. Measuring transcript expression following EMT, we identified down-regulation of numerous antioxidant genes concomitant with up-regulation of NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGFβ treatment increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased glutathione redox potential. Our findings suggest that the decreased nucleophilic tone during EMT coincides with the acquisition of a mesenchymal phenotype over too long a time scale to enable enhanced Smad3 phosphorylation during initiation of EMT. We further challenged the mesenchymal phenotype following EMT through antioxidant and TGFβ inhibitor treatments, which failed to induce a mesenchymal-epithelial transition (MET). Our characterization of multivariate phenotype dynamics during EMT indicates that the decrease in nucleophilic tone occurs alongside EMT; however, maintenance of the mesenchymal phenotype following EMT is independent of both the nascent

Topics: Blotting, Western; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Fluorescence; Glutathione; Humans; Hydrogen Peroxide; Luminol; Lung Neoplasms; NADPH Oxidase 4; NADPH Oxidases; Oxidation-Reduction; Smad2 Protein; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastasis is present at diagnosis. Because transforming growth factor-β (TGFβ) has been shown to promote metastasis in many solid tumors, we investigated the effect of the natural TGFβ/Smad signaling inhibitor Smad7 and the TβRI inhibitor SD-208 on osteosarcoma behavior.. By using a mouse model of osteosarcoma induced by paratibial injection of cells, we assessed the impact of Smad7 overexpression or SD-208 on tumor growth, tumor microenvironment, bone remodeling, and metastasis development.. First, we demonstrated that TGFβ levels are higher in serum samples from patients with osteosarcoma compared with healthy volunteers and that TGFβ/Smad3 signaling pathway is activated in clinical samples. Second, we showed that Smad7 slows the growth of the primary tumor and increases mice survival. We furthermore demonstrated that Smad7 expression does not affect in vitro osteosarcoma cell proliferation but affects the microarchitectural parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of osteolytic factors such as RANKL, suggesting that Smad7 overexpression affects the "vicious cycle" established between tumor cells and bone cells by its ability to decrease osteoclast activity. Finally, we showed that Smad7 overexpression in osteosarcoma cells and the treatment of mice with SD208 inhibit the development of lung metastasis.. Taken together, these results demonstrate that the inhibition of the TGFβ/Smad signaling pathway may be a promising therapeutic strategy against tumor progression of osteosarcoma, specifically against the development of lung metastasis.

Topics: Animals; Bone Neoplasms; Bone Resorption; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; Heterografts; Humans; Lung Neoplasms; Mice; Osteoclasts; Osteosarcoma; Pteridines; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Tumor Burden

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

Signaling pathway(s) responsible for transforming growth factor β (TGFβ)-induced epithelial mesenchymal transition (EMT), invasion and migration of H460 cells (non-small cell lung cancer/NSCLC) was identified in the study. The results showed that TGFβ-induced p(38)/β-catenin/PPARγ signaling pathway played a critical role in the promotion of EMT, invasion and migration of H460 cells. All these pathological outcomes attributed to PPARγ-increased expression of p-EGFR, p-c-MET and Vimentin and the decrease of E-cadherin. Transforming growth factor β and p(38)-induced β-catenin not only stimulated the expression of PPARγ but also physically interacted with it. Blocking the ligand binding domain of PPARγ (with GW9662) could significantly interfere the binding between PPARγ and β-catenin, and interrupt the nuclear infiltration of both factors. These findings suggested that β-catenin was an upstream regulator and a ligand of PPARγ, and the binding between these two molecules was critical for their nuclear infiltration. Transforming growth factor β-induced tumor invasion and migration was also seen in U373 cells (brain glioma, with high inducible PPARγ) in a PPARγ-dependent manner, but not in CH27 cells (squamous NSCLC, with low PPARγ). PPARγ shRNA, GW9662, JW67 and 2,4-diaminoquinazoline were all revealed to have important values in the control of the intrinsic and TGFβ-induced EMT, tumor invasion and migration of H460 cells. The results further suggested that PPARγ and β-catenin may be the potential markers for the early diagnosis and/or treatment of metastatic tumors.

Topics: Apoptosis; beta Catenin; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Humans; Immunoprecipitation; Lung Neoplasms; p38 Mitogen-Activated Protein Kinases; PPAR gamma; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

The "seed and soil" hypothesis emphasizes the importance of interactions between tumor cells and their microenvironment. CAFs (Cancer associated fibroblasts) are important components of the tumor microenvironment. They were widely involved in cancer cells growth and metastasis. Fibroblasts may also play a role in inflammatory disease. The phenotype conversion of fibroblasts in lung diseases has not been investigated previously. We hypothesized that fibroblasts phenotypes may vary among different types of lung disease.. The study included six types of lung tissues, ranging from normal lung to lung adenocarcinoma with lymphatic metastasis. Para-carcinoma tissues which were 2-cm-away from the tumor focus were also included in the analysis. The expression of target proteins including alpha-SMA (smooth muscle actin), FAP (fibroblast activation protein), vimentin, E-cadherin, and CK-19 (cytokeratin-19) were examined by immunohistochemistry. TGF-beta(transforming growth factor) and Twist were detected simultaneously in all samples.. A progressive increase in the levels of alpha-SMA, vimentin and CK-19 was observed in correlation to the degree of malignancy from normal lung tissue to lung adenocarcinoma with lymphatic metastasis, whereas E-cadherin expression showed the opposite trend. TGF-beta and Twist were detected in cancer tissues and inflammatory pseudotumors. None of the proteins were detected in para-carcinoma tissues.. Fibroblast phenotypes varied according to the type and degree of lung malignancy and fibroblasts phenotypic conversion occurs as a gradual process with specific spatiotemporal characteristics. Similar fibroblast phenotypes in inflammatory diseases and cancer tissues suggested a correlation between inflammation and cancer and implied a common mechanism underlying the formation of fibroblasts in inflammatory diseases and lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adolescent; Adult; Aged; Cadherins; Endopeptidases; Female; Fibroblasts; Gelatinases; Humans; Immunohistochemistry; Lung; Lung Diseases; Lung Neoplasms; Lymphatic Metastasis; Male; Membrane Proteins; Middle Aged; Nuclear Proteins; Phenotype; Serine Endopeptidases; Transforming Growth Factor beta; Twist-Related Protein 1; Young Adult

Transforming growth factor beta (TGFβ) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFβ receptor (TβRII) for signaling. Previously, we have shown that deletion of TβRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFβ signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFβ signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.. All studies were performed on MMTV-Neu mice with and without dominant-negative TβRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.. Attenuated TGFβ signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.. Our studies provide insights into a novel mechanism by which epithelial TGFβ signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFβ pathway in vivo during tumor progression remain controversial. The targeting of TGFβ signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFβ signaling.

Topics: Animals; Carcinogenesis; Chemokines; Female; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice, Transgenic; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The chemokine CXCL1 has been reported to be expressed in lung airway epithelium and non-small cell lung cancer biopsy specimens. In this study, we investigated the effects of TNF-α, an abundant cytokine detected in inflammation and various cancers, on CXCL1 release by human A549 lung carcinoma epithelial cells.. CXCL1 expression was determined by ELISA and RT-PCR. TNF-α signaling was examined by western blotting. Monocyte migration was assayed by a Transwell migration system.. TNF-α stimulated CXCL1 release and mRNA expression, and this release was inhibited by inhibitors of JNK, p38 MAPK, PI-3K/Akt and AP-1 transcription factor. TNF-α treatment was followed by JNK, p38 MAPK and PI3K/Akt activation. However, only the JNK inhibitor could reduce the CXCL1 mRNA level, suggesting that JNK is required mainly for CXCL1 mRNA synthesis, whereas p38 MAPK and PI-3K/Akt might be responsible for CXCL1 secretion. Dexamethasone (dex) and TGF-β reduced CXCL1 secretion, with dex upregulating the expression of MAP kinase phosphatase-1 and TGF-β causing smad2/3 activation and nuclear translocation. A functional analysis showed that the released CXCL1 enhanced monocyte migration and could be abolished by a CXCL1 neutralizing antibody and CXCR antagonist.. We demonstrate that TNF-α induces CXCL1 expression through the JNK, p38 MAPK and PI-3K/Akt signaling pathways in human pulmonary epithelial cells.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Chemokine CXCL1; Epithelial Cells; Humans; JNK Mitogen-Activated Protein Kinases; Lung; Lung Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcription Factor AP-1; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Although tumor cell lysate (TCL) is a type of immunocyte stimulator, its immunosuppressive function must not be ignored. The present study reported that TCL prepared from a Lewis lung cancer cell was able to induce the development of immunosuppressive macrophages (MΦ) and tolerogenic dendritic cells. In addition, TCL upregulated the expression of CD69 in mouse splenocytes, and cell apoptosis and the percentage of regulatory T cells in mouse splenocytes simultaneously increased. Furthermore, the present study found that the immunosuppressive factor, hyaluronan, and the apoptosis inducers, Fas ligand and transforming growth factor-β, are present in TCL. These components may be associated with the emergence of immunosuppressive cells or splenocyte apoptosis. Thus, the present study has enriched our understanding of the composition of TCL and its negative regulatory effect on immunocytes.

Topics: Animals; Apoptosis; Cell Line, Tumor; Dendritic Cells; Fas Ligand Protein; Female; Hyaluronic Acid; Immune Tolerance; Immunosuppression Therapy; Lung Neoplasms; Macrophages; Mice; Mice, Inbred C57BL; Spleen; T-Lymphocytes; Transforming Growth Factor beta

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

Topics: Animals; beta Catenin; Blotting, Western; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation; Humans; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice; Mice, Nude; Nuclear Proteins; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Wnt3A Protein; Xenograft Model Antitumor Assays; Zinc Finger Protein Gli2

Regulatory T cells (Tregs) and B cells (Bregs) play an important role in the development of lung cancer. The present study aimed to investigate the phenotype of circulating Tregs and Bregs in patients with lung cancer and explore potential mechanism by which lung cancer cells act on the development of both.. Patients with lung cancer (n = 268) and healthy donors (n = 65) were enrolled in the study. Frequencies of Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD45RA, CD19, CD24, CD27 and IL-10 before and after co-cultures. qRT-PCR was performed to evaluate the mRNA levels of RANTES, MIP-1α, TGF-β, IFN-γ and IL-4.. We found a lower frequency of Tregs and a higher frequency of Bregs in patients with lung cancer compared to healthy donors. Co-culture of lung cancer cells with peripheral blood mononuclear cells could polarize the lymphocyte phenotype in the similar pattern. Lipopolysaccharide (LPS)-stimulated lung cancer cells significantly modulated regulatory cell number and function in an in vitro model.. We provide initial evidence that frequencies of peripheral Tregs decreased or Bregs increased in patients with lung cancer, which may be modulated directly by lung cancer cells. It seems cancer cells per se plays a crucial role in the development of tumor immunity.

Topics: B-Lymphocytes, Regulatory; Cell Line, Tumor; Chemokine CCL3; Coculture Techniques; Humans; Interferon-gamma; Interleukin-4; Lipopolysaccharides; Lung Neoplasms; Lymphocyte Activation; Lymphocyte Count; NF-kappa B; RNA, Messenger; Signal Transduction; Tissue Donors; Transforming Growth Factor beta

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

Topics: Aniline Compounds; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dasatinib; Drug Resistance, Neoplasm; Humans; Intracellular Space; Lung Neoplasms; Molecular Docking Simulation; Nitriles; Protein Conformation; Protein Serine-Threonine Kinases; Pyrimidines; Quinolines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Thiazoles; Transforming Growth Factor beta

Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of JARID2 was increased during TGF-ß-induced EMT of these cell lines and knockdown of JARID2 inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. JARID2 knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB family and microRNA-200 family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of CDH1 and microRNA-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.

Topics: Antigens, CD; Cadherins; Colonic Neoplasms; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Histones; HT29 Cells; Humans; Lung Neoplasms; MicroRNAs; Polycomb Repressive Complex 2; Transforming Growth Factor beta

The establishment of lethal metastases depends on the capacity of a small number of cancer cells to regenerate a tumor after entering a target organ. Stankic and colleagues have identified a role for the inhibitor of differentiation protein, ID1, as a critical regulator of breast cancer stem-like properties and metastatic colonization. Under the control of tumor growth factor-beta signaling, ID1 induces mesenchymal-epithelial transition at the metastatic site by antagonizing the activity of the basic helix-loop-helix transcription factor Twist1. This study sheds light on mechanisms that initiate metastatic outgrowth, and strengthens the concept that epithelial-mesenchymal plasticity is crucial at different stages of metastasis.

Topics: Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; Nuclear Proteins; Transforming Growth Factor beta; Twist-Related Protein 1

EMT (epithelial-mesenchymal transition) is crucial for cancer cells to acquire invasive phenotypes. In A549 lung adenocarcinoma cells, TGF-β elicited EMT in Smad-dependent manner and TNF-α accelerated this process, as confirmed by cell morphology, expression of EMT markers, capacity of gelatin lysis and cell invasion. TNF-α stimulated the phosphorylation of Smad2 linker region, and this effect was attenuated by inhibiting MEK or JNK pathway. Comprehensive expression analysis unraveled genes differentially regulated by TGF-β and TNF-α, such as cytokines, chemokines, growth factors and ECM (extracellular matrices), suggesting the drastic change in autocrine/paracrine signals as well as cell-to-ECM interactions. Integrated analysis of microRNA signature enabled us to identify a subset of genes, potentially regulated by microRNAs. Among them, we confirmed TGF-β-mediated induction of miR-23a in lung epithelial cell lines, target genes of which were further identified by gene expression profiling. Combined with in silico approaches, we determined HMGN2 as a downstream target of miR-23a. These findings provide a line of evidence that the effects of TGF-β and TNF-α were partially mediated by microRNAs, and shed light on the complexity of molecular events elicited by TGF-β and TNF-α.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Computer Simulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HMGN2 Protein; Humans; Lung Neoplasms; MicroRNAs; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Changes to innate cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), during aging in healthy or tumor-bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6-8 weeks) C57BL/6J mice to those from healthy geriatric (24-28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co-expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF-β in response to IL-4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell-derived supernatants. While both supernatants skewed macrophages toward the M2-phenotype regardless of age, only geriatric-derived macrophages produced IL-4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric- and young-derived macrophages induced allogeneic T-cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T-cell IFN-γ production. We examined the potential of an IL-2/agonist anti-CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL-2-/CD40-activated macrophages rescued T-cell production of IFN-γ in geriatric mice. Therefore, targeting macrophages with IL-2/anti-CD40 antibody may improve innate and T-cell immunity in aging hosts.

Topics: Aging; Animals; CD4-Positive T-Lymphocytes; CD40 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Culture Media, Conditioned; CX3C Chemokine Receptor 1; Immunity, Innate; Interferon-gamma; Interleukin-2; Interleukin-4; Lung Neoplasms; Lymphocyte Activation; Macrophages; Mesothelioma; Mice; Mice, Inbred C57BL; Receptors, Chemokine; Transforming Growth Factor beta; Tumor Microenvironment

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

The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. Transforming growth factor-βeta (TGF-β) and Chemokine (C-X-C Motif) Ligand-1 (CXCL1) are cytokines involved in the colonization of distant sites by CTCs in several pre-clinical animal models. However, their role is poorly-investigated in patients with metastatic cancer. Here, we investigated whether circulating levels of TGF-β and CXCL1 are predictors of CTC seeding in preferential distant sites in patients with metastatic breast cancer.. CTCs were isolated from the peripheral blood of 61 patients with metastatic breast cancer by immunomagnetic separation. Plasma samples were collected from the same patients and assayed for TGF-β and CXCL1 by enzyme-linked immunoassay.. Patients were grouped in CK1+/- (N<10), CK2+ (N ≥ 10<50) and CK3+ (N ≥ 50), according to the number (N) of cytokeratin 7/8-positive CTCs: the highest number of CK7/8-positive CTCs was detected in patients with negative Human epidermal growth factor receptor-2 (HER-2/NEU) status (p<0.0001) antigen, identified by the monoclonal antibody Ki-67 (Ki-67) ≥ 15% (p=0.003), Carcinoma antigen 15-3 (CA-15.3) ≥ 40 U/ml (p=0.004) and those with lung metastases (p=0.01). We found that elevated plasma concentrations of TGF-β and CXCL1 are predictive for the detection of CTCs. In particular, patients with CK3+ CTCs and plasma concentrations of TGF-β and CXCL1 higher than the median value had a poor prognosis in comparison to patients with CK1+/- CTCs and TGF-β and CXCL1 concentrations below the median value.. Our study shows that elevated circulating levels of TGF-β and CXCL1 are associated with a poor prognosis, and higher detection of CTCs and propensity of these cells to seed lung metastases in patients with breast cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Chemokine CXCL1; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Lung Neoplasms; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Prognosis; Survival Rate; Transforming Growth Factor beta; Young Adult

In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumour cells (DTCs) are kept dormant, and what wakes them up, are fundamental problems in tumour biology. To address these questions, we used metastasis assays in mice and showed that dormant DTCs reside on microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumour-promoting factors derived from endothelial tip cells. Our work reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth.

Topics: Animals; Bone Marrow Neoplasms; Brain Neoplasms; Breast Neoplasms; Cell Adhesion Molecules; Endothelium, Vascular; Female; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Mice; Neoplasm, Residual; Neovascularization, Pathologic; Pericytes; Stem Cell Niche; Thrombospondin 1; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment; Zebrafish

Natural killer (NK) cells hold great potential for improving the immunotherapy of cancer. However, existing data indicate that tumor cells can effectively escape NK cell-mediated apoptosis through immunosuppressive effect in the tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressant. The present study was intended to develop a treatment strategy through adoptive transfer of TGF-β insensitive NK-92 cells. To block TGF-β signaling pathway, NK-92 cells were genetically modified with dominant negative TGF-β type II receptor (DNTβRII) by optimizing electroporation using the Amaxa Nucleofector system. These genetically modified NK-92 cells were insensitive to TGF-β and able to resist the suppressive effect of TGF-β on Calu-6 lung cancer cells in vitro. To determine the antitumor activity in vivo, recipient mice were challenged with a single subcutaneous injection of Calu-6 cells. Adoptive transfer of TGF-β insensitive NK-92 cells decreased tumor proliferation, reduced lung metastasis, produced more IFN-γ, and increased the survival rate of nude mice bearing established Calu-6 cells. Hence, we have demonstrated that blocking transforming growth factor-β signaling pathway in NK cells provides a novel therapeutic strategy and warrants further investigation.

Topics: Animals; Cell Line, Tumor; Cytotoxicity, Immunologic; Humans; Immunosuppression Therapy; Immunotherapy, Adoptive; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transgenes; Tumor Microenvironment; Xenograft Model Antitumor Assays

Lung cancer is the most frequent and one of the most deadly cancer types and is classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Transforming growth factor beta (TGFβ) regulates a wide array of cell functions and plays a major role in lung diseases, including NSCLC. TGFβ signals through the complex of TGFβ type I and type II receptors, triggering Smad and non-Smad signaling pathways such as PI3K/Akt and MEK1/ERK. We investigated the role of TGFβ1 on the progression of the murine lung adenocarcinoma cell line LP07. Furthermore, we undertook a retrospective study with tissue samples from stage I and II NSCLC patients to assess the clinical pathologic role and prognostic significance of TβRI expression. We demonstrated that although lung cancer cell monolayers responded to TGFβ1 anti-mitogenic effects and TGFβ1 pulse (24 h treatment) delayed tumor growth at primary site; a switch towards malignant progression upon TGFβ1 treatment was observed at the metastatic site. In our model, TGFβ1 modulated in vitro clonogenicity, protected against stress-induced apoptosis and increased adhesion, spreading, lung retention and metastatic outgrowth. PI3K and MEK1 signaling pathways were involved in TGFβ1-mediated metastasis stimulation. Several of these TGFβ responses were also observed in human NSCLC cell lines. In addition, we found that a higher expression of TβRI in human lung tumors is associated with poor patient's overall survival by univariate analysis, while multivariate analysis did not reach statistical significance. Although additional detailed analysis of the endogenous signaling in vivo and in vitro is needed, these studies may provide novel molecular targets for the treatment of lung cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Animals; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Follow-Up Studies; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Staging; Phosphatidylinositol 3-Kinases; Prognosis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Retrospective Studies; Survival Rate; Transforming Growth Factor beta

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.

Topics: Animals; Carcinogenesis; Collagen; Enzyme Inhibitors; Female; Fibroblasts; Focal Adhesion Kinase 1; Humans; In Situ Hybridization; Keratin-14; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Microscopy, Atomic Force; Myeloid Cells; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Lysine 6-Oxidase; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Adenoid cystic carcinoma (ACC) is one of the most common malignancies of salivary glands, characterized by poor prognosis, particularly due to pulmonary metastasis. We previously reported that transforming growth factor (TGF)-β1 promoted ACC cell migration and invasion via the Smad pathway in vitro. The aim of this study was to establish the underlying mechanisms.. TGF-β1, phospho-Smad2 and β-catenin expression in ACC tissues derived from patients was evaluated by immunohistochemistry. The role of TGF- β1 on the invasive capacity of ACC cells was determined by transwell assays in SACC-83 cells transfected with TGF-β1 and TGF-β type II dominant-negative receptor (TβRIIDN) plasmids or silenced by TGF-β1 siRNA. Expression of the epithelial-mesenchymal transition (EMT) markers, β-catenin, E-cadherin and Nectin-1, was determined by real-time PCR and immunochemistry. In vivo investigations were performed by inoculating nude mice with the transfected ACC cells and examining metastasis in bilateral lung tissues by immunohistochemistry.. Overexpression of TGF-β1 and phospho-Smad2, and reduced expression of membrane β-catenin, were closely associated with lung metastasis in ACC. Furthermore, the EMT markers were downregulated. In vitro, cells transfected with TGF-β1 exhibited altered morphology and increased invasive capacity compared to TβRIIDN-transfected cells or TGF-β1 siRNA silenced cells. In vivo, mice inoculated with TGF-β1 transfected ACC cells exhibited more metastases than other cells.. TGF-β1, phospho-Smad2 and β-catenin were significantly correlated with ACC metastasis. Blockade of TGF-β signaling by TβRIIDN or siRNA may offer potential gene therapies against pulmonary metastasis in patients with ACC.

Topics: Animals; beta Catenin; Cadherins; Carcinoma, Adenoid Cystic; Cell Adhesion Molecules; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Nectins; Neoplasms, Experimental; Real-Time Polymerase Chain Reaction; Salivary Gland Neoplasms; Smad2 Protein; Transforming Growth Factor beta

Cancer stem cells (CSCs) are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, the identification of molecules involved in CSC self-renewal is a necessary step toward more effective therapies. To this aim, through the transcription profiling of the murine ErbB2(+) tumor cell line TUBO vs. derived CSC-enriched mammospheres, Toll-like receptor 2 (TLR2) was identified as 2-fold overexpressed in CSCs, as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA-MB-231 (50%), HCC1806 (60%), and MCF7 (50%) cells. In CSC, TLR2 was activated by endogenous high-mobility-group box 1 (HMGB1), inducing IκBα phosphorylation, IL-6 and TGFβ secretion, and, consequently, STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2-fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively, these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self-renewal, tumorigenesis, and metastatic ability. These findings, while providing evidence against the controversial use of TLR2 agonists in antitumor therapy, lay out new paths toward the design of anticancer treatments.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Female; HMGB1 Protein; Humans; I-kappa B Kinase; Interleukin-6; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Neovascularization, Pathologic; Smad3 Protein; STAT3 Transcription Factor; Toll-Like Receptor 2; Transcription, Genetic; Transforming Growth Factor beta

Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor β (TGF-β) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

Topics: Apoptosis; Benzamides; Cell Line, Tumor; Cell Survival; Cisplatin; Cytokines; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Janus Kinases; Lung Neoplasms; Molecular Targeted Therapy; Naphthols; Paclitaxel; Phosphatidylinositol 3-Kinases; Receptor, IGF Type 1; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Sirtuin 1; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha; Tyrphostins

Members of the 18 glycosyl hydrolase (GH 18) gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1), which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2) and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses.

Topics: Animals; Apoptosis; Chitinase-3-Like Protein 1; Glycoproteins; Humans; Inflammasomes; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Lung Neoplasms; Macrophages; MAP Kinase Signaling System; Melanoma; Mice; Mice, Inbred C57BL; Oxidative Stress; Protein Binding; Transforming Growth Factor beta; Wnt Signaling Pathway

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

Fibroblasts are key components of the tumor microenvironment. We clarified the role of transforming growth factor (TGF)-β and interleukin (IL)-6 in the interaction between fibroblasts and non-small-cell lung cancer (NSCLC) cells.. We used NSCLC cells (A549, NCI-H358) and normal human lung fibroblast (NHLF) cells to evaluate phenotypic changes in the presence of human IL-6, TGF-β1, and conditioned media (CM) from these cells. Possible pathways were evaluated with SB431542, a TGF-β receptor inhibitor, or an anti-human IL-6 receptor neutralizing antibody (IL-6R-Ab).. A549 and NCI-H358 cells incubated with IL-6 (50 ng/mL) and TGF-β1 (2 ng/mL) showed significantly increased epithelial-mesenchymal transition (EMT) signaling compared to those treated with TGF-β1 alone. Furthermore, NHLF cells were synergistically activated by IL-6 and TGF-β1. IL-6 increased the expression of TGF-β type I receptors on the surface of A549, NCI-H358 and NHLF cells and enhanced TGF-β signaling. TGF-β1 induced phenotypic changes were attenuated by IL-6R-Ab. NHLF cells were activated and A549 cells showed induction of EMT in response to CM from the other cell type. These activities were attenuated by SB431542 or IL-6R-Ab, suggesting that interplay between NSCLC cells and NHLF may lead to increased EMT signaling in NSCLC cells and activation of NHLF cells through TGF-β and IL-6 signaling. Subcutaneous co-injection of A549 and NHLF cells into mice resulted in a high rate of tumor formation compared with injection of A549 cells without NHLF cells. SB431542 or IL-6R-Ab also attenuated the tumor formation enhanced by co-injection of the two cell types.. IL-6 enhanced epithelial cell EMT and stimulated tumor progression by enhancing TGF-β signaling. IL-6 and TGF-β may play a contributing role in maintenance of the paracrine loop between these two cytokines in the communication between fibroblasts and NSCLC cells for tumor progression.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Interleukin-6; Lung Neoplasms; Mice; Receptors, Interleukin-6; Signal Transduction; Transforming Growth Factor beta

We reported previously that tumor-evoked regulatory B cells (tBregs) play an essential role in breast cancer lung metastasis by inducing TGF-β-dependent conversion of metastasis-promoting Foxp3(+) regulatory T cells (Tregs). In this article, we show that resveratrol (RSV), a plant-derived polyphenol, at low and noncytotoxic doses for immune cells, can efficiently inhibit lung metastasis in mice. The mechanism of this process is that RSV inactivates Stat3, preventing the generation and function of tBregs, including expression of TGF-β. As a result, it frees antitumor effector immune responses by disabling tBreg-induced conversion of Foxp3(+) Tregs. We propose that low doses of RSV may also benefit humans by controlling cancer escape-promoting tBregs/Tregs without nonspecific inactivation of effector immune cells.

Topics: Animals; B-Lymphocytes, Regulatory; Female; Forkhead Transcription Factors; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Resveratrol; STAT3 Transcription Factor; Stilbenes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

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

Transforming growth factor β (TGFβ) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFβ-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via β-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFβ can modulate both β-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFβ-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFβ stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFβ-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of β-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFβ-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFβ-induced malignant phenotypes in lung cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Gene Expression; Heterografts; Humans; Lung Neoplasms; Mice; Mutation; Phenotype; Phosphorylation; Protein Interaction Domains and Motifs; PTEN Phosphohydrolase; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Burden

ID genes are required for breast cancer colonization of the lungs, but the mechanism remains poorly understood. Here, we show that Id1 expression induces a stem-like phenotype in breast cancer cells while retaining epithelial properties, contrary to the notion that cancer stem-like properties are inextricably linked to the mesenchymal state. During metastatic colonization, Id1 induces a mesenchymal-to-epithelial transition (MET), specifically in cells whose mesenchymal state is dependent on the Id1 target protein Twist1, but not at the primary site, where this state is controlled by the zinc finger protein Snail1. Knockdown of Id expression in metastasizing cells prevents MET and dramatically reduces lung colonization. Furthermore, Id1 is induced by transforming growth factor (TGF)-β only in cells that have first undergone epithelial-to-mesenchymal transition (EMT), demonstrating that EMT is a prerequisite for subsequent Id1-induced MET during lung colonization. Collectively, these studies underscore the importance of Id-mediated phenotypic switching during distinct stages of breast cancer metastasis.

Topics: Breast Neoplasms; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; MCF-7 Cells; Neoplastic Stem Cells; Nuclear Proteins; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

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

Our previous studies demonstrated that autocrine motility factor/phosphoglucose isomerase (AMF/PGI) possesses tumorigenic activities through the modulation of intracellular signaling. We then investigated the effects of ursolic acid (UA), oleanolic acid (OA), tangeretin, and nobiletin against AMF/PGI-mediated oncogenesis in cultured stable Huh7 and Hep3B cells expressing wild-type or mutated AMF/PGI and in a mouse model in this study. The working concentrations of the tested compounds were lower than their IC10 , which was determined by Brdu incorporation and colony formation assay. Only UA efficiently suppressed the AMF/PGI-induced Huh7 cell migration and MMP-3 secretion. Additionally, UA inhibited the AMF/PGI-mediated protection against TGF-β-induced apoptosis in Hep3B cells, whereas OA, tangeretin, and nobiletin had no effect. In Huh7 cells and tumor tissues, UA disrupted the Src/RhoA/PI 3-kinase signaling and complex formation induced by AMF/PGI. In the Hep3B system, UA dramatically suppressed AMF/PGI-induced anti-apoptotic signaling transmission, including Akt, p85, Bad, and Stat3 phosphorylation. AMF/PGI enhances tumor growth, angiogenesis, and pulmonary metastasis in mice, which is correlated with its enzymatic activity, and critically, UA intraperitoneal injection reduces the tumorigenesis in vivo, enhances apoptosis in tumor tissues and also prolongs mouse survival. Combination of sub-optimal dose of UA and cisplatin, a synergistic tumor cell-killing effects was found. Thus, UA modulates intracellular signaling and might serve as a functional natural compound for preventing or alleviating hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cell Proliferation; Cisplatin; Disease Models, Animal; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Flavones; Fluorescent Antibody Technique; Glucose-6-Phosphate Isomerase; Humans; Immunoprecipitation; Liver Neoplasms; Luciferases; Lung Neoplasms; Male; Matrix Metalloproteinase 3; Mice; Mice, Nude; Neovascularization, Pathologic; Oleanolic Acid; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta; Triterpenes; Tumor Cells, Cultured; Ursolic Acid

Epithelial-mesenchymal transition (EMT) programs are essential in promoting breast cancer invasion, systemic dissemination and in arousing proliferative programs in breast cancer micrometastases, a reaction that is partially dependent on focal adhesion kinase (FAK). Many functions of FAK are shared by its homolog, protein tyrosine kinase 2 (Pyk2), raising the question as to whether Pyk2 also participates in driving the metastatic outgrowth of disseminated breast cancer cells. In addressing this question, we observed Pyk2 expression to be (i) significantly upregulated in recurrent human breast cancers; (ii) differentially expressed across clonal isolates of human MDA-MB-231 breast cancer cells in a manner predictive for metastatic outgrowth, but not for invasiveness; and (iii) dramatically elevated in ex vivo cultures of breast cancer cells isolated from metastatic lesions as compared with cells that produced the primary tumor. We further show that metastatic human and murine breast cancer cells robustly upregulate their expression of Pyk2 during EMT programs stimulated by transforming growth factor-β (TGF-β). Genetic and pharmacological inhibition of Pyk2 demonstrated that the activity of this protein tyrosine kinase was dispensable for the ability of breast cancer cells to undergo invasion in response to TGF-β, and to form orthotopic mammary tumors in mice. In stark contrast, Pyk2-deficiency prevented TGF-β from stimulating the growth of breast cancer cells in 3D-organotypic cultures that recapitulated pulmonary microenvironments, as well as inhibited the metastatic outgrowth of disseminated breast cancer cells in the lungs of mice. Mechanistically, Pyk2 expression was inversely related to that of E-cadherin, such that elevated Pyk2 levels stabilized β1 integrin expression necessary to initiate the metastatic outgrowth of breast cancer cells. Thus, we have delineated novel functions for Pyk2 in mediating distinct elements of the EMT program and metastatic cascade regulated by TGF-β, particularly the initiation of secondary tumor outgrowth by disseminated cells.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Focal Adhesion Kinase 2; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Signal Transduction; Smad4 Protein; src-Family Kinases; Transforming Growth Factor beta

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antigens, Neoplasm; Biomarkers; Cell Line; Cell Movement; Cell Proliferation; Coculture Techniques; Female; Humans; Integrins; Lung Neoplasms; Macaca fascicularis; Matrix Metalloproteinase 9; Mice; Neoplasms; Protein Binding; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer-associated fibroblasts (CAFs) contribute to tumor progression through multiple pathways. However, the effect of CAFs on gene expression in lung cancer has been largely unknown. Here we systematically compared the gene expression changes in lung cancer cells induced by normal fibroblasts and CAFs.. Wound healing and cell proliferation assays were used to identify the property of CAFs used in this study. We used cDNA microarray analysis to compare gene expression in lung cancer cells cultured with either conditioned medium (CM) from lung CAFs or normal lung fibroblasts, the result of which was confirmed by RT-PCR and Western blot analysis. Immunohistochemistry on tissue sections from lung cancers was conducted to further confirm the results of cDNA microarray analysis.. The expression of many genes was upregulated in cancer cells by CAF CM, particularly cell adhesion molecules, integrins, and anti-apoptotic protein Bcl-2. Expression of integrins appeared to be upstream from Bcl-2. We identified transforming growth factor-β as a candidate factor that induced the expression of those genes in cancer cells. Immunohistochemical studies of clinical lung cancer tissues revealed that integrins and Bcl-2 were more highly expressed in the leading cells (LCs) than in the following cells, at the invasive front of cancer nests, which are adjacent to or in proximity to the stroma. Furthermore, the expression of integrins and Bcl-2 in LCs had a tendency to correlate with the clinical stage of cancer progression, including lymph node metastasis.. Our results suggest that CAFs promote lung cancer progression partly through the direct regulation of gene expression in the LCs of invasive cancer nests.

Topics: Adenocarcinoma; Cell Movement; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Integrins; Lung Neoplasms; Microarray Analysis; Neoplasm Invasiveness; Neoplastic Stem Cells; Stem Cell Niche; Transforming Growth Factor beta; Tumor Cells, Cultured

The epithelial-mesenchymal transition (EMT) is a crucial step in epithelial cancer invasion and metastasis. The aims of this study were to investigate and validate unidentified micro RNAs (miRNAs) that regulate EMT and to reveal their clinical relevance in epithelial cancer patients. By applying miRNA array screening in a natural epithelial-mesenchymal phenotype cell line pair and in a transforming growth factor β-induced EMT cell model, we found miR-153 was markedly downregulated in the cells that underwent an EMT. A close association was confirmed between inhibition of miR-153 and the EMT phenotype, as well as the invasive ability of epithelial cancer cells. Ectopic expression of miR-153 in mesenchymal-like cells resulted in an epithelial morphology change with decreased cellular invasive ability. On the contrary, transfection of a miR-153 inhibitor in epithelial-like cells led to a mesenchymal phenotype change. In vivo ectopic expression of miR-153 significantly inhibited tumor cell metastasis formation. Data from the dual-luciferase reporter gene assay showed, for the first time, that SNAI1 and ZEB2 were direct targets of miR-153. Inverse correlations were also observed between miR-153 and SNA1 and ZEB2 levels in oral cancer patients' samples. Furthermore, low expression level of miR-153 was found to be significantly related to metastasis and poor prognosis in oral cancer patients. These data demonstrate that miR-153 is a novel regulator of EMT by targeting SNAI1 and ZEB2 and indicate its potential therapeutic value for reducing cancer metastasis.

Topics: 3' Untranslated Regions; Animals; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Kaplan-Meier Estimate; Luciferases, Firefly; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms, Glandular and Epithelial; Repressor Proteins; RNA Interference; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Burden; Zinc Finger E-box Binding Homeobox 2

Curcumin has been shown to have potent anticancer activities like inhibition of cell proliferation, induction of apoptosis, and suppression of angiogenesis. Transforming growth factor-β (TGF-β) signaling plays a complex role in tumor suppression and promotion depending on the tumor type and stage. However, the effect of curcumin on TGF-β signaling in cancer cells and the role of TGF-β signaling in curcumin-induced anticancer activities have not been determined. Here, we investigate the role of curcumin on TGF-β signaling, and whether TGF-β signaling is involved in the antitumor activities of curcumin.. Human non-small cell lung cancer (NSCLC) cell lines, ACC-LC-176 (without TGF-β signaling), H358, and A549 (with TGF-β signaling) were treated with curcumin to determine cell growth, apoptosis, and tumorigenicity. Antitumor activities of curcumin were determined using these cell lines and an in vivo mouse model. We also tested the effect of curcumin on TGF-β/Smad signaling by western blotting and by luciferase assays.. Curcumin inhibited cell growth and induced apoptosis of all three NSCLC cell lines in vitro and in vivo. It significantly reduced subcutaneous tumor growth by these three cell lines irrespective of TGF-β signaling status. Curcumin inhibited TGF-β-induced Smad2/3 phosphorylation and transcription in H358 and A549 cells, but not in ACC-LC-176 cells.. Curcumin reduces tumorigenicity of human lung cancer cells in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. These results suggest that TGF-β signaling is not directly involved in curcumin-mediated growth inhibition, induction of apoptosis, and inhibition of tumorigenicity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Curcumin; Female; Flow Cytometry; Humans; Lung Neoplasms; Mice; Mice, Nude; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Epithelial-to-mesenchymal transition (EMT) is controlled by cellular signaling pathways that trigger the loss of cell-cell adhesion and lead to the restructuring of the cell cytoskeleton. Transforming growth factor β (TGF-β) has been shown to regulate cell plasticity through the phosphorylation of Par6 on a conserved serine residue (S345) by the type II TGF-β receptor. We show here that atypical protein kinase C (aPKC) is an essential component to this signaling pathway in non-small-cell lung cancer (NSCLC) cells. We show that the aPKC, PKCι, interacts with TGF-β receptors through Par6 and that these proteins localize to the leading edge of migrating cells. Furthermore, Par6 phosphorylation on serine 345 by TGF-β receptors is enhanced in the presence of aPKC. aPKC kinase activity, as well as an association with Par6, were found to be important for Par6 phosphorylation. In effect, small interfering RNA-targeting aPKC reduces TGF-β-induced RhoA and E-cadherin loss, cell morphology changes, stress fiber production, and the migration of NSCLC cells. Interestingly, reintroduction of a phosphomimetic Par6 (Par6-S345E) into aPKC-silenced cells rescues both RhoA and E-cadherin loss with TGF-β stimulation. In conclusion, our results suggest that aPKCs cooperate with TGF-β receptors to regulate phospho-Par6-dependent EMT and cell migration.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung; Lung Neoplasms; Phosphorylation; Protein Interaction Mapping; Protein Kinase C; Rats; Receptors, Transforming Growth Factor beta; rhoA GTP-Binding Protein; RNA Interference; Signal Transduction; Transforming Growth Factor beta

Cancer-associated fibroblasts (CAF) play a vital role in lung cancer initiation and progression. Although mesenchymal stem cells (MSC) are considered progenitor cells of fibroblasts and show cancer modulating abilities themselves, analyses on their presence and properties in lung cancer are lacking so far.. We performed a comparative molecular and functional analysis of MSC derived from non-small cell lung cancer (NSCLC) and corresponding normal lung tissue (NLT) of a total of 15 patients. MSC were identified and selected according to their mesenchymal multilineage differentiation capability and surface marker profile.. Compared to NLT-MSC, NSCLC-MSC showed accelerated growth kinetics and reduced sensitivity to cisplatin. Karyotyping, comparative genomic hybridization and multiplex fluorescence in situ hybridization revealed no chromosomal aberrations. However, gene expression profiling of NSCLC- and NLT-MSC indicated variable expression of 62 genes involved in proliferation, DNA repair, apoptosis, extracellular matrix synthesis, tissue remodeling and angiogenesis. Differential expression of the selected candidate genes butyrylcholinesterase, clusterin and quiescin Q6 sulfhydryl oxidase 1 was validated by quantitative real-time PCR and, on protein level, by immunohistochemical analyses of original tumor tissue. Upon exposure to tumor cell-conditioned medium or transforming growth factor-β, both, NSCLC-MSC and NLT-MSC acquired expression of α-smooth muscle actin (α-SMA), a major characteristics of CAF.. This study indicates that NSCLC tissue contains MSC with specific molecular and functional properties. These cells might represent a progenitor reservoir for CAF and thus crucially contribute to lung cancer progression.

Topics: Actins; Apoptosis; Butyrylcholinesterase; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Proliferation; Cells, Cultured; Clusterin; Comparative Genomic Hybridization; Culture Media, Conditioned; Fibroblasts; Gene Expression Profiling; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Lung; Lung Neoplasms; Mesenchymal Stem Cells; Muscle, Smooth; Oligonucleotide Array Sequence Analysis; Oxidoreductases Acting on Sulfur Group Donors; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

The human suppressive T cells that stably express transcription factor FOXP3, or regulatory T cells (Tregs), are thought to suppress antitumor immune responses. The most specific marker for human Tregs is the demethylation of CpG dinucleotides located in the first intron of FOXP3 (FOXP3i1). FOXP3i1 is completely methylated in other hematopoietic cells, including nonsuppressive T cells that transiently express FOXP3 after activation. Previously, we and others reported estimations of the frequency of Tregs in the blood of melanoma patients using a FOXP3i1 methylation-specific qPCR assay. Here, we attempted to quantify Tregs inside tumor samples using this assay. However, we found demethylated FOXP3i1 sequences in the melanoma cells themselves. This demethylation was not associated with substantial FOXP3 mRNA or protein expression, even though the demethylation extended to the promoter and terminal regions of the gene in some melanoma cells. Our results imply that analyzing Treg frequencies by quantification of demethylated FOXP3i1 will require that tumor-infiltrating T cells be separated from melanoma cells.

Topics: Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; CpG Islands; DNA Methylation; Epigenesis, Genetic; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Introns; Lung Neoplasms; Male; Melanoma; Phosphorylation; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Smad2 Protein; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The tumor microenvironment comprises various cellular components and associated subcellular molecules with antitumor and protumor effects. Because respective targeted treatment strategies are arising, it is important to characterize the exact role of these parameters. This study provides a comprehensive analysis of key immunologic factors in the tumor microenvironment of 383 surgically resected non-small cell lung cancer specimens. CD4, CD8, forkhead box protein P3, transforming growth factor β, Casitas B-cell lymphoma-b, programed death 1, T-cell-restricted intracellular antigen 1, granzyme B, mast cell tryptase, and stromal cell-derived factor 1 were analyzed by immunohistochemistry using a standardized tissue microarray platform. Extensive clinical data enabled detailed clinicopathologic correlations over a postoperative follow-up period of 15 years. Among the immunologic variables focused on, transforming growth factor β expression was the only prognostically relevant factor. Transforming growth factor β was more frequently expressed in adenocarcinoma as compared with other histologic subtypes. Expression of transforming growth factor β in tumor-infiltrating lymphocytes or in tumor cells was associated with significantly reduced postoperative survival time especially in patients with squamous cell carcinoma (P = .035 and P = .046, respectively). In these patients, the amount of transforming growth factor β-positive tumor-infiltrating lymphocytes represented the only independent immunologic parameter with prognostic significance by multivariat analysis (P = .021; hazard ratio, 2.602; 95% confidence interval, 1.159-5.844). These results should help to identify patients who are most suitable for therapeutic strategies aiming to block the transforming growth factor β signaling pathway.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Austria; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; ROC Curve; Survival Rate; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Microenvironment; Young Adult

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Topics: Adenocarcinoma; Adipose Tissue; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Chromatography, Liquid; Culture Media, Conditioned; Extracellular Matrix Proteins; Humans; Lung Neoplasms; Lysophospholipids; Mesenchymal Stem Cells; Paracrine Communication; Proteome; Proteomics; Receptors, Lysophosphatidic Acid; rho-Associated Kinases; Tandem Mass Spectrometry; Transforming Growth Factor beta; Tumor Cells, Cultured

Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-β (TGF-β) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-β-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-β-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1β. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-β, TNF-α, and IL-1β, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-β-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-β-induced EMT in A549 cells, which is transduced in part by NFκB signalling.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Cytokines; Epithelial-Mesenchymal Transition; Humans; Inflammation; Lung Neoplasms; Macrophages; Mice; Signal Transduction; Transforming Growth Factor beta

We employ a microfluidic chip with three culture chambers to investigate the interactions among lung cancer cells, macrophages and myofibroblasts. By mixing the conditioned media of macrophages and myofibroblasts in this chip, we confirm that these two stromal cells have synergistic effects in accelerating the migration of cancer cells. However, as the myofibroblasts are pretreated with the conditioned medium of macrophages, the myofibroblasts' ability to enhance the migration of cancer cells is lowered. The tumour necrosis factor-α produced by macrophages reduces the expression of α-smooth muscle actin and the secretion of transforming growth factor-β1 in myofibroblasts. Once the tumour necrosis factor-α in the macrophage conditioned medium is neutralized, the macrophage medium-pretreated myofibroblasts can still accelerate the migration of cancer cells.

Topics: Adenocarcinoma; Cell Communication; Cell Line, Tumor; Cell Movement; Coculture Techniques; Culture Media, Conditioned; Humans; Lung Neoplasms; Macrophages; Microfluidics; Microscopy, Confocal; Myofibroblasts; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor, which can be recurrently activated by gene amplification in adult lung cancer. We have discovered the first microRNA (i.e., miR-365) that directly regulates TTF-1 by interacting with its 3'-untranslated region. By gene expression profiling, we identified other putative targets of miR-365 and miR-365*. In line with the microRNA/target relationship, the expression patterns of miR-365 and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature miR-365 (i.e., mir-365-1). This implies the existence of genetic selection pressure to lose the repressive miR-365 that would otherwise suppress amplified TTF-1. We detected a signaling loop between transforming growth factor beta (TGFb) and miR-365 and this loop reinforced suppression of TTF-1 via miR-365. Mir-365 also targeted an epithelial mesenchymal transition (EMT)-promoting gene HMGA2. In summary, these data connect the lung transcriptional program to the microRNA network.

Topics: 3' Untranslated Regions; Cell Line; Gene Expression Profiling; Gene Expression Regulation; HMGA2 Protein; Humans; Lung Neoplasms; MicroRNAs; Nuclear Proteins; Signal Transduction; Thyroid Nuclear Factor 1; Transcription Factors; Transforming Growth Factor beta

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-β (TGF-β) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease α-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-β-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process.

Topics: Actins; Animals; Cell Line, Tumor; Culture Media, Conditioned; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Deoxyadenosines; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Humans; Lung; Lung Neoplasms; Mice; Phosphorylation; Thionucleosides; Transforming Growth Factor beta

Epidemiologic studies have shown that most cases of lung cancers (85%-90%) are directly attributable to cigarette smoking. Although much information has been gained about the effects of cigarette smoking on various signaling pathways causing lung cancer, nothing is known about the effect of cigarette smoking on the TGF-β-induced tumor suppressor function in lung cancer. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated with cigarette smoke condensate (CSC) and dimethyl sulfoxide (as a control) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in a decrease in Smad3 and Smad4 complex formation and TGF-β-mediated transcription due to reduced expression of Smad3. Long-term CSC treatment reduced apoptosis, increased cell viability, decreased TGF-β-mediated growth inhibition, and enhanced tumorigenicity. The decrease in apoptosis is due to the upregulation of Bcl-2, which is a downstream target of Smad3. Re-expression of Smad3 in the CSC-treated cells restored TGF-β signaling, increased apoptosis, and decreased cell viability and tumorigenicity. Withdrawal of CSC treatment resulted in the restoration of Smad3 expression, reduction in cell viability, and increased TGF-β-mediated growth inhibition. Expression of Smad3 is lower in lung tumors of current smokers than that observed in never-smokers. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity partly by abrogating TGF-β-mediated growth inhibition and apoptosis by reducing expression of Smad3.

Topics: Acetylation; Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Bronchi; Carcinoma, Squamous Cell; Cell Proliferation; Cells, Cultured; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Histones; Humans; Immunoenzyme Techniques; Immunoprecipitation; Lung Neoplasms; Mice; Mice, Nude; Phosphorylation; Protein Binding; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad3 Protein; Small Cell Lung Carcinoma; Smoking; Survival Rate; Tissue Array Analysis; Transcription, Genetic; Transforming Growth Factor beta

Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.. We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).. Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.. Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.

Topics: Adenocarcinoma; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Macrophages; Mice; Neoplasm Invasiveness; Teratocarcinoma; Transforming Growth Factor beta

The aim of the study was to evaluate the role of Serpin B3/B4 in advanced idiopathic pulmonary fibrosis (IPF) patients, mainly focusing on epithelial proliferation.. Lungs from 48 IPF patients (including cases with cancer or high-grade epithelial dysplasia) were studied and compared with other diffuse parenchymal diseases and normal lungs. Immunohistochemistry for Serpin B3/B4 and Ki-67 was quantified in all cases, distinguishing stained metaplastic cells. In IPF patients correlations between Serpin expression and several clinicopathological data, including fibrotic remodelling [fibrosis extension and transforming growth factor β expression (TGF-β)] were performed. Molecular analysis was used for Serpin isoform characterisation.. In IPF patients Serpin B3/B4 and Ki-67 were significantly overexpressed in many metaplastic cells (mainly squamous type) compared to control cases. Higher Serpin B3/B4 was found in older patients and cases with more impaired respiratory function. Serpin B3/B4 expression was related to both TGF-β and Ki-67 and was higher in patients with cancer/high-grade dysplasia. Serpin B3 was expressed in all cases, whereas Serpin B4 was expressed only in IPF.. Serpin B3/B4, particularly Serpin B4, appears to play an important role in aberrant epithelial proliferation. Evaluation of Serpin B3/B4 could have prognostic value in predicting disease progression, especially in patients with increased susceptibility to lung cancer.

Topics: Adult; Aged; Antigens, Neoplasm; Carcinoma in Situ; Cell Proliferation; Female; Humans; Idiopathic Pulmonary Fibrosis; Ki-67 Antigen; Lung Neoplasms; Lung Transplantation; Male; Middle Aged; Prognosis; Protein Isoforms; Respiratory Mucosa; Serpins; Transforming Growth Factor beta

The present study examined the effects of types of liver resection on the growth of liver and lung metastases.. Experimental liver metastases were established by spleen injection of the Colon 26 murine adenocarcinoma cell line expressing green fluorescent protein (GFP) into transgenic nude mice expressing red fluorescent protein. Experimental lung metastases were established by tail-vein injection with Colon 26-GFP. Three days after cell injection, groups of mice underwent (35% + 35% repeated minor resection versus 70% major resection versus 35% minor resection). Metastatic tumor growth was measured by color-coded fluorescence imaging of the GFP-expressing cancer cells and red fluorescent protein-expressing stroma.. Although major and repeated minor resection removed the same total volume of liver parenchyma, the 2 procedures had very different effects on metastatic tumor growth. Major resection stimulated liver and lung metastatic growth and recruitment of host-derived stroma compared with repeated minor resection. Repeated minor resection did not stimulate metastasis or stromal recruitment. No significant difference was found in liver regeneration between the 2 groups. Host-derived stroma density, which was stimulated by major resection compared with repeated minor resection, might stimulate growth in the liver-metastatic tumor. Transforming growth factor-β is also preferentially stimulated by major resection and might play a role in stromal and metastasis stimulation.. The results of the present study indicate that when liver resection is necessary, repeated minor liver resection will be superior to major liver resection, because major resection, unlike repeated minor resection, stimulates metastasis. This should be taken into consideration in clinical situations that require liver resection.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Green Fluorescent Proteins; Hepatectomy; Hepatocyte Growth Factor; Liver Neoplasms; Luminescent Proteins; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Neoplasm Transplantation; Red Fluorescent Protein; Spleen; Stromal Cells; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Small cell lung cancer (SCLC) kills at least one person every 2 hr in the United Kingdom. Some patients do relatively well but most have rapidly progressive disease. There is no effective treatment and overall 2-year survival is less than 5%. Patients with SCLC have poorly understood local and systemic immune defects and can be immunocompromised. As CD4(+) T lymphocytes coordinate and regulate immunity, a better understanding of interactions between SCLC tumour cells and CD4(+) T cells may lead to effective molecular immunotherapy. We show that some, but not all, SCLC tumour cell lines secrete molecules that induce IL-10 secretion by and de novo differentiation of functional CD4(+)CD25(+)FOXP3(+)CD127(lo)Helios(-) regulatory T (Treg) cells in healthy blood lymphocytes. FOXP3(+) T cells were found in SCLC tumour biopsies, and patients with higher ratios of FOXP3(+) cells in tumour infiltrates have a worse survival rate. The inhibitory effect of SCLC tumour cells was not affected by blocking IL-10 receptor or TGF-β signalling but was partially reversed by blocking IL-15, which is reported to be involved in human Treg cells induction. IL-15 was secreted by SCLC cells that inhibited CD4(+) T-cell proliferation and was present in SCLC biopsy tumour cells. These novel findings demonstrate that SCLC tumour cells can induce CD4(+) T-cell-mediated immunosuppression. This gives a potential mechanism by which SCLC tumour cells may downregulate local and systemic immune responses and contribute to poor patient survival. Our data suggest that IL-15 and Treg cells are potential new therapeutic targets to improve immune response and patient survival in SCLC.

Topics: Carcinoma, Small Cell; Cell Cycle; Cell Line, Tumor; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-15; Lung Neoplasms; Lymphocyte Activation; T-Lymphocytes, Regulatory; 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

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-β/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-β1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-β1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-β-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.

Topics: Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gefitinib; Humans; Lung Neoplasms; MicroRNAs; Quinazolines; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient's likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients.

Topics: Actin Cytoskeleton; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Separation; Cells, Cultured; Down-Regulation; Doxycycline; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Luciferases; Lung Neoplasms; Mice; Mice, 129 Strain; MicroRNAs; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; rho GTP-Binding Proteins; Signal Transduction; Statistics, Nonparametric; Transcription Factors; Transcriptome; Transforming Growth Factor beta; Tumor Suppressor Proteins; Zinc Finger E-box-Binding Homeobox 1

Lysophosphatidic acid (LPA) is involved in mesenchymal stem cell-stimulated tumor growth in vivo. However, the molecular mechanism by which mesenchymal stem cells promote tumorigenesis remains elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induced the expression of ADAM12, a disintegrin and metalloproteases family member, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated ADAM12 expression was abrogated by pretreatment of hASCs with the LPA receptor 1 inhibitor Ki16425 or by small interfering RNA-mediated silencing of LPA receptor 1, suggesting a key role for the LPA-LPA receptor 1 signaling axis in A549 CM-stimulated ADAM12 expression. Silencing of ADAM12 expression using small interfering RNA or short hairpin RNA abrogated LPA-induced expression of both α-smooth muscle actin, a marker of carcinoma-associated fibroblasts, and ADAM12 in hASCs. Using a xenograft transplantation model of A549 cells, we demonstrated that silencing of ADAM12 inhibited the hASC-stimulated in vivo growth of A549 xenograft tumors and the differentiation of transplanted hASCs to α-smooth muscle actin-positive carcinoma-associated fibroblasts. LPA-conditioned medium from hASCs induced the adhesion of A549 cells and silencing of ADAM12 inhibited LPA-induced expression of extracellular matrix proteins, periostin and βig-h3, in hASCs and LPA-conditioned medium-stimulated adhesion of A549 cells. These results suggest a pivotal role for LPA-stimulated ADAM12 expression in tumor growth and the differentiation of hASCs to carcinoma-associated fibroblasts expressing α-smooth muscle actin, periostin, and βig-h3.

Topics: Actins; ADAM Proteins; ADAM12 Protein; Adenocarcinoma; Adenocarcinoma of Lung; Adipose Tissue; Animals; Cell Adhesion; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Extracellular Matrix Proteins; Fibroblasts; Gene Silencing; Humans; Lung Neoplasms; Lysophospholipids; Membrane Proteins; Mesenchymal Stem Cells; Mice; Mice, Nude; Neoplasms; Receptors, Lysophosphatidic Acid; RNA, Small Interfering; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β)-mediated epithelial mesenchymal transition (EMT) of human lung cancer cells may contribute to lung cancer metastasis. It has been reported that EGCG can inhibit tumorigenesis and cancer cell growth in lung cancer; however, the effect of EGCG on EMT in nonsmall cell lung cancer (NSCLC) cells has not been investigated. In this study, we found that NSCLC cells A549 and H1299 were converted to the fibroblastic phenotype in response to TGF-β. Epithelial marker E-cadherin was down-regulated, and mesenchymal marker vimentin was up-regulated simultaneously. Our results illustrated that TGF-β was able to induce EMT in NSCLC cells, and EGCG would reverse TGF-β-induced morphological changes, up-regulate the expression of E-cadherin, and down-regulate the expression of vimentin. Immunofluorescent staining also demonstrated that E-cadherin was up-regulated and that vimentin was down-regulated by EGCG pretreatment. Moreover, wound-healing and the in vitro invasion assay showed that EGCG could inhibit TGF-β-induced migration and invasion of NSCLC cells. By using the dual-luciferase reporter assay, we demonstrated that EGCG inhibited TGF-β-induced EMT at the transcriptional level. EGCG decreased the phosphorylation of Smad2 and Erk1/2, inhibited the nuclear translocation of Smad2, and repressed the expression of transcription factors ZEB1, Snail, Slug, and Twist, and up-regulated the expression of E-cadherin. In summary, our results suggest that EGCG can inhibit TGF-β-induced EMT via down-regulation of phosphorylated Smad2 and Erk1/2 in NSCLC cells.

Topics: Carcinoma, Non-Small-Cell Lung; Catechin; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; MAP Kinase Signaling System; Plant Extracts; Smad2 Protein; Transforming Growth Factor beta

Although the role of TGF-β in tumor progression has been studied extensively, its impact on drug delivery in tumors remains far from understood. In this study, we examined the effect of TGF-β blockade on the delivery and efficacy of conventional therapeutics and nanotherapeutics in orthotopic mammary carcinoma mouse models. We used both genetic (overexpression of sTβRII, a soluble TGF-β type II receptor) and pharmacologic (1D11, a TGF-β neutralizing antibody) approaches to block TGF-β signaling. In two orthotopic mammary carcinoma models (human MDA-MB-231 and murine 4T1 cell lines), TGF-β blockade significantly decreased tumor growth and metastasis. TGF-β blockade also increased the recruitment and incorporation of perivascular cells into tumor blood vessels and increased the fraction of perfused vessels. Moreover, TGF-β blockade normalized the tumor interstitial matrix by decreasing collagen I content. As a result of this vessel and interstitial matrix normalization, TGF-β blockade improved the intratumoral penetration of both a low-molecular-weight conventional chemotherapeutic drug and a nanotherapeutic agent, leading to better control of tumor growth.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Neutralizing; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Doxorubicin; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Tissue Distribution; Transforming Growth Factor beta; Treatment Outcome; Tumor Burden; Xenograft Model Antitumor Assays

Brain metastasis (BM) from non-small cell lung cancer (NSCLC) is relatively common, but identifying which patients will develop brain metastasis has been problematic. We hypothesized that genotype variants in the TGF-β signaling pathway could be a predictive biomarker of brain metastasis.. We genotyped 33 SNPs from 13 genes in the TGF-β signaling pathway and evaluated their associations with brain metastasis risk by using DNA from blood samples from 161 patients with NSCLC. Kaplan-Meier analysis was used to assess brain metastasis risk; Cox hazard analyses were used to evaluate the effects of various patient and disease characteristics on the risk of brain metastasis.. The median age of the 116 men and 45 women in the study was 58 years; 62 (39%) had stage IIIB or IV disease. Within 24 months after initial diagnosis of lung cancer, brain metastasis was found in 60 patients (37%). Of these 60 patients, 16 had presented with BM at diagnosis. Multivariate analysis showed the GG genotype of SMAD6: rs12913975 and TT genotype of INHBC: rs4760259 to be associated with a significantly higher risk of brain metastasis at 24 months follow-up (hazard ratio [HR] 2.540, 95% confidence interval [CI] 1.204-5.359, P = 0.014; and HR 1.885, 95% CI 1.086-3.273, P = 0.024), compared with the GA or CT/CC genotypes, respectively. When we analyzed combined subgroups, these rates showed higher for those having both the GG genotype of SMAD6: rs12913975 and the TT genotype of INHBC: rs4760259 (HR 2.353, 95% CI 1.390-3.985, P = 0.001).. We found the GG genotype of SMAD6: rs12913975 and TT genotype of INHBC: rs4760259 to be associated with risk of brain metastasis in patients with NSCLC. This finding, if confirmed, can help to identify patients at high risk of brain metastasis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; DNA, Neoplasm; Female; Genotype; Humans; Inhibin-beta Subunits; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis; Smad6 Protein; Survival Rate; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-β induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the 'metastable' nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

Topics: Blotting, Western; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Microscopy, Confocal; Oncostatin M; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor beta

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

Tumor-associated macrophages (TAMs) constitute a major component of the immune cell infiltrate observed in the tumor microenvironment (TME). Factors present in the TME, including tumor growth factor-β (TGF-β), allow tumors to circumvent host-mediated immune responses to promote tumor progression. However, the molecular mechanism(s) involved are not clear. Toll-like receptors (TLRs) are important mediators of innate immune responses by immune cells, whose activation triggers the production of molecules required for anti-tumoral responses. Interleukin (IL) receptor-associated kinase (IRAK)-M is an inactive serine/threonine kinase, predominantly expressed in macrophages and is a potent negative regulator of TLR signaling. In this study, we show that TAMs express significantly higher levels of IRAK-M compared with peritoneal macrophages in a syngeneic mouse model of lung cancer. Subcutaneous implantation of Lewis lung carcinoma cells in IRAK-M(-/-) mice resulted in a five-fold reduction in tumor growth as compared with tumors in wild-type (WT) animals. Furthermore, compared with WT TAMs, TAMs isolated from IRAK-M(-/-) mice displayed features of a classically activated (M1) rather than alternatively activated (M2) phenotype, as manifest by greater expression of IL-12, interferon-γ (IFN-γ) and inducible nitric oxide synthase. Human lung cancer cells induced IRAK-M expression in human peripheral blood mononuclear cells (PBMCs) when co-cultured together. Tumor cell-induced expression of IRAK-M was dependent on the activation of TGF-β pathway. Similarly, treatment of human PBMCs or mouse macrophage cell line, RAW 264.4, with TGF-β, induced IRAK-M expression. Interestingly, IRAK-M gene expression in 439 human lung adenocarcinoma tumors correlated with poor survival in patients with lung cancer. Together, our data demonstrates that TGF-β-dependent induction of IRAK-M expression is an important, clinically relevant mechanism by which tumors may circumvent anti-tumor responses of macrophages.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Interleukin-1 Receptor-Associated Kinases; Interleukin-12; Lung Neoplasms; Macrophages; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Transforming Growth Factor beta

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

The skeleton is the most common site of prostate cancer metastasis, which often results in osteoblastic lesions. The role of transforming growth factor-beta (TGFβ) signaling in prostate cancer-induced osteoblastic metastasis is not clear. We investigated the role of TGFβ signaling in prostate cancer-induced bone metastasis using a novel human prostate cancer cell line, PacMetUT1.. We injected PacMetUT1/Luc-GFP cells in male nude mice by intracardiac and intratibia injections and then investigated the effect of TGFβ signaling abrogation on osteoblastic tumor growth and incidence in vivo by using fluorescence and bioluminescence imaging analysis and quantifying bone and tumor volume by histomorphometry analysis. Osteoclasts were counted using TRAP assay.. Osteoblastic bone metastasis in skull, rib, and femur was detected after 10-16 weeks of intracardiac injection of the PacMetUT1 cells. Stable knockdown of TGFβ1 with an shRNA resulted in decreased tumor incidence and bone formation when the cells were directly injected into the tibiae. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan TGFβ binding protein (BG(E) RII) also decreased bone tumor growth and osteoblastic bone formation in vivo after 7 weeks of treatment.. Our results for the first time indicate that blockade of TGFβ signaling in the PacMetUT1 model significantly inhibits osteoblastic bone formation and tumor incidence. Thus, TGFβ signaling pathway may be a viable target for the prevention and treatment of prostate cancer-induced bone metastasis.

Topics: Animals; Base Sequence; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Molecular Sequence Data; Osteoblasts; Prostatic Neoplasms; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Severe Acute Respiratory Syndrome (SARS) is a severe respiratory illness caused by a novel virus, the SARS coronavirus (SARS-CoV). 3C-like protease (3CLpro) of SARS-CoV plays a role in processing viral polypeptide precursors and is responsible of viral maturation. However, the function of 3CLpro in host cells remains unknown. This study investigated how the 3CLpro affected the secretion of cytokines in the gene-transfected cells.. From immunofluorescence microscopy, the localization of c-myc tagged 3CLpro was detected both in the cytoplasm and nucleus of transfected A549 cells. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly decreased in 3CLpro-transfected cells by both RT-PCR and ELISA, but without changes in other cytokines, i.e., IL-1β, IL-6, IL-8, IL12p40, TNF-α, and TGF-β. Furthermore, the protein levels of NF-kB decreased in 3CLpro-transfected A549 cells when compared to EGFP transfected cells.. Our results suggest that the 3CLpro may suppress expression of GM-CSF in transfected A549 cells through down-regulation of NF-kB production.

Topics: Blotting, Western; Cell Line, Tumor; Coronavirus 3C Proteases; Cysteine Endopeptidases; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Granulocyte-Macrophage Colony-Stimulating Factor; Green Fluorescent Proteins; Humans; Interleukin-12 Subunit p40; Interleukin-1beta; Interleukin-6; Interleukin-8; Lung Neoplasms; Microscopy, Fluorescence; Mutation; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Viral Proteins

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

Epidemiological studies have demonstrated a close correlation between nickel exposure and the incidence of lung cancer. Several studies have suggested that nickel contributes to tumor progression of human lung cancer. In this in vitro study, we found that nickel, as nickel chloride, could significantly enhance the invasive potential of human lung cancer cells, accompanied by elevated expression of IL-8, TGF-β, MMP2 and MMP9 in human lung cancer cells. Importantly, we demonstrated that nickel could activate TLR4 signaling in human lung cancer cells. Further studies showed that the TLR4/MyD88 signaling conferred the enhanced invasive potential of human lung cancer cells induced by nickel. Finally, we revealed that the p38MAPK pathway and NF-kB pathway were necessary for the enhanced invasive potential of human lung cancer cells induced by nickel. Our data provide a mechanistic explanation for nickel induced invasion of human lung cancer, and they suggest new strategies for nickel-related lung cancer clinical biotherapies.

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Myeloid Differentiation Factor 88; Neoplasm Invasiveness; NF-kappa B; Nickel; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGF-β) contribute to the growth of some skeletal metastases through autocrine stimulation. Secreted phosphoprotein 24 kDa (spp24) has been shown to bind to both BMP-2 and TGF-β and to markedly inhibit the osteogenic properties of rhBMP-2. We hypothesized that the addition of spp24 would sequester autocrine growth factors (especially BMP-2) and reduce tumor growth in a system (A549 human non-small cell lung cancer cell line) where autocrine stimulation by BMP-2 is known to be important. A549 cells were injected into two sites (subcutaneous and intraosseus) in SCID mice with and without the co-injection of BMP-2 and spp24. Tumor growth after 8 weeks was assessed through gross examination, radiological imaging, and histological analysis. Spp24 attenuated the tumor growth enhancing effects of rhBMP-2 and reduced the tumor growth when added to tumor cells that were not treated with BMP-2. We conclude that spp24 can reduce A549 cell tumor growth in both soft tissue and intraosseus environments. We hypothesize that the mechanism for this inhibition is interruption of autocrine stimulation through the sequestration of BMP-2. Spp24 can be developed into a therapeutic agent that can be employed in clinical situations where the inhibitions of BMPs and related proteins is advantageous.

Topics: Animals; Autocrine Communication; Bone Morphogenetic Protein 2; Bone Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cystatins; Humans; Lung Neoplasms; Male; Mice; Mice, SCID; Phosphoproteins; Recombinant Proteins; Transforming Growth Factor beta

The magnitude of benefit is variable for advanced non-small cell lung cancer (NSCLC) patients receiving platinum-based chemotherapy. The purpose of this study is to determine whether genetic variations in the transforming growth factor-beta (TGF-β) pathway are associated with clinical outcomes in NSCLC patients receiving first-line platinum-based chemotherapy. Five hundred and ninety-eight advanced-stage NSCLC patients who received first-line platinum-based chemotherapy with or without radiotherapy were recruited at the MD Anderson Cancer Center between 1995 and 2007. DNA from blood was genotyped for 227 single nucleotide polymorphisms (SNPs) in 23 TGF-β pathway-related genes to evaluate their associations with overall survival. In individual SNP analysis, 22 variants were significantly associated with overall survival, of which the strongest associations were found for BMP2:rs235756 [hazard ratio (HR) = 1.45; 95% confidence interval (CI), 1.11-1.90] and SMAD3:rs4776342 (HR = 1.25; 95% CI, 1.06-1.47). Fifteen and 18 genetic loci displayed treatment-specific associations for chemotherapy and chemoradiation, respectively, identifying a majority of the cases who would be predicted to respond favorably to a specific treatment regimen. BMP2:rs235753 and a haplotype in SMAD3 were associated with overall survival for both treatment modalities. Cumulative effect analysis showed that multiple risk genotypes had a significant dose-dependent effect on overall survival (P(trend) = 2.44 x 10(-15)). Survival tree analysis identified subgroups of patients with dramatically different median survival times of 45.39 versus 13.55 months and 18.02 versus 5.89 months for high- and low- risk populations when treated with chemoradiation and chemotherapy, respectively. These results suggest that genetic variations in the TGF-β pathway are potential predictors of overall survival in NSCLC patients treated with platinum-based chemotherapy with or without radiation.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Female; Genetic Variation; Haplotypes; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Survival Analysis; Transforming Growth Factor beta

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.. The numbers of both CD39(+)Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.. Both CD39(+)Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39(+)Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39(+)Tregs could express latency-associated peptide on their surface. When naïve CD4(+) T cells were cocultured with CD39(+)Tregs, Th17 cell numbers decreased as CD39(+)Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39(+)Tregs.. Therefore, the above results indicate that CD39(+)Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.

Topics: Adult; Aged; Antigens, CD; Apyrase; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Flow Cytometry; Humans; Immunophenotyping; Interleukin-1beta; Interleukin-6; Lung Neoplasms; Middle Aged; Peptides; Pleural Effusion, Malignant; Protein Precursors; Signal Transduction; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells. CR also decreased proliferation and angiogenesis and increased apoptosis in tumors. CR reduced levels of insulin, leptin, insulin-like growth factor 1, insulin-like growth factor binding protein 3 and increased adiponectin in tumors. We also demonstrated that tumors from CR mice possessed lower levels of transforming growth factor-β, lower intratumor deposition of collagen IV and reduced invasiveness due to a decrease in tumor secretion of active matrix metalloproteinase 9. Our results suggest that CR-induced metabolic and signaling changes affect the stroma and the tumor cells resulting in a microenvironment that prevents proliferation of breast tumors and their metastases.

Topics: Animals; Apoptosis; Body Weight; Caloric Restriction; Cell Line, Tumor; Cell Proliferation; Collagen; Eating; Female; Lung Neoplasms; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; 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

Fermitin family member 1 (FERMT1, Kindlin-1) is an epithelial-specific regulator of integrin functions and is associated with Kindler syndrome, a genetic disorder characterized by skin blistering, atrophy, and photosensitivity. However, the possible role of kindlin-1 in cancer remains unknown.. Kindlin-1 expression was quantified in several human cancers using quantitative real-time polymerase chain reaction and published microarray datasets. The association between kindlin-1 expression and patient metastasis-free survival (N = 516) was assessed with Kaplan-Meier analyses. Effects of ectopic expression or silencing of kindlin-1 on cell signaling, migration, and invasion were assessed in human breast cancer cell lines using western blotting, immunofluorescence, wound healing assays, and invasion on Matrigel or type I collagen substrates. Breast tumor growth and lung metastasis were evaluated in 12-week-old female BALB/c mice (10 controls and six Kindlin-1-knockdown mice). All statistical tests were two-sided.. Kindlin-1 expression was consistently higher in tumors than in normal tissues in various cancer types metastasizing to the lungs, including colon and bladder cancer. Kindlin-1 expression was associated with metastasis-free survival in both breast and lung adenocarcinoma (breast cancer: hazard ratio of lung metastasis = 2.55, 95% confidence intervals [CI] = 1.39 to 4.69, P = .001; lung cancer: hazard ratio of metastasis = 1.96, 95% CI = 1.25 to 3.07, P = .001). Overexpression of kindlin-1 induced changes indicating epithelial-mesenchymal transition and transforming growth factor beta (TGFβ) signaling, constitutive activation of cell motility, and invasion (number of migrating cells, Kindlin-1 cells vs control, mean = 164.66 vs. 19.00, difference = 145.6, 95% CI = 79.1 to 212.2, P = .004; invasion rate, Kindlin-1-cells vs control = 9.65% vs. 1.92%, difference = 7.73%, 95% CI = 4.75 to 10.70, P < .001). Finally, Kindlin-1 depletion in an orthotopic mouse model statistically significantly inhibited breast tumor growth (P < .001) and lung metastasis (P = .003).. These results suggest a role for kindlin-1 in breast cancer lung metastasis and lung tumorigenesis and advance our understanding of kindlin-1 as a regulator of TGFβ signaling, offering new avenues for therapeutic intervention against cancer progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Proliferation; Female; Fluorescent Antibody Technique; Focal Adhesions; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Proportional Hazards Models; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell-cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24-30 h after exposure to TGF-β1.

Topics: Animals; Biosensing Techniques; Breast Neoplasms; Cell Differentiation; Cells, Cultured; Electric Impedance; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Mammary Glands, Animal; Mice; Microscopy, Fluorescence; Transforming Growth Factor beta

Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. Here we demonstrate that neutrophils accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H(2)O(2) and tumor secreted CCL2 is a critical mediator of optimal antimetastatic entrainment of G-CSF-stimulated neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, whereas tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site.

Topics: Animals; Bone Marrow Cells; Breast Neoplasms; Cell Line, Tumor; Chemokine CCL2; Cytotoxicity, Immunologic; Female; Granulocyte Colony-Stimulating Factor; Humans; Hydrogen Peroxide; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Invasiveness; Neutrophils; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta

Acquisition of mesenchymal phenotype by epithelial cells by means of epithelial-mesenchymal transition (EMT) is considered as an early event in the multistep process of tumor metastasis. Therefore, inhibition of EMT might be a rational strategy to prevent metastasis.. Using the global gene expression profile from a cell culture model of transforming growth factor-β (TGF-β)-induced EMT, we identified potential EMT inhibitors. We used a publicly available database (www.broad.mit.edu/cmap) comprising gene expression profiles obtained from multiple different cell lines in response to various drugs to derive negative correlations to EMT gene expression profile using Connectivity Map, a pattern matching tool.. Experimental validation of the identified compounds showed rapamycin as a novel inhibitor of TGF-β signaling along with 17-AAG, a known modulator of TGF-β pathway. Both of these compounds completely blocked EMT and the associated migratory and invasive phenotype. The other identified compound, LY294002, demonstrated a selective inhibition of mesenchymal markers, cell migration and invasion, without affecting the loss of E-cadherin expression or Smad phosphorylation.. Our data reveal that rapamycin is a novel modulator of TGF-β signaling, and along with 17-AAG and LY294002, could be used as therapeutic agent for inhibiting EMT. This study demonstrates the potential of a systems approach in identifying novel modulators of a complex biological process.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Benzoquinones; Biomarkers, Tumor; Blotting, Western; Cadherins; Cell Movement; Chromones; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Gene Expression Profiling; HSP90 Heat-Shock Proteins; Humans; Immunosuppressive Agents; Lactams, Macrocyclic; Lung Neoplasms; Morpholines; Oligonucleotide Array Sequence Analysis; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

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

Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII). However, little is known about the mechanism of down-regulation of TβRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-β-induced tumor suppressor function. Here we show that HDIs restore TβRII expression and that DNA hypermethylation has no effect on TβRII promoter activity in lung cancer cell lines. TGF-β-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TβRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TβRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TβRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TβRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TβRII in lung cancer and that TGF-β tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TβRII expression.

Topics: Animals; Blotting, Western; Butadienes; CCAAT-Binding Factor; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Nitriles; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clusterin; Epithelial Cells; Extracellular Space; Female; Gene Expression Profiling; Humans; Immunoglobulin G; Lung Neoplasms; Mesoderm; Mice; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis; Phenotype; Reproducibility of Results; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

TGF-beta has been postulated to play an important role in the maintenance of epithelial homeostasis and the development of epithelium-derived cancers. However, most of previous studies are mainly focused on the function of TGF-beta in immune cells to the development of allergic asthma and how TGF-beta signaling in airway epithelium itself in allergic inflammation is largely unknown. Furthermore, the in vivo TGF-beta function specifically in the airway epithelium during lung cancer development has been largely elusive.. To evaluate the in vivo contribution of TGF-beta signaling in lung epithelium to the development of allergic disease and lung cancer, we generated a transgenic mouse model with Smad7, an intracellular inhibitor of TGF-beta signaling, constitutively expressed in mouse airway Clara cells using a mouse CC10 promoter. The mice were subjected to the development of OVA-induced allergic asthma and urethane-induced lung cancer. The Smad7 transgenic animals significantly protected from OVA-induced asthma, with reduced airway inflammation, airway mucus production, extracellular matrix deposition, and production of OVA-specific IgE. Further analysis of cytokine profiles in lung homogenates revealed that the Th2 cytokines including IL-4, IL-5 and IL-13, as well as other cytokines including IL-17, IL-1, IL-6, IP10, G-CSF, and GM-CSF were significantly reduced in the transgenic mice upon OVA induction. In contrast, the Smad7 transgenic animals had an increased incidence of lung carcinogenesis when subjected to urethane treatment.. These studies, therefore, demonstrate for the first time the in vivo function of TGF-beta signaling specifically in airway epithelium during the development of allergic asthma and lung cancer.

Topics: Animals; Asthma; Cytokines; Disease Models, Animal; Genetic Therapy; Inflammation; Lung Neoplasms; Mice; Mice, Transgenic; Ovalbumin; Respiratory Mucosa; Signal Transduction; Smad7 Protein; Th2 Cells; Transforming Growth Factor beta; Urethane

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

SnoN is an important negative regulator of transforming growth factor-beta (TGF-beta) signaling that was originally identified as a transforming oncogene in chicken embryonic fibroblasts. Both pro-oncogenic and antioncogenic activities of SnoN have been reported, but its function in normal epithelial cells has not been defined. In the mouse mammary gland, SnoN is expressed at relatively low levels, but it is transiently upregulated at late gestation before being downregulated during lactation and early involution. To assess the effects of elevated levels of SnoN, we generated transgenic mice expressing a SnoN fragment under the control of the mouse mammary tumor virus promoter. In this model system, SnoN elevation increased side-branching and lobular-alveolar proliferation in virgin glands, while accelerating involution in postlactation glands. Increased proliferation stimulated by SnoN was insufficient to induce mammary tumorigenesis. In contrast, elevated levels of SnoN cooperated with polyoma middle T antigen to accelerate the formation of aggressive multifocal adenocarcinomas and to increase the formation of pulmonary metastases. Our studies define functions of SnoN in mammary epithelial cell proliferation and involution, and provide the first in vivo evidence of a pro-oncogenic role for SnoN in mammalian tumorigenesis.

Topics: Adenocarcinoma; Animals; Blotting, Western; Female; Humans; Intracellular Signaling Peptides and Proteins; Lactation; Lung Neoplasms; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morphogenesis; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Lung cancer is one of the most prevalent neoplasias in developed countries. Advances in patient survival have been limited and the identification of prognostic molecules is needed. Resistance to treatment is strongly related to tumor cell adhesion to the extracellular matrix and alterations in the quantity and nature of molecules constituting the tumor cell niche. Recently, transforming growth factor beta-induced protein (TGFBI), an extracellular matrix adaptor protein, has been reported to be differentially expressed in transformed tissues. Loss of TGFBI expression has been described in several cancers including lung carcinoma, and it has been suggested to act as a tumor suppressor gene.. To address the importance of TGFBI expression in cancer progression, we determined its expression in NSCLC clinical samples using immunohistochemistry. We identified a strong association between elevated TGFBI expression and the response to chemotherapy. Furthermore, we transiently over-expressed and silenced TGFBI in human NSCLC cell lines. Cells over-expressing TGFBI displayed increased sensitivity to etoposide, paclitaxel, cisplatin and gemcitabine. We observed that TGFBI-mediated induction of apoptosis occurred through its binding to alphavbeta3 integrin. We also determined that full-length TGFBI did not induce caspase 3/7 activation but its proteolytic fragments that were < 3 kDa in size, were able to activate caspase 3, 7 and 8. This pro-apoptotic effect was blocked by anti-alphavbeta3 integrin antibodies.. The results shown here indicate that TGFBI is a predictive factor of the response to chemotherapy, and suggest the use of TGFBI-derived peptides as possible therapeutic adjuvants for the enhancement of responses to chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Enzyme Activation; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Lung Neoplasms; Prognosis; Transforming Growth Factor beta

Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy seems to arise from tumor cells that disseminated early in the course of disease but did not develop into clinically apparent lesions. These long-term surviving, disseminated tumor cells maintain a state of dormancy, but may be triggered to proliferate through largely unknown factors. We now show that the induction of fibrosis, associated with deposition of type I collagen (Col-I) in the in vivo metastatic microenvironment, induces dormant D2.0R cells to form proliferative metastatic lesions through beta1-integrin signaling. In vitro studies using a three-dimensional culture system modeling dormancy showed that Col-I induces quiescent D2.0R cells to proliferate through beta1-integrin activation of SRC and focal adhesion kinase, leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Blocking beta1-integrin, Src, ERK, or myosin light chain kinase by short hairpin RNA or pharmacologic approaches inhibited Col-I-induced activation of this signaling cascade, cytoskeletal reorganization, and proliferation. These findings show that fibrosis with Col-I enrichment at the metastatic site may be a critical determinant of cytoskeletal reorganization in dormant tumor cells, leading to their transition from dormancy to metastatic growth. Thus, inhibiting Col-I production, its interaction with beta1-integrin, and downstream signaling of beta1-integrin may be important strategies for preventing or treating recurrent metastatic disease.

Topics: Actins; Animals; Cell Growth Processes; Collagen Type I; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Female; Focal Adhesion Kinase 1; Integrin beta1; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Myosin-Light-Chain Kinase; Phosphorylation; Pulmonary Fibrosis; Rats; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-beta-mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-beta-dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-beta are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Topics: Animals; Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Neoplasm; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; Mesoderm; Mice; Mice, Nude; Mutation; Protein Kinase Inhibitors; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transforming Growth Factor beta

Cancer metastasis is a major cause of death in cancer patients, with invasion as a first step greatly contributing to the failure of clinical treatments. Any compounds with an inhibitory influence on this process are therefore of prime interest. Momordica charantia (bitter melon) is widely consumed as a vegetable and especially as a folk medicine in Asia. Here, we investigated the anti-invasive effects of bitter melon leaf extract (BMLE) on a rat prostate cancer cell line (PLS10) in vitro and in vivo. The results indicated that non-toxic concentrations of BMLE significantly inhibited the migration and invasion of cells in vitro. The results of zymography showed that BMLE inhibited the secretion of MMP-2, MMP-9 and urokinase plasminogen activator (uPA) from PLS10. Real-time RT-PCR revealed that BMLE not only significantly decreased gene expression of MMP-2 and MMP-9, but also markedly increased the mRNA level of TIMP-2, known to have inhibitory effects on the activity of MMP-2. An EnzChek gelatinase/collagenase assay showed that collagenase type IV activity was partially inhibited by BMLE. In the in vivo study, intravenous inoculation of PLS10 to nude mice resulted in a 100% survival rate in the mice given a BMLE-diet as compared with 80% in the controls. The incidence of lung metastasis did not show any difference, but the percentage lung area occupied by metastatic lesions was slightly decreased in the 0.1% BMLE treatment group and significantly decreased with 1% BMLE treatment as compared with the control. Thus, the results indicate for the first time an anti-metastatic effect of BMLE both in vitro and in vivo.

Topics: Animals; Cell Line, Tumor; Disease Progression; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Momordica charantia; Phytotherapy; Plant Extracts; Plant Leaves; Prostatic Neoplasms; Rats; Transforming Growth Factor beta

The role of transforming growth factor-beta (TGF-beta) in the development of hepatic metastasis from colon cancer is not clearly elucidated. The aim of this study was to determine the role of TGF-beta in the development of such metastasis.. Two human colon cancer cell lines were utilized: FET-alpha cells (intact TGF-beta inhibitory response), and CBS cells (defects in TGF-beta inhibitory response caused by a deficiency in type II receptor activity). The ability of these cell lines to metastasize was analysed in an orthotopic colon cancer mouse model.. FET-alpha cells did not metastasize to the liver, but showed lung metastasis in 10% of the animals, whereas CBS cells gave rise to metastasis in 65%. Following the elimination of TGF-beta activity by transfection and overexpression of dominant negative type II receptor, FET-alpha cells demonstrated liver and lung metastasis in 70% of the animals. Similarly, after the restoration of type II receptor activity by ectopic expression, CBS cells formed metastasis in fewer (10%) animals.. The results of our study demonstrate for the first time that TGF-beta displays selective metastasis suppressor activity. These abnormal pathways can serve as selective targets for future development of targeted therapies.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Burden

The epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of epithelial to mesenchymal transition has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in-vitro three-dimensional culture system to address MT in melanoma cells upon transforming growth factor-β/ tumor necrosis factor-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of 12 (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) melanoma cell lines. However, after cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) melanoma cell lines. Data support the transforming growth factor-β production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT after cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

Topics: Antigens, CD; Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Melanoma; Phenotype; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The oncoprotein c-Ski has been implicated in the negative regulation of transforming growth factor-β (TGF-β) signaling owing to its ability to repress Smad transcriptional activity via recruitment of a transcriptional corepressor complex containing histone deacetylases. However, c-Ski has also been shown to localize to the cytoplasm, raising the interesting possibility that it might disable TGF-β signaling through alternative mechanisms. Here, we provide evidence that c-Ski can restrict TGF-β signaling by interacting directly with the activated TGF-β type I receptor (TβRI). We explored the physiologic relevance of the c-Ski/TβRI interaction and found that it can culminate in a constitutive association of TβRI with a nonfunctional R-Smad/Smad4 complex. Based on these findings, we hypothesize that the interaction between c-Ski and TβRI might interfere with nuclear translocation of the R-Smad/Smad4 complex, thereby attenuating TGF-β signaling. Such a mechanism may play a crucial role in tumor progression, because many tumors that express high levels of c-Ski also display impaired nuclear accumulation of Smads.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Female; Humans; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Smad Proteins, Receptor-Regulated; Smad2 Protein; Smad3 Protein; Tissue Array Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured

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

CD137 (4-1BB) is a TNFR superfamily member that mediates the costimulatory signal resulting in T cells and NK cells proliferation and cytokines production, but the effects of CD137 signaling on CD3(+)CD56(+) cell subpopulation have not been well-documented. The aim of this study was to investigate the effects of CD137 signaling on regulation of CD3(+)CD56(+) cell function. Anti-CD137 mAb or mouse IgG1 isotype control was added to CIK cell culture to determine the effects of proliferation and anti-tumor effects on CD3(+)CD56(+) cells. We observed that anti-CD137 mAb could dramatically promote proliferation of CIK cells. And CD137-CIK cells and CD3(+)CD56(+) cell subpopulation within them possessed higher ability to kill tumor cell line A549. The SCID mice engrafted with A549 cells and treated with CD137-CIK cells have prolonged survival. Further studies revealed that the percentages of CD3(+)CD56(+) cells were elevated significantly in CD137-CIK cells. The expression of NKG2D was up-regulated on CD3(+)CD56(+) cells from CD137-CIK cells. The expression of IFN-gamma, IL-2 and TNF-alpha increased significantly whereas the production of TGF-beta(1), IL-4 and IL-10 decreased in CD3(+)CD56(+) cells from CD137-CIK cells. In addition, anti-CD137 mAb can elevate the capacity of CD3(+)CD56(+) cells to induce CD4(+) Th1 responses. We further showed that the anti-CD137 mAb also had the same effects on CD3(+)CD56(+) cells expanded from the PBMCs of patients with NSCLC. We concluded that CD137 signaling could enhance the abilities of CIK cells to kill tumor cells in vitro and in vivo via increasing the proportion of CD3(+)CD56(+) cells and their cytotoxicity. Furthermore, CD137 signaling can elevate the capacity of CD3(+)CD56(+) cells to induce CD4(+) Th1 responses which may enhance their anti-tumor activity indirectly. Taken together, our studies could be considered as valuable in CIK cells-based cancer immunotherapy.

Topics: Adult; Aged; Animals; Antibodies, Monoclonal; Carcinoma, Non-Small-Cell Lung; CD3 Complex; CD4 Antigens; CD56 Antigen; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Humans; Interferon-gamma; Interleukins; Leukocytes, Mononuclear; Lung Neoplasms; Male; Mice; Mice, SCID; Middle Aged; Neoplasms, Experimental; Th1 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor Receptor Superfamily, Member 9; Tumor Necrosis Factor-alpha

Thyroid transcription factor-1 (TTF-1) is expressed in lung cancer, but its functional roles remain unexplored. TTF-1 gene amplification has been discovered in a part of lung adenocarcinomas, and its action as a lineage-specific oncogene is highlighted. Epithelial-to-mesenchymal transition (EMT) is a crucial event for cancer cells to acquire invasive and metastatic phenotypes and can be elicited by transforming growth factor-beta (TGF-beta). Mesenchymal-to-epithelial transition (MET) is the inverse process of EMT; however, signals that induce MET are largely unknown. Here, we report a novel functional aspect of TTF-1 that inhibits TGF-beta-mediated EMT and restores epithelial phenotype in lung adenocarcinoma cells. This effect was accompanied by down-regulation of TGF-beta target genes, including presumed regulators of EMT, such as Snail and Slug. Moreover, silencing of TTF-1 enhanced TGF-beta-mediated EMT. Thus, TTF-1 can exert a tumor-suppressive effect with abrogation of cellular response to TGF-beta and attenuated invasive capacity. We further revealed that TTF-1 down-regulates TGF-beta2 production in A549 cells and that TGF-beta conversely decreases endogenous TTF-1 expression, suggesting that enhancement of autocrine TGF-beta signaling accelerates the decrease of TTF-1 expression and vice versa. These findings delineate potential links between TTF-1 and TGF-beta signaling in lung cancer progression through regulation of EMT and MET and suggest that modulation of TTF-1 expression can be a novel therapeutic strategy for treatment of lung adenocarcinoma.

Topics: Adenocarcinoma; Animals; Cadherins; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cloning, Molecular; DNA-Binding Proteins; Epithelial Cells; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Mesoderm; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Tranilast (N-[3,4-dimethoxycinnamonyl]-anthranilic acid) is a drug of low toxicity that is orally administered, and has been used clinically in Japan as an antiallergic and antifibrotic agent. Its antifibrotic effect is thought to depend on the inhibition of transforming growth factor-beta (TGF-beta). It has also been shown to exert antitumor effects, but its mode of action is unclear. Here, we explored the antitumor effects of tranilast in vitro and in vivo. Tranilast inhibited the proliferation of several tumor cell lines including mouse mammary carcinoma (4T1), rat mammary carcinoma stem cell (LA7), and human breast carcinoma (MDA-MB-231 and MCF-7). Tranilast blocked cell-cycle progression in vitro. In the highly metastatic 4T1 cell line, tranilast inhibited phospho-Smad2 generation, consistent with a blockade of TGF-beta signaling. It also inhibited the activation of MAP kinases (extracellularly regulated kinase 1 and 2 and JNK), which have been linked to TGF-beta-dependent epithelial-to-mesenchymal transition and, indeed, it blocked epithelial-to-mesenchymal transition. Although tranilast only partially inhibited TGF-beta production by 4T1 tumor cells, it potently inhibited the production of TGF-beta, interferon-gamma, IL-6, IL-10, and IL-17 by lymphoid cells, suggesting a general anti-inflammatory activity. In vivo, female BALB/c mice were inoculated with syngeneic 4T1 cells in mammary fat pads and treated with tranilast by gavage. Tranilast reduced (>50%) the growth of the primary tumor. However, its effects on metastasis were more striking, with more than 90% reduction of metastases in the lungs and no metastasis in the liver. Thus, tranilast has potential activity as an antimetastatic agent in breast cancer.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Cell Transdifferentiation; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Humans; Liver Neoplasms; Lung Neoplasms; Lymphoma; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Organ Specificity; ortho-Aminobenzoates; Rats; Rats, Sprague-Dawley; Smad2 Protein; Species Specificity; Thymoma; Thymus Neoplasms; Transforming Growth Factor beta

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-beta (TGF-beta)-induced growth arrest. As TGF-beta has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-beta-induced transcriptional activity, which is associated with increased TGF-beta-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-beta-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Pancreatic Neoplasms; Prognosis; Proto-Oncogene Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

We present a multiscale agent-based non-small cell lung cancer model that consists of a 3D environment with which cancer cells interact while processing phenotypic changes. At the molecular level, transforming growth factor beta (TGFbeta) has been integrated into our previously developed in silico model as a second extrinsic input in addition to epidermal growth factor (EGF). The main aim of this study is to investigate how the effects of individual and combinatorial change in EGF and TGFbeta concentrations at the molecular level alter tumor growth dynamics on the multi-cellular level, specifically tumor volume and expansion rate. Our simulation results show that separate EGF and TGFbeta fluctuations trigger competing multi-cellular phenotypes, yet synchronous EGF and TGFbeta signaling yields a spatially more aggressive tumor that overall exhibits an EGF-driven phenotype. By altering EGF and TGFbeta concentration levels simultaneously and asynchronously, we discovered a particular region of EGF-TGFbeta profiles that ensures phenotypic stability of the tumor system. Within this region, concentration changes in EGF and TGFbeta do not impact the resulting multi-cellular response substantially, while outside these concentration ranges, a change at the molecular level will substantially alter either tumor volume or tumor expansion rate, or both. By evaluating tumor growth dynamics across different scales, we show that, under certain conditions, therapeutic targeting of only one signaling pathway may be insufficient. Potential implications of these in silico results for future clinico-pharmacological applications are discussed.

Topics: Computational Biology; Epidermal Growth Factor; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

Aminoacyl-transfer ribonucleic acid (tRNA) synthetases-interacting multifunctional protein (AIMP) 2 is a factor associated with the macromolecular protein synthesis machinery consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors. However, it was shown to work as a multifaceted regulator through the versatile interactions with diverse signal mediators. For instance, it can mediate pro-apoptotic response to DNA damage and tumor necrosis factor-alpha (TNF-alpha) stimulus and growth-arresting signal by transforming growth factor (TGF)-beta. Considering that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to work as a potent tumor suppressor with broad coverage against different cancer types. Here we investigated whether AIMP2 would give gene dosage effect on its pro-apoptotic and anti-proliferative activities using the wild-type, hetero- and homozygous AIMP2 cells and whether AIMP2 would be critical in preventing tumorigenesis using different in vivo tumor models. Both the apoptotic responses to DNA damage and TNF-alpha and sensitivity to growth arresting TGF-beta signal were reduced in AIMP2 hetero- and homozygous cells compared with the wild-type cells in dose-dependent manner. In all the in vivo carcinogenesis experiments, reduction of AIMP2 level in heterozygous AIMP2 mice provided higher susceptibility to tumor formation. Thus, this work proves the functional significance of AIMP2 in determination of cell proliferation and death, and as a haploinsufficient tumor suppressor.

Topics: Amino Acyl-tRNA Synthetases; Animals; Apoptosis; Cells, Cultured; Colonic Neoplasms; Disease Susceptibility; DNA Damage; Heterozygote; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasms; NF-kappa B; Papilloma; Skin Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

TGF-beta blockade significantly slows tumor growth through many mechanisms, including activation of CD8(+) T cells and macrophages. Here, we show that TGF-beta blockade also increases neutrophil-attracting chemokines, resulting in an influx of CD11b(+)/Ly6G(+) tumor-associated neutrophils (TANs) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of proinflammatory cytokines. Accordingly, following TGF-beta blockade, depletion of these neutrophils significantly blunts antitumor effects of treatment and reduces CD8(+) T cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8(+) T cells intratumorally. Together, these data suggest that TGF-beta within the tumor microenvironment induces a population of TAN with a protumor phenotype. TGF-beta blockade results in the recruitment and activation of TANs with an antitumor phenotype.

Topics: Animals; Antigens, Ly; Azabicyclo Compounds; CD11b Antigen; CD8-Positive T-Lymphocytes; Cell Line, Transformed; Cell Line, Tumor; Cell Polarity; Cell Transformation, Viral; Cytokines; Immunohistochemistry; Lung Neoplasms; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Neutrophils; Phenotype; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

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

While adjuvant therapy of early-stage non-small-cell lung cancer (NSCLC) is widely accepted, literature data concerning neoadjuvant treatment provide contradictory results with both improved and unaffected survival rates. Also, data concerning potential effects of neo-adjuvant therapy on cellular level are scarce.. The aim of present study was to analyze the effect of chemotherapy followed by surgical resection on several key biological markers of tumor growth (TGF-beta, VEGF), apoptosis (sAPO-1/Fas/CD95) and invasiveness (TIMP-1) assessed in the sera of NSCLC early-stage patients (IB-IIIA). -. Measurements were performed by ELISA method in blood serum from 24 NSCLC patients (I-IIIA) collected prior therapy, one day before surgery and 3 days after.. TGF-beta serum concentrations were significantly lower after both chemotherapy (P<0.05) and surgery (P<0.01) in comparison to the baseline. VEGF levels decreased following NEO therapy with subsequent significant up-regulation after surgery (P<0.001). Interestingly, post-surgery serum VEGF strongly correlated with TGF-beta concentration (r = 0.52, P = 0.014). No significant differences were observed for serum sAPO-1/CD95/FAS as well as TIMP-1 concentrations at any of three evaluated time-points.. Neoadjuvant treatment of early-stage NSCLC affects mostly mechanisms responsible for tumor growth and vascularization. Its effect on cancer cells apoptotic activity needs further evaluation.

Topics: Adult; Aged; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Staging; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase III as Topic; Humans; Lung Neoplasms; Neoplasm Staging; Oligonucleotides, Antisense; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The clinical heterogeneity of HCC, and the lack of good diagnostic markers and treatment strategies, has rendered the disease a major challenge. Patients with HCC have a highly variable clinical course, indicating that HCC comprises several biologically distinctive subgroups reflecting a molecular heterogeneity of the tumors. Transforming growth factor beta (TGF-beta) is known to exhibit tumor stage dependent suppressive (that is, growth inhibition) and oncogenic (that is, invasiveness) properties. Here, we asked if a TGF-beta specific gene expression signature could refine the classification and prognostic predictions for HCC patients. Applying a comparative functional genomics approach we demonstrated that a temporal TGF-beta gene expression signature established in mouse primary hepatocytes successfully discriminated distinct subgroups of HCC. The TGF-beta positive cluster included two novel homogeneous groups of HCC associated with early and late TGF-beta signatures. Kaplan-Meier plots and log-rank statistics indicated that the patients with a late TGF-beta signature showed significantly (P < 0.005) shortened mean survival time (16.2 +/- 5.3 months) compared to the patients with an early (60.7 +/- 16.1 months) TGF-beta signature. Also, tumors expressing late TGF-beta-responsive genes displayed invasive phenotype and increased tumor recurrence. We also showed that the late TGF-beta signature accurately predicted liver metastasis and discriminated HCC cell lines by degree of invasiveness. Finally, we established that the TGF-beta gene expression signature possessed a predictive value for tumors other than HCC.. These data demonstrate the clinical significance of the genes embedded in TGF-beta expression signature for the molecular classification of HCC.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Knockout; Neoplasm Recurrence, Local; Phenotype; Predictive Value of Tests; Prognosis; Sensitivity and Specificity; Signal Transduction; Transforming Growth Factor beta

We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFNgamma were reduced, and the levels of CD4(+)Foxp3(+) regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-beta. Importantly, we found a synergistic up-regulation of IFNgamma with simultaneous blockade of B7-H1 and TGF-beta that was much greater than observed with TDLN. In vitro activation of cTDLN with anti-B7-H1 and anti-TGF-beta and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-beta as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host.

Topics: Animals; Antigens, Surface; Apoptosis Regulatory Proteins; B7-1 Antigen; B7-H1 Antigen; Immune Tolerance; Intestinal Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Models, Biological; Neoplasm Transplantation; Neoplasms, Second Primary; Peptides; Programmed Cell Death 1 Receptor; Sarcoma; Signal Transduction; Skin Neoplasms; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Escape

Doxorubicin is a DNA-damaging drug, commonly used for treatment of cancer patients. Doxorubicin causes not only cytotoxic and cytostatic effects, but also inhibits metastasis formation, while TGFbeta1 (Transforming Growth Factor-beta1) is a cytokine that is often up-regulated in human cancers and can promote metastasis formation. We have studied the influence of Doxorubicin on TGFbeta signaling in tumor cells. Here we have demonstrated that Doxorubicin inhibited TGFbeta-signaling in human lung adenocarcinoma A549 cells, namely, it blocked TGFbeta1-induced activation of Smad3-responsive CAGA(12)-Luc reporter, but did not affect c-myc-Luc reporter. That effect was observed as early as after 1-3 h of treating these cells with Doxorubicin, while the other drugs cisplatin or methotrexate did not alter activation of CAGA(12)-Luc reporter under the same conditions. Besides, after 1 h action, Doxorubicin abrogated TGFbeta-induced translocation of Smad3-protein from the cytoplasm to the nucleus. Down-regulation of expression of Smad2, Smad3, and Smad4 proteins, and up-regulation of inhibitory Smad7 protein upon Doxorubicin treatment, were found after 12-24 h of Doxorubicin treatment. Phosphorylation of Smad2/3 proteins was also affected by Doxorubicin. Summarizing, we have found that human tumor cells treatment with Doxorubicin resulted in the inhibition of TGFbeta-signaling at both early (1 h) and later (12 h) stages of the drug action. Such inhibition can be a new potential mechanism for Doxorubicin action towards tumor cells.

Topics: Active Transport, Cell Nucleus; Antibiotics, Antineoplastic; Doxorubicin; Humans; Lung Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Dendritic cells (DCs) have been widely used as cancer vaccines. However, their functional abilities have often been suppressed by tumor-secreted immunosuppressants such as transforming growth factor-beta (TGF-beta). We developed a new strategy using a TGF-beta insensitive DC as cancer vaccine. The effect of this vaccine was tested in a murine pulmonary metastases model of renal carcinoma (Renca). Tumor lysate-pulsed DCs (TP-DCs) were infected with retrovirus containing gene of dominant negative TGF-beta type II receptor (TbetaRIIDN) and thus made TGF-beta insensitive. Vaccination of the mice bearing Renca pulmonary metastases with the TbetaRIIDN TP-DC induced powerful tumor-specific cytotoxic T lymphocyte (CTL) responses, suppressed pulmonary metastases, and prolonged survival times. These results suggest TGF-beta-insensitive TP-DC vaccine can be used to enhance the antitumor efficacy of DC vaccine.

Topics: Animals; Blotting, Western; Dendritic Cells; Female; Flow Cytometry; Kidney Neoplasms; Lung Neoplasms; Mice; Mice, Inbred BALB C; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

NSCLC cells with a mesenchymal phenotype have shown a marked reduction in sensitivity to EGFR inhibitors, though the molecular rationale has remained obscure. Here we find that in mesenchymal-like tumor cells both tyrosine phosphorylation of EGFR, ErbB2, and ErbB3 signaling networks and expression of EGFR family ligands were decreased. While chronic activation of EGFR can promote an EMT-like transition, once having occurred EGFR family signaling was attenuated. We investigated the mechanisms by which mesenchymal-like cells bypass EGFR signaling and acquire alternative routes of proliferative and survival signaling. Mesenchymal-like NSCLC cells exhibit aberrant PDGFR and FGFR expression and autocrine signaling through these receptors can activate the MEK-ERK and PI3K pathways. Selective pharmacological inhibition of PDGFR or FGFR receptor tyrosine kinases reduced cell proliferation in mesenchymal-like but not epithelial NSCLC cell lines. A metastable, reversible EMT-like transition in the NSCLC line H358 was achieved by exogenous TGFbeta, which served as a model EMT system. The H358/TGFbeta cells showed many of the attributes of established mesenchymal-like NSCLC cells including a loss of cell-cell junctions, a loss of EGF-family ligand expression, a loss of ErbB3 expression, increased EGFR-independent Mek-Erk pathway activation and reduced sensitivity to EGFR inhibition. Notably an EMT-dependent acquisition of PDGFR, FGFR and TGFbeta receptors in H358/TGFbeta cells was also observed. In H358/TGFbeta cells both PDGFR and FGFR showed functional ligand stimulation of their intrinsic tyrosine kinase activities. The findings of kinase switching and acquired PDGFR and FGFR signaling suggest investigation of new inhibitor combinations to target NSCLC metastases.

Topics: Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Chromatography, Liquid; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Humans; Immunoblotting; Lung Neoplasms; Mesoderm; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinazolines; Quinolines; Receptor, ErbB-2; Receptor, ErbB-3; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Thiophenes; Transforming Growth Factor beta; Tumor Cells, Cultured

Vimentin, nuclear factor-kappaB (NF-kappaB) p105, fascin, E-cadherin, TGF-beta, Par6 and atypical PKC are molecular markers that play an important role in cell differentiation. Herein, we investigate their prognostic impact in primary non-small-cell carcinoma (NSCLC). Tumour tissue samples from 335 resected patients with stage I-IIIA were used. Tissue microarrays were constructed from duplicate cores of both neoplastic cells and stromal cells and were immunohistochemically evaluated. In univariate analyses, high tumour epithelial cell expressions of NF-kappaB p105 (P=0.02) and E-cadherin (P=0.03) were positive prognostic indicators for disease-specific survival (DSS), whereas high tumour epithelial cell expression of vimentin (P=0.001) was a negative prognostic indicator. High expression of NF-kappaB p105 (P=0.001) and Par6 (P=0.0001) in the stromal compartment correlated with a good prognosis. In multivariate analyses, the tumour epithelial cell expression of NF-kappaB p105 (P=0.0001) and vimentin (P=0.005) and the stromal cell expression of NF-kappaB p105 (P=0.007) and Par6 (P=0.0001) were independent prognostic factors for DSS. High expression of NF-kappaB p105 and low expression of vimentin in tumour epithelial cells are independent predictors of better survival in primary NSCLC. In stromal cells, high expressions of NF-kappaB p105 and Par6 are both favourable independent prognostic indicators.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Cadherins; Carcinoma, Non-Small-Cell Lung; Epithelial Cells; Female; Humans; Immunohistochemistry; Isoenzymes; Lung Neoplasms; Male; Middle Aged; NF-kappa B p50 Subunit; Proportional Hazards Models; Protein Kinase C; Stromal Cells; Tissue Array Analysis; Transforming Growth Factor beta; Vimentin

The malignant transformation in several types of cancer, including lung cancer, results in a loss of growth inhibition by transforming growth factor-beta (TGF-beta). Here, we show that SMAD6 expression is associated with a reduced survival in lung cancer patients. Short hairpin RNA (shRNA)-mediated knockdown of SMAD6 in lung cancer cell lines resulted in reduced cell viability and increased apoptosis as well as inhibition of cell cycle progression. However, these results were not seen in Beas2B, a normal bronchial epithelial cell line. To better understand the mechanism underlying the association of SMAD6 with poor patient survival, we used a lentivirus construct carrying shRNA for SMAD6 to knock down expression of the targeted gene. Through gene expression analysis, we observed that knockdown of SMAD6 led to the activation of TGF-beta signaling through up-regulation of plasminogen activator inhibitor-1 and phosphorylation of SMAD2/3. Furthermore, SMAD6 knockdown activated the c-Jun NH2-terminal kinase pathway and reduced phosphorylation of Rb-1, resulting in increased G0-G1 cell arrest and apoptosis in the lung cancer cell line H1299. These results jointly suggest that SMAD6 plays a critical role in supporting lung cancer cell growth and survival. Targeted inactivation of SMAD6 may provide a novel therapeutic strategy for lung cancers expressing this gene.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Phosphorylation; RNA, Small Interfering; Signal Transduction; Smad6 Protein; Transduction, Genetic; Transforming Growth Factor beta

Locally produced transforming growth factor-beta (TGF-beta) promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This article explores the potential for increased efficacy when combining immunotherapies with TGF-beta suppression using the TGF-beta type I receptor kinase inhibitor SM16. Adenovirus expressing IFN-beta (Ad.IFN-beta) was injected intratumorally once in established s.c. AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a Kras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing human papillomavirus-E7 (HPV-E7; Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine expression and cell populations were measured in tumors and spleens by real-time PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.IFN-beta increased the number of intratumoral leukocytes (including macrophages, natural killer cells, and CD8(+) cells) and increased the percentage of T cells expressing the activation marker CD25. SM16 also augmented the antitumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8(+) T cells in the spleens but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-beta signaling pathway augments the antitumor effects of Ad.IFN-beta immune-activating or Ad.E7 vaccination therapy. The addition of TGF-beta blocking agents in clinical trials of immunotherapies may increase efficacy.

Topics: Adenoviridae; Animals; Azabicyclo Compounds; Cancer Vaccines; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Cytokines; Epitopes, T-Lymphocyte; Female; Immunogenetics; Immunotherapy; Intercellular Adhesion Molecule-1; Interferon-beta; Lung Neoplasms; Male; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oncogene Proteins, Viral; RNA, Messenger; Transforming Growth Factor beta

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

Several genes encoding different cytokines may play crucial roles in host susceptibility to lung cancer, since cytokine production capacity varies among individuals and depends on cytokine gene polymorphisms. The association between cytokine gene polymorphisms with primary lung carcinoma was investigated. DNA samples were obtained from a Turkish population of 44 patients with primary lung cancer, and 59 healthy control subjects. All genotyping (IFN-gamma, TGF-beta1, TNF-alpha, IL-6 and IL-10) experiments were performed using sequence-specific primers (SSP)-PCR. When compared to the healthy controls, the frequencies of high/intermediate producing genotypes of IL-10 and low producing genotype of TNF-alpha were significantly more common in the patient group. It is noteworthy that lung cancer patients with the TGF-beta T/T genotype in codon 10 had statistically longer survival compared to those having the C/C genotype (Kaplan-Meier survival function test, log rank significance = 0.014). These results suggest that IL-10, TNF-alpha and TGF-beta1 gene polymorphisms may affect host susceptibility to lung cancer and the outcome of the patients.

Topics: Adult; Alleles; Case-Control Studies; Codon; Cytokines; Female; Gene Frequency; Genotype; Humans; Interferon-gamma; Interleukin-10; Interleukin-6; Lung Neoplasms; Male; Middle Aged; Phenotype; Polymorphism, Genetic; Racial Groups; Regression Analysis; Survival Analysis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Turkey

Transforming growth factor beta1 (TGF-beta1) suppresses tumor development at early stages of cancer, but enhances tumor invasion and formation of metastasis. TGF-beta1-mediated tumor invasion is associated with epithelial to mesenchymal transition (EMT) and matrix proteolysis. The mechanisms of these TGF-beta1 responses in normal and tumor cells are not well understood. Recently, we have reported that TGF-beta1 increases expression of high-molecular weight tropomyosins (HMW-tropomyosins) and formation of actin stress fibers in normal epithelial cells. The present study investigated the role of tropomyosin in TGF-beta1-mediated cell motility and invasion. We found that TGF-beta1 restricts motility of normal epithelial cells although it promotes EMT and formation of actin stress fibers and focal adhesions. Cell motility was enhanced by siRNA-mediated suppression of HMW-tropomyosins. TGF-beta1 stimulated migration and matrix proteolysis in breast cancer MDA-MB-231 cells that express low levels of HMW-tropomyosins. Tet-Off-regulated expression of HMW-tropomyosin inhibited cell migration and matrix proteolysis without affecting expression of matrix metalloproteinases. Tropomyosin increased cell adhesion to matrix by enhancing actin fibers and focal adhesions. Finally, tropomyosin impaired the ability of tumor cells to form lung metastases in SCID mice. Thus, these results suggest that HMW-tropomyosins are important for TGF-beta-mediated control of cell motility and acquisition of the metastatic potential.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Movement; Collagen; Drug Combinations; Humans; Immunoblotting; Laminin; Lung Neoplasms; Mammary Glands, Animal; Matrix Metalloproteinases; Mice; Mice, SCID; Molecular Weight; Neoplasm Invasiveness; Proteoglycans; Rats; Transforming Growth Factor beta; Tropomyosin; Tumor Cells, Cultured; Wound Healing

Non small cell lung carcinomas (NSCLC) are known to secrete various cytokines such as VEGF (vascular endothelial growth factor), G-CSF (granulocyte-colony stimulating factor) and TGF-beta (transforming growth factor-beta) that may stimulate bone marrow activity.. This study reports on the relationship between serum levels of VEGF, G-CSF, TGF-beta and FDG uptake by the bone marrow in NSCLC patients.. Thirty-three patients suffering from newly diagnosed NSCLC who were successively referred to undergo an FDG PET scan as a part of their routine staging procedure and that did not suffer from bone metastases were included in the study. FDG bone marrow activity was determined in all patients and related to pre-treatment VEGF, G-CSF and TGF-beta serum levels.. Mean standardized uptake values (SUV mean) of the bone marrow ranged from 0.1 to 2.1 (mean 1.1). G-CSF, VEGF and TGF-beta serum levels ranged from 13.5 to 110 pg/ml (mean 41.4 pg/ml), from 95 to 3,221 pg/ml (mean 1,111 pg/ml) and from 269 to 615 pg/ml (mean 387 pg/ml), respectively. SUV mean values of the bone marrow significantly correlated with TGF-beta serum measurements (r = 0.621, p < 0.0001), but not with VEGF and G-CSF measurements.. FDG uptake by bone marrow in newly diagnosed NSCLC patients correlates with serum levels of TGF-beta, but not with VEGF or G-CSF levels.

Topics: Aged; Aged, 80 and over; Bone Marrow; Carcinoma, Non-Small-Cell Lung; Female; Fluorodeoxyglucose F18; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; Male; Middle Aged; Radionuclide Imaging; Radiopharmaceuticals; Statistics as Topic; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of betaig-h3/TGFBI (transforming growth factor, beta-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the betaig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of betaig-h3 expression dramatically reduced metastasis. Mechanistically, betaig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of the integrin alphavbeta5-Src signaling pathway. Thus, cancers associated with overexpression of betaig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients.

Topics: Amino Acid Sequence; Animals; Antigens, CD; Cadherins; Cell Line, Tumor; Colonic Neoplasms; Endothelial Cells; Endothelium, Vascular; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Integrins; Intercellular Junctions; Lung Neoplasms; Mice; Mice, SCID; Molecular Sequence Data; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligopeptides; Receptors, Vitronectin; Transforming Growth Factor beta

The mouse renal cell carcinoma line, Renca, is insensitive to transforming growth factor-beta (TGF-beta) in vitro. The present study was conducted to determine whether removal of TGF-beta from these tumor cells would inhibit tumor progression in vivo.. TGF-beta elimination was accomplished either by administration of neutralizing TGF-beta antibody into mice receiving intravenous injection of Renca tumor cells or infection of TGF-beta antisense expression vector into these tumor cells before subcutaneous injection into recipient mice.. Although a low dose of TGF-beta antibody (5 mg/kg every 3 days) was without any effect, a high dose of TGF-beta antibody (50 mg/kg every 3 days), administered to recipient mice, resulted in a significant reduction in lung metastasis and was accompanied by increased apoptosis in the tumor cells. When the tumor cells were transfected with a TGF-beta1 antisense expressing vector, a significant reduction occurred in the tumor incidence, as well as the tumor burden. However, in nude mice, cells with reduced TGF-beta1 production grew almost as well as did the unmodified Renca cells, suggesting that the host's immune system might play an antitumor role.. These results indicate that progression of Renca tumor can be inhibited by eliminating TGF-beta from the tumor cells. Our results also suggest that, although insensitive to TGF-beta under in vitro conditions, Renca tumors could be inhibited by TGF-beta removal through the systemic host environment.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; In Vitro Techniques; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Transfection; Transforming Growth Factor beta

The transfusion of allogeneic blood products containing white cells (WBCs) has been reported to reduce resistance to infection, stimulate the growth of some types of tumors in animal models, and prevent abortion of allogeneic embryos in the CBAxDBA/2 murine model.. In this study, the issue explored was whether allogeneic BALB/c whole blood given to C57Bl/6 mice by tail vein after injection of syngeneic FSL-10 fibrosarcoma cells increased the number of lung nodules enumerated on Day 21. The effect on the tumor growth-promoting effect produced by allogeneic BALB/c whole blood was then examined by exposure of the allogeneic BALB/c blood to various monoclonal antibodies (MoAbs). The antibodies added to the BALB/c blood included anti-murine CD200 antibodies, anti-lymphoid dendritic cell (DC) antibodies (DEC205), or anti-myeloid DC (anti-CD11c) antibodies.. The tumor growth-promoting effect of the allogeneic BALB/c blood was abrogated by the addition to the BALB/c blood of MoAb either to myeloid DCs (anti-CD11c) or to the CD200 tolerance signaling molecule, but not by adding MoAb to lymphoid DCs (DEC205). BALB/c blood also was shown to increase the percentage of transforming growth factor (TGF)-beta+ splenocytes detected in recipient mice, on Day 12 after transfusion. This effect was abrogated by adding anti-CD200 antibody to the BALB/c donor blood. Moreover, physiologic concentrations of TGF-beta, but not interleukin-10, were shown to stimulate, in cell culture experiments, the proliferation of syngeneic FSL-10 sarcoma cells.. These data support the hypothesis that the mechanism of the tumor growth-promoting effect of allogeneic blood is mediated by a highly potent population of peripheral blood DCs expressing the CD200 tolerance signaling molecule. These data also indicate that tumor cell growth can be mediated by the stimulation of TGF-beta-producing cells and that TGF-beta may act by tumor cell growth stimulation, rather than by host immunosuppression.

Topics: Animals; Antigens, CD; CD11c Antigen; Dendritic Cells; Female; Fibrosarcoma; Immune Tolerance; Immunologic Factors; Isoantigens; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Signal Transduction; Spleen; Transforming Growth Factor beta; Transfusion Reaction

Cooperation between the Neu/ErbB-2 and transforming growth factor beta (TGF-beta) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-beta induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-beta-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-beta-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-beta-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-beta-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Movement; Female; Focal Adhesions; Gene Expression; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Invasiveness; Phosphotyrosine; Point Mutation; Receptor, ErbB-2; RNA, Small Interfering; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Transforming Growth Factor beta

To observe the effects of ginsenoside (Gs) and berberine (Ber), two kinds of active components of traditional Chinese herbal medicine, on transforming growth factor-beta1 (TGF-beta1) and prostaglandin E2 (PGE2) in PG cells.. Co-culture system of human lung carcinoma cell line PG and human T lymphocyte cell line Jurkat was established. PG cells were treated with Gs (100 microg/ml) and Ber (10 mug/ml) for twenty-four hours, and then cocultured with Jurkat cells. After 24-hour coculture, the state of Jurkat cells was observed with inverted microscope. The viable count of Jurkat cells was detected by trypan blue staining after 6- and 24-hour coculture, and the apoptosis of Jurkat cells was evaluated by flow cytometry. PG cells were treated with 100, 50, 25 microg/ml Gs and 10, 5, 2.5 microg/ml Ber respectively, and the content of TGF-beta1 and PGE(2) in PG cells was detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) method.. After coculture with PG cells treated with Gs and Ber, the number of Jurkat cells was less than blank control group, and the apoptosis rates of Jurkat cells in Gs- and Ber-treated groups were higher than blank control group. Gs and Ber could promote the secretion of TGF-beta1 in PG cells, but could not change the level of PGE(2).. Gs and Ber can promote the growth inhibition and apoptosis of Jurkat cells induced by PG cells, which may be related to the up-regulation of Gs and Ber on TGF-beta1 secretion in PG cells.

Topics: Apoptosis; Berberine; Coculture Techniques; Dinoprostone; Ginsenosides; Humans; Jurkat Cells; Lung Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

Topics: Animals; Bone Marrow Cells; Breast Cyst; Cell Differentiation; Cell Survival; Disease Progression; Hyperplasia; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Neoplastic Stem Cells; Precancerous Conditions; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Stromal Cells; Transforming Growth Factor beta

Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage-related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-beta (TGF-beta) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-beta blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-beta-dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization.

Topics: Animals; Bone Neoplasms; Bone Resorption; Carcinoma, Large Cell; Carcinoma, Squamous Cell; Cell Line, Tumor; Extracellular Matrix; Gene Expression Profiling; Humans; Lung Neoplasms; Matrix Metalloproteinases; Mice; Mice, Nude; Osteoclasts; Stromal Cells; Transforming Growth Factor beta

The microenvironment of a tumor is known to influence tumor progression and spread to distant sites (metastasis). Padua et al. (2008) now show that transient exposure of breast cancer cells to the signaling molecule transforming growth factor beta (TGFbeta) promotes their extravasation from blood vessels and entry into the lung by upregulation of the adipokine angiopoietin-like 4 (ANGPTL4). Their work shows that the later stages of metastasis can be influenced by transient signals produced in the primary tumor microenvironment.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Breast Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Endothelial Cells; Female; Gene Expression Profiling; Humans; Intercellular Junctions; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neoplasms, Experimental; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Topics: Animals; Anoikis; Antimetabolites, Antineoplastic; Apoptosis; Cell Movement; Deoxycytidine; Drug Therapy, Combination; Gemcitabine; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Pancreatic Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Ribonucleotide Reductases; Signal Transduction; Smad2 Protein; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Lung metastasis is the most crucial event affecting the therapeutic outcome of osteosarcoma. The prevention of lung metastasis is therefore important in improving the prognosis of patients with osteosarcoma. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The biological role of decorin in osteosarcoma has yet to be clarified. The aim of this study was to examine the potential of decorin as a novel biological target for the treatment of osteosarcoma. In this study, the LM8 murine osteosarcoma cell line (LM8) with high metastatic potential to the lung was used. The two cell lines established were LM8-DCN which stably expressed human decorin (hDCN) and LM8-mock, established as a control. The LM8-DCN cell line was subcutaneously injected into the backs of mice. Significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated with LM8 and LM8-mock (P<0.001). In addition, the mice in the LM8-DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group, based on the Kaplan-Meier survival analysis and log-rank tests (P<0.005). The effect of decorin on the growth rates, motility and invasion ability of LM8 was investigated in vitro. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Movement; Decorin; Extracellular Matrix Proteins; Genetic Vectors; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Osteosarcoma; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated whether TGF-beta induced by anticancer therapies accelerates tumor progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, we show that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-beta1 as well as increased circulating tumor cells and lung metastases. These effects were abrogated by administration of a neutralizing pan-TGF-beta antibody. Circulating polyomavirus middle T antigen-expressing tumor cells did not grow ex vivo in the presence of the TGF-beta antibody, suggesting autocrine TGF-beta is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack the type II TGF-beta receptor, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-beta on the cancer cells. These data implicate TGF-beta induced by anticancer therapy as a pro-metastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-beta inhibitors.

Topics: Animals; Antibodies, Blocking; Antigens, Polyomavirus Transforming; Cell Line, Tumor; Female; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Neoplasms, Radiation-Induced; Neoplastic Cells, Circulating; Retroviridae Infections; Signal Transduction; Transforming Growth Factor beta; Tumor Virus Infections

The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, alpha-smooth muscle actin, and fibroblast activation protein, but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumor-associated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was up-regulated by IFN-gamma. Production of cytokines and chemokines, including IFN-gamma, monokine induced by IFN-gamma, IFN-gamma-inducible protein-10, RANTES, and TGF-beta1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-beta1-mediated suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases, blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment.

Topics: Antigens, CD; B7-1 Antigen; B7-H1 Antigen; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Chemokines; Coculture Techniques; Cytokines; Fibroblasts; Humans; Interferon-gamma; Lung Neoplasms; Lymphocyte Activation; Programmed Cell Death 1 Ligand 2 Protein; Receptors, Antigen, T-Cell; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Survival; Homeodomain Proteins; Humans; Liver Neoplasms; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

The pathogenesis of cancer anorexia-cachexia syndrome (CACS) is very complicated. In development of this syndrome play a role: metabolism of glucose, tumor necrosis factor (TNF), interleukin 1, interleukin 6, interferon a, interferon y, the oncological treatment and many other factors. Vascular endothelial growth factor (VEGF) is the most important proangiogenic factor. It promotes new vessels development, enhances vascular permeability and recruits monocytes. Many factors play a role in regulation of VEGF level. One of the most important is transforming growth factor beta (TGF-beta). Vascular endothelial growth factor and TGF-beta play a role in inflammation reaction too. The aim of this study was the evaluation of correlation between VEGF and TGF-beta in CACS in lung cancer.. We measured the serum level of VEGF and TGF-beta in 40 patients with lung cancer (20 with and 20 without CACS) and in control group. The serum level was measured by ELISA method.. The correlation between VEGF and TGF-beta was not statistically significant in patients with CACS (p = 0.67), but the statistical significance was in patients without CACS (p = 0.006) and in control group (p = 0.035).. Results suggest, that VEGF regulation in CACS may be more complicated.

Topics: Anorexia; Cachexia; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

TGF-beta is a potent immunosuppressant. High levels of TGF-beta produced by cancer cells have a negative inhibition effect on surrounding host immune cells and leads to evasion of the host immune surveillance and tumor progression. In the present study, we report a distinct ability of tumor reactive, TGF-beta-insensitive CD8+ T cells to infiltrate into established tumors, secrete relevant cytokines, and induce apoptosis of tumor cells.. CD8+ T cells were isolated from the spleens of C57BL/6 mice, which were primed with irradiated mouse prostate cancer cells, the TRAMP-C2 cells. After ex vivo expansion, these tumor reactive CD8+ cells were rendered TGF-beta-insensitive by infection with a retroviral (MSCV)-mediated dominant negative TGF-beta type II receptor (TbetaRIIDN). Control CD8+ cells consist of those transfected with the GFP-only empty vector and naïve CD8+ T cells. Recipient mice were challenged with a single injection of TRAMP-C2 cells 21 days before adoptive transfer of CD8+ T cells was performed. Forty days after the adoptive transfer, all animals were sacrificed. The presence of pulmonary metastases was evaluated pathologically. Serial slides of malignant tissues were used for immunofluorescent staining for different kinds of immune cell infiltration, cytokines, and apoptosis analysis.. Pulmonary metastases were either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells (3 out of 12) when compared to GFP controls (9 out of 12), and naïve CD8+ T cells (12 out of 12). Results of immunofluorescent studies demonstrated that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis when compared to CD4+ T cells, NK cells, and B cells. A large amount of cytokines such as perforin, nitric oxide, IFN-gamma, IL-2, TNF-alpha were secreted in tumor tissue treated with tumor-reactive TGF-beta-insensitive CD8+ T cells. No immune cells infiltration and cytokine secretion were detected in tumor tissues treated with naïve T cells and GFP controls.. Our results demonstrate the mechanism of anti-tumor effect of tumor-reactive TGF-beta-insensitive CD8+ T cells that adoptive transfer of these CD8+ T cells resulted in infiltration of these immune cells into the tumor parenchyma, secretion of relevant cytokines, and induction of apoptosis in tumor cells. These results support the concept that tumor-reactive TGF-beta-insensitive CD8+ T cells may prove beneficial in the treatment of advanced cancer patients.

Topics: Animals; Apoptosis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytokines; Immunohistochemistry; In Situ Nick-End Labeling; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Mice; Mice, Inbred C57BL; Prostatic Neoplasms; Specific Pathogen-Free Organisms; Transforming Growth Factor beta

The POS-1 murine model of osteolytic osteosarcoma was used to elucidate the molecular and cellular mechanisms involved in the development of primary bone tumors and associated lung metastasis. The POS-1 cell line is derived from an osteosarcoma tumor which develops spontaneously in C3H mice. The POS-1 cell line was characterized in vitro by mineralization capacity and expression of bone markers by semi-quantitative RT-PCR, compared to primary osteoblasts and bone marrow cells. POS-1 cells showed no mineralization capacity and exhibited an undifferentiated phenotype, expressing both osteoblastic and unexpected osteoclastic markers (TRAP, cathepsin K and RANK). Thereby, experiments were performed to determine whether RANK was functional, by studying the biological activity of murine RANKL through the receptor RANK expressed on POS-1 cells. Results revealed a RANKL-induced increase in ERK phosphorylation, as well as BMP-2 induction at the mRNA and protein levels, and a decrease of POS-1 cell proliferation in the presence of 10 ng/ml RANKL. BMP-2 induction is dependent on the ERK 1/2 signal transduction pathway, as its expression is abolished in the presence of UO126, a specific synthetic inhibitor of the ERK 1/2 pathway. Moreover, a 2-fold molar excess of soluble RANK blocks the RANKL-induced BMP-2 expression, demonstrating that the biological effects of RANKL observed in POS-1 cells are mediated by RANK. This is the first report describing a functional RANK expressed on osteosarcoma cells, as shown by its ability to induce signal transduction pathways and biological activity when stimulated by RANKL.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Carrier Proteins; Cell Proliferation; Gene Expression Profiling; Humans; Lung Neoplasms; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Betaig-h3 as a secreted protein induced by transforming growth factor-beta has been suggested to modulate cell adhesion and tumor formation. Although we have previously shown that downregulation of Betaig-h3 gene is involved in the cellular transformation of human bronchial epithelial cells induced by radiation, its regulation in primary human lung cancers is not clearly understood. In this study, Betaig-h3 expression was studied in 130 primary human lung carcinomas by immunohistochemistry. Betaig-h3 protein was absent or reduced by more than two-fold in 45 of 130 primary lung carcinomas relative to normal lung tissues examined. Recovery of Betaig-h3 expression in H522 lung cancer cells lacking endogenous Betaig-h3 protein significantly suppressed their in vitro cellular growth and in vivo tumorigenicity. In addition, parental H522 cancer cells are resistant to the etoposide induced apoptosis compared with normal human bronchial epithelial cells. However, recovery of Betaig-h3 expression in H522 cancer cells results in significantly higher sensitivity to apoptotic induction than parental tumor cells. IGFBP3 is upregulated in Betaigh3-transfected H522 cells that may mediate the apoptotic sensitivity and antitumor function of Betaig-h3 gene. These observations demonstrate that downregulation of Betaig-h3 gene is a frequent event and related to the tumor progression in human lung cancer.

Topics: Adenocarcinoma; Animals; Apoptosis; Carcinogenicity Tests; Carcinoma, Large Cell; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Lung Neoplasms; Mice; Mice, Nude; Phenotype; Transfection; Transforming Growth Factor beta; Up-Regulation

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.

Topics: Animals; Carcinoma, Lewis Lung; Cell Communication; Cell Line, Tumor; Immunotherapy, Adoptive; Interleukin-12; Killer Cells, Natural; Ligands; Lung Neoplasms; Lymphoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, Natural Killer Cell; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) functions as a suppressor of tumor initiation by inhibiting cellular proliferation or by promoting cellular differentiation or apoptosis in the early phase of cancer development. Variations in the DNA sequence in the TGF-beta1 gene may lead to altered TGF-beta1 production and/or activity, and so this can modulate an individual's susceptibility to lung cancer. To test this hypothesis, we investigated the association of the TGF-beta1 -509C > T and 869T > C (L10P) polymorphisms and their haplotypes with the risk of lung cancer in a Korean population.. The TGF-beta1 genotypes were determined in 432 lung cancer patients and in 432 healthy control subjects who were frequency-matched for age and gender. The TGF-beta1 haplotypes were predicted using a Bayesian algorithm in the Phase program.. Individuals with at least one -509T allele were at a significantly decreased risk of adenocarcinoma (AC) and small cell carcinoma (SM), as compared with carriers with the -509CC genotype [adjusted odds ratio (OR), 0.63; 95% confidence interval (CI), 0.42-0.96; P = 0.04; and adjusted OR, 0.45; 95% CI, 0.27-0.76; P = 0.002; respectively]. For the 869T > C polymorphism, the combined TC + CC genotype was associated with a significantly decreased risk of SM compared with the TT genotype (adjusted OR, 0.52; 95% CI, 0.31-0.88; P = 0.01). Consistent with the results of the genotyping analyses, the -509T/869C haplotype was associated with a significantly decreased risk of AC and SM as compared with the -509C/869T haplotype (adjusted OR, 0.75; 95% CI, 0.57-0.98; P = 0.04; and adjusted OR, 0.67; 95% CI, 0.47-0.96; P = 0.02; respectively).. The TGF-beta1 -509C > T and 869T > C polymorphisms and their haplotypes may contribute to genetic susceptibility to AC and SM of the lung.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Case-Control Studies; Female; Genetic Predisposition to Disease; Haplotypes; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Gene silencing by CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. It has been shown previously that Betaig-h3 gene, which encodes an extracellular matrix protein involved in cell adhesion and tumorigenesis, is down-regulated or silenced in a variety of human cancer cell lines. To unravel the underlying molecular mechanism(s) for this phenomenon, DNA methylation patterns of Betaig-h3 CpG island were examined in normal, immortalized, and cancer cell lines derived from lung, prostate, mammary, and kidney. A good correlation was observed between promoter hypermethylation and lost expression of Betaig-h3 gene, which was supported by the data that demethylation of promoter by 5-aza-2'-deoxycytidine reactivated Betaig-h3 and restored its expression in Betaig-h3-silenced tumor cell lines. This result was further substantiated by a luciferase reporter assay, showing the restoration of promoter activities and increased response to transforming growth factor-beta treatment in Betaig-h3-negative 293T cells when transfected with unmethylated Betaig-h3 promoter. In contrast, activity of Betaig-h3 promoter was completely inactivated by in vitro methylation. Furthermore, CpG methylation of Betaig-h3 promoter was also shown in primary lung tumors that expressed decreased level of Betaig-h3 protein. These results suggest that promoter methylation plays a critical role in promoter silencing of the Betaig-h3 gene in human tumor cells.

Topics: Azacitidine; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Epigenesis, Genetic; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Male; Neoplasms; Promoter Regions, Genetic; Prostatic Neoplasms; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Movement; Chromatography, Liquid; Epithelial Cells; Humans; Image Processing, Computer-Assisted; Lung Neoplasms; Mass Spectrometry; Mesoderm; Neoplasm Invasiveness; Phenotype; Proteins; Proteomics; Software; Transforming Growth Factor beta

Recent studies found that peroxiredoxin-I (Prx-I) is secreted from A549 cells although it does not contain a signal peptide and is known to be a cytosolic protein. Transforming growth factor-beta1 (TGF-beta1) treatment dramatically enhanced Prx-I secretion from A549 cells, and this effect was not inhibited by brefeldin A. Further investigation revealed that A549 cells constitutively secrete TGF-beta1. Furin, a TGF-beta1-converting enzyme, was also highly activated in A549 cells. Ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-PDX), a potent furin inhibitor, blocked both TGF-beta1 activation and Prx-I secretion. Our findings collectively suggest that non-classical secretion of Prx-I is induced by TGF-beta1, which is constitutively activated by furin in A549 cells.

Topics: Adenocarcinoma; Cell Line, Tumor; Dose-Response Relationship, Drug; Furin; Humans; Lung Neoplasms; Peroxidases; Peroxiredoxins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The development and progression of malignancies is a complex multistage process that involves the contribution of a number of genes giving growth advantage to cells when transformed. The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex with tumor-suppressor or prooncogenic activities depending on the cell type and the stage of the disease. We have previously reported the identification of a novel WD-domain protein, STRAP, that associates with both TGF-beta receptors and that synergizes with the inhibitory Smad, Smad7, in the negative regulation of TGF-beta-induced transcription. Here, we show that STRAP is ubiquitously expressed and is localized in both cytoplasm and nucleus. STRAP is up-regulated in 60% colon and in 78% lung carcinomas. Stable expression of STRAP results in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and in down-regulation of the cyclin-dependent kinase inhibitor p21(Cip1), which results in retinoblastoma protein hyperphosphorylation. In addition, we have observed that Smad2/3 phosphorylation, TGF-beta-mediated transcription, and growth inhibition are induced in STRAP-knockout mouse embryonic fibroblasts compared with wild-type cells. Ectopic expression of STRAP in A549 lung adenocarcinoma cell line inhibits TGF-beta-induced growth inhibition and enhances anchorage-independent growth of these cells. Moreover, overexpression of STRAP increases tumorigenicity in athymic nude mice. Knockdown of endogenous STRAP by small interfering RNA increases TGF-beta signaling, reduces ERK activity, increases p21(Cip1) expression, and decreases tumorigenicity. Taken together, these results suggest that up-regulation of STRAP in human cancers may provide growth advantage to tumor cells via TGF-beta-dependent and TGF-beta-independent mechanisms, thus demonstrating the oncogenic function of STRAP.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Transformation, Neoplastic; Chlorocebus aethiops; Colorectal Neoplasms; COS Cells; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mink; Mitogen-Activated Protein Kinase Kinases; Neoplasm Proteins; NIH 3T3 Cells; Oncogenes; Phosphorylation; Proteins; Retinoblastoma Protein; RNA-Binding Proteins; Transforming Growth Factor beta

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

Escape from TGF-beta inhibition of proliferation is a hallmark of multiple cancers including lung cancer. We explored the role of ELF, crucial TGF-beta adaptor protein identified from endodermal progenitor cells, in lung carcinogenesis and cell-cycle regulation. Interestingly, elf-/- mice develop multiple defects that include lung, liver, and cardiac abnormalities. Four out of 6 lung cancer and mesothelioma cell lines displayed deficiency of ELF expression with increased CDK4 expression. Immunohistochemistry and Western blot analysis of primary human lung cancers also showed decreased ELF expression and overexpression of CDK4. Moreover, rescue of ELF in ELF-deficient cell lines decreased the expression of CDK4 and resulted in accumulation of G1/S checkpoint arrested cells. These results suggest that disruption in TGF-beta signaling mediated by loss of ELF in lung cancer leads to cell-cycle deregulation by modulating CDK4 and ELF highlights a key role of TGF-beta adaptor protein in suppressing early lung cancer.

Topics: Animals; Carcinoma; Carrier Proteins; Cell Cycle; Cyclin-Dependent Kinase 4; G1 Phase; Humans; Lung Neoplasms; Mice; Microfilament Proteins; S Phase; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.

Topics: Activin Receptors, Type I; Animals; Bone Neoplasms; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Enzyme Inhibitors; Female; Humans; Lung Neoplasms; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-beta, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-gamma production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-beta. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-gamma production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-beta and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-beta are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Forkhead Transcription Factors; Immunity, Innate; Immunosuppression Therapy; Interleukin-10; Lung Neoplasms; Lymphocyte Count; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred BALB C; Mice, Transgenic; RNA, Messenger; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Histone deacetylase (HDAC) inhibitors have been actively exploited as potential anticancer agents. To identify gene targets of HDAC inhibitors, we found that HDAC inhibitors such as sodium butyrate, scriptaid, apicidin and oxamflatin induced the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a potential cyclooxygenase-2 (COX-2) antagonist and tumor suppressor, in a time and concentration dependent manner in A549 and H1435 lung adenocarcinoma cells. Detailed analyses indicated that HDAC inhibitors activated the 15-PGDH promoter-luciferase reporter construct in transfected A549 cells. A representative HDAC inhibitor, scriptaid, and its negative structural analog control, nullscript, were further evaluated at the chromatin level. Scriptaid but not nullscript induced a significant accumulation of acetylated histones H3 and H4 which were associated with the 15-PGDH promoter as determined by chromatin immunoprecipitation assay. Transforming growth factor-beta1 (TGF-beta1) also induced the expression of 15-PGDH in a time and concentration dependent manner in A549 and H1435 cells. Induction of 15-PGDH expression by TGF-beta1 was synergistically stimulated by the addition of Wnt3A which was inactive by itself. However, combination of TGF-beta and an HDAC inhibitor, scriptaid, only resulted in an additive effect. Together, our results indicate that 15-PGDH is one of the target genes that HDAC inhibitors and TGF-beta may induce to exhibit tumor suppressive effects.

Topics: Adenocarcinoma; Enzyme Induction; Enzyme Inhibitors; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxyprostaglandin Dehydrogenases; Lung Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

The present study was conducted to investigate the association between single nucleotide polymorphisms (SNPs) of the transforming growth factor-beta 1 (TGFB1) gene and susceptibility to lung cancer and the clinical effect of the SNPs on lung cancer progression in a Korean population. Two polymorphisms in the promoter region of the TGFB1 (T-1572C, C-509T), and one SNP in codon 10 (T+869C) were determined using a SNaPshot primer extension assay in 194 Korean lung cancer patients and 283 normal controls. The polymorphic allele frequencies of A-1572G, C-509T, and T+869C were similar among lung cancer patients (0.52, 0.47, and 0.47, respectively) and controls (0.54, 0.46, and 0.44, respectively). When the data was stratified for smoking history, patients who smoked heavily and had heterozygous C-509T and T+869C genotypes showed an increased lung cancer risk (odds ratio OR = 3.77, confidence interval 95% CI = 1.25-11.30, P = 0.017; OR = 3.61, 95% CI = 1.21-10.74, P = 0.021 for each), after adjustment for age and sex. When heterozygous and homozygous variants for each SNPs were analyzed together, patients who were smokers and had variant genotypes also showed increased risk compared to the reference group. Further analyses to test the effect of the SNPs on the clinical parameters did not reveal an association of each polymorphic allele to the tumor stage or response to treatment. In addition, DNA fragments containing polymorphic genotype of the promoter region (-509T) showed increased transcriptional activity in luciferase assays using non-small cell lung cancer cell lines. In conclusion, this study suggests that heavy smokers in this Korean population who have specific polymorphic variants, which have been associated with increased transcriptional activity of TGFB1, might be more vulnerable to lung cancer.

Topics: Base Sequence; Case-Control Studies; DNA Primers; Genotype; Humans; Korea; Lung Neoplasms; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Risk Factors; Transforming Growth Factor beta

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase (MMP) family. Although it is expressed in a number of tissues, no biological substrates or functions for this enzyme have been identified yet. We have expressed recombinant epilysin in A549 lung adenocarcinoma cells and found that this resulted in stable and irreversible epithelial to mesenchymal transition (EMT) accompanied by loss of cell surface E-cadherin, proteolytic processing of latent TGF-beta-complexes and increased levels of active TGF-beta. The cascade of events leading to the onset of EMT is prevented by the MMP inhibitor GM6001 or antibodies neutralizing the activity of TGF-beta. Once EMT had occurred the cell phenotype could, however, not be reversed by the MMP-inhibitor. Importantly, the expression of epilysin also resulted in upregulation of MT1-MMP and gelatinase-B (MMP-9) and in the collagen invasive activity of A549 cells. Further, we found that epilysin and the recombinant hemopexin domain were targeted to the surface of epithelial cells. This cell surface interaction was sensitive to the proteolytic activity of MT1-MMP, and was lost after EMT. Current results indicate that epilysin can induce EMT and cell invasion through a TGF-beta-dependent mechanism suggesting novel biological roles for this enzyme in the regulation of epithelial cell function and in the induction of carcinogenesis.

Topics: Animals; Cell Movement; Collagen; Culture Media, Conditioned; Dogs; Epithelial Cells; Hemopexin; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Mesoderm; Neoplasm Invasiveness; Phenotype; Protein Structure, Tertiary; Recombinant Proteins; Transforming Growth Factor beta; Up-Regulation

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

Topics: Analysis of Variance; Biomarkers; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Radiation Pneumonitis; Time Factors; Transforming Growth Factor beta

Topics: Biomarkers; Carcinoma, Non-Small-Cell Lung; Centrifugation, Density Gradient; Humans; Lung Neoplasms; Radiation Pneumonitis; Reference Values; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta is a potent immunosuppressant. Overproduction of TGF-beta by tumor cells may lead to tumor evasion from the host immune surveillance and tumor progression. The present study was conducted to develop a treatment strategy through adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells. The mouse TRAMP-C2 prostate cancer cells produced large amounts of TGF-beta1 and were used as an experimental model. C57BL/6 mice were primed with irradiated TRAMP-C2 cells. CD8+ T cells were isolated from the spleen of primed animals, were expanded ex vivo, and were rendered TGF-beta insensitive by infecting with a retrovirus containing dominant-negative TGF-beta type II receptor. Results of in vitro cytotoxic assay revealed that these CD8+ T cells showed a specific and robust tumor-killing activity against TRAMP-C2 cells but were ineffective against an irrelevant tumor line, B16-F10. To determine the in vivo antitumor activity, recipient mice were challenged with a single injection of TRAMP-C2 cells for a period up to 21 days before adoptive transfer of CD8+ T cells was done. Pulmonary metastasis was either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells. Results of immunofluorescent studies showed that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. Furthermore, transferred tumor-reactive TGF-beta-insensitive CD8+ T cells were able to persist in tumor-bearing hosts but declined in tumor-free animals. These results suggest that adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells may warrant consideration for cancer therapy.

Topics: Adoptive Transfer; Animals; Apoptosis; CD8-Positive T-Lymphocytes; Fluorescent Antibody Technique; Genes, Dominant; Green Fluorescent Proteins; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retroviridae; Skin Neoplasms; Spleen; T-Lymphocytes, Cytotoxic; Transfection; Transforming Growth Factor beta

Differentially Expressed Nucleolar TGF-beta1 Target (DENTT) is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) superfamily whose mRNA is induced by TGF-beta1 in TGF-beta1-responsive human lung cancer cells. Monkey DENTT mRNA contains a 2085-bp open reading frame that encodes a predicted polypeptide of 695 amino acids with five nuclear localization signals, two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. RT-PCR amplification and Western blot analyses showed DENTT mRNA and protein in adult monkey tissues, including the adrenal gland, cerebral cortex, and ovary. Immunohistochemical staining showed that numerous neurons were intensely immunoreactive for DENTT, as were anterior pituitary secretory cells, thyroid follicular cells, and smooth muscle cells of arteries and lung bronchial walls. DENTT expression was also prominent in monkey bronchiolar-alveolar adenomas and cell lines. While the addition of TGF-beta1 or retinoic acid to monkey normal lung bronchial 12MBr6 cells and human lung cancer NCI-H727 cells increased DENTT protein production, TGF-beta1 together with retinoic acid resulted in a more sustained increase in DENTT production than with TGF-beta1 or retinoic acid alone. Transient transfection studies showed that ectopic DENTT expression significantly increased TGF-beta1-responsive 3TP-Lux and CAGA12-Lux reporter transcription in 12MBr6 and NCI-H727 cells with TGF-beta1 addition, while ectopic DENTT expression had no significant effect on the transcription of a retinoic acid-responsive element reporter in the presence of retinoic acid or TGF-beta1. These findings suggest new possibilities for DENTT as a TGF-beta1-regulated, but not a retinoic acid-regulated member of the TTSN superfamily in primate physiology.

Topics: Adrenal Glands; Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; Blotting, Western; Cell Line; Cell Nucleus; Cerebral Cortex; Chlorocebus aethiops; Cytoplasm; Disease Models, Animal; DNA Primers; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Macaca; Molecular Sequence Data; Myocytes, Smooth Muscle; Nuclear Proteins; Ovary; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology; Thyroid Gland; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin

Endostatin is a potent antiangiogenesis protein with little or no toxicity that has potential to enhance radiotherapy. The major goal of this study was to evaluate the combination of radiation and endostatin gene therapy in a preclinical lung cancer model.. Plasmid pXLG-mEndo, constructed in our laboratory, includes the mouse endostatin gene cloned into the pWS4 vector. The kinetics of endostatin expression and efficacy of the pXLG-mEndo and radiation ((60)Co gamma-rays) combination was evaluated in the C57BL/6 mouse-Lewis lung carcinoma (LLC) model. The LLC cells were implanted s.c. and pXLG-mEndo was injected intratumorally 12-14 days later without any transfection agent; a dose of 10 Gy radiation was applied approximately 16 h thereafter. Some groups received each modality twice. Endostatin, vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were quantified in plasma and tumors, and tumor vasculature was examined.. Endostatin expression within LLC tumors peaked on Day 7 after pXLG-mEndo injection. Addition of radiation to pXLG-mEndo significantly enhanced the level of tumor endostatin compared with plasmid alone (p < 0.05). Tumor growth was significantly delayed in mice receiving pXLG-mEndo plus radiation compared with no treatment (p < 0.005), radiation (p < 0.05), and control plasmid (p < 0.05). The number of LLC tumor vessels was reduced after combined treatment (p < 0.05), and significant treatment-related changes were observed in both VEGF and TGF-beta1.. The data demonstrate that delivery of endostatin by pXLG-mEndo as an adjuvant to radiation can significantly enhance the antitumor efficacy of radiotherapy in the LLC mouse tumor model and support further investigation of this unique combination therapy.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; Endostatins; Female; Genetic Therapy; Lung Neoplasms; Mice; Mice, Inbred BALB C; Plasmids; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Topics: Biomarkers; Carcinoma, Non-Small-Cell Lung; Centrifugation, Density Gradient; Humans; Lung Neoplasms; Radiation Pneumonitis; Reference Values; Transforming Growth Factor beta

The bone morphogenetic proteins (BMP) are phytogenetically conserved proteins, which are essential for embryonic development. Bone morphogenetic protein-2 (BMP-2) was recently shown to be expressed in a small sample of lung carcinomas. Studies have suggested that BMP-2 may enhance tumor growth. The present study examined which BMP family members are expressed in non-small cell lung carcinomas (NSCLC). Furthermore, the frequency of BMP-2 overexpression and the types of lung carcinomas expressing BMP-2 were determined.. Tissue samples were obtained from the operating room and frozen in liquid nitrogen. Samples included metastatic NSCLC, benign lung tumors, adenocarcinoma, squamous cell carcinoma, bronchioloalveolar, and neuroendocrine carcinomas. Paired normal lung tissues served as the controls. The BMP-2, BMP-4, BMP-6, BMP-7, and growth differentiation factor 5 (GDF-5) expressions were examined by Western blot analysis.. The BMP-4, BMP-6, BMP-7, and GDF-5 were infrequently expressed in NSCLC. The BMP-2 was expressed in 41 of 42 NSCLC with minimal expression in normal lung tissue; BMP-2 was expressed 17 fold higher than that of normal lung tissue. The BMP-2 was over-expressed in all subtypes of NSCLC, including neuroendocrine carcinomas. The BMP-2 expression was similar between squamous cell carcinomas and adenocarcinomas; however, bronchioloalveolar carcinomas tended to have a lower level of expression. The BMP-2 was not significantly expressed in benign lung tumors.. Bone morphogenetic protein-2 is the predominant family member expressed in NSCLC. The BMP-2 is overexpressed in the majority of human lung carcinomas independent of cell type.

Topics: Adenocarcinoma; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Neuroendocrine; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Growth Differentiation Factor 5; Humans; Lung Neoplasms; Transforming Growth Factor beta

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Humans; Lung Neoplasms; Neuroendocrine Tumors; Transforming Growth Factor beta

Insulin-like growth factor binding protein-3 (IGFBP-3) is a mediator of growth suppression signals and a putative tumor suppressor gene. The growth suppression mechanisms of IGFBP-3 have not been well clarified. We examined the expression of IGFBP-3 transcripts in human hepatocellular carcinoma (HCC) and the relationship between IGFBP-3 expression and the transforming growth factor-beta (TGF-beta) and/or retinoblastoma (Rb) signaling pathways. In situ hybridization revealed IGFBP-3 transcripts in cancer cells in 6 of 57 (10%) HCCs, including moderately and poorly differentiated HCCs with intrahepatic metastasis. In contrast, all lung metastatic nodules of 4 HCCs showed IGFBP-3 transcripts in cancer cells. The cDNA microarray showed that genes for the TGF-beta pathway and Rb were up-regulated in IGFBP-3-expressing HCCs. In 6 HCCs presenting IGFBP-3, immunohistochemical analyses showed abnormalities in the TGF-beta and/or Rb pathways; the loss of phosphorylated-Smad2 was observed in 2, and overexpression of phosphorylated-Rb was observed in the remaining 4 HCCs. The present study suggests that IGFBP-3 mediates growth suppression signals via the TGF-beta and/or Rb pathways in HCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Case-Control Studies; Cell Differentiation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Insulin-Like Growth Factor Binding Protein 3; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Cell migration is essential for invasive and metastatic phenotypes of cancer cells. Potential chemopreventive agents of cancer-sulindac sulfide, caffeic acid phenethyl ester (CAPE), curcumin, and (+)-catechin-have been reported to interfere with several types of intracellular signaling. In this study, we examined the effects of these agents on transforming growth factor-beta(TGF-beta)-induced motility and Akt phosphorylation in A549 cells. Judged by gold particle phagokinesis assay, sulindac sulfide, CAPE, and curcumin suppressed the motility of A549 cells promoted by TGF-beta. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase(PI3K)/Akt signaling, also suppressed TGF-beta-induced motility and Akt phosphorylation. Sulindac sulfide and CAPE, but not curcumin, suppressed TGF-beta-induced Akt phosphorylation. We conclude that sulindac sulfide and CAPE suppress the motility promoted by TGF-beta in lung adenocarcinoma cells through the suppression of Akt. Our observations raise the possibility that these agents, except for (+)-catechin, can be applied not only as chemopreventive agents but also as anti-metastatic therapy.

Topics: Adenocarcinoma; Antineoplastic Agents; Caffeic Acids; Catechin; Cell Movement; Curcumin; Humans; Lung Neoplasms; Neovascularization, Pathologic; NF-kappa B; Phenylethyl Alcohol; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sulindac; Transforming Growth Factor beta; Tumor Cells, Cultured

To assess, in lung cancer patients, the effects of thoracic radiotherapy (RT) on the concentrations of transforming growth factor-beta(1) (TGF-beta(1)) and interleukin-6 (IL-6) in the bronchoalveolar lavage (BAL) fluid.. Eleven patients with lung cancer requiring RT as part of their treatment were studied. BAL was performed bilaterally before, during, and 1, 3, and 6 months after RT. Before each BAL session, the patient's status was assessed clinically using pulmonary function tests and an adapted late effects on normal tissue-subjective, objective, management, analytic (LENT-SOMA) scale, including subjective and objective alterations. The National Cancer Institute Common Toxicity Criteria were used to grade pneumonitis. The TGF-beta(1) and IL-6 levels in the BAL fluid were determined using the Easia kit.. The TGF-beta(1) and IL-6 concentrations in the BAL fluid recovered from the irradiated areas were significantly increased by thoracic RT. The increase in TGF-beta(1) levels tended to be greater in the group of patients who developed severe pneumonitis. In the BAL fluid from the nonirradiated areas, the TGF-beta(1) and IL-6 concentrations remained unchanged.. The observed increase in TGF-beta(1) and IL-6 concentrations in the BAL fluid recovered from the irradiated lung areas demonstrated that these cytokines may contribute to the process leading to a radiation response in human lung tissue.

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Follow-Up Studies; Humans; Interleukin-6; Lung Neoplasms; Male; Middle Aged; Pilot Projects; Radiation Pneumonitis; Radiotherapy Dosage; Transforming Growth Factor beta; Transforming Growth Factor beta1

Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by stimulating osteoblastic production of IL-11. The subsequent release of PGE(2) followed by inhibition of GM-CSF production by cells within the bone microenvironment plays an important part in mediating the effects of breast cancer cells on OCL formation and their resorptive activity.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Coculture Techniques; Colorectal Neoplasms; Dinoprostone; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-11; Interleukin-6; Lung Neoplasms; Mice; Osteoclasts; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Lymphangioleiomyomatosis (LAM) is a rare disorder of unknown etiology, affecting almost exclusively women of childbearing age, that is associated with the proliferation of spindle cells and cystic changes in the affected lung. The underlying processes that contribute to this disease are poorly understood. Transforming growth factor (TGF)-beta(1) is a potent cytokine that promotes mesenchymal cell proliferation and regulates the synthesis of extracellular matrix (ECM) components, particularly fibronectins. Herein, we evaluate the expression of TGF beta(1) and matrix-associated fibronectin in lung specimens demonstrating LAM.. Lung biopsy specimens that were confirmed to contain pathologic LAM cells were obtained from 13 patients. The specimens were submitted to immunohistochemical evaluation for TGF beta(1) and fibronectin, as well as the typical markers of LAM cells. Healthy lung parenchyma surrounding resected neoplasms was studied in a parallel fashion as control tissues.. In all 13 LAM cases and in healthy lung parenchyma, we demonstrated that TGF beta(1) localized consistently to airway epithelial cells. However, in LAM tissues, matrix-associated TGF beta(1) was also consistently found in regions containing pathologic LAM cells. Notably, more abundant TGF beta(1) was observed in highly cellular areas compared to the walls of chronic cystic regions in LAM tissues. Fibronectin, a matrix component that is strongly expressed in response to active TGF beta(1) was found to consistently colocalize with this protein in these highly cellular regions, supporting TGF beta(1) activity in these regions. The markers of proliferating LAM cells, including proliferating cell nuclear antigen, were also markedly present in these highly cellular LAM regions.. These studies suggest that the proliferation of aberrant LAM cells may be associated with altered regional expression of TGF beta(1) and related ECM proteins.

Topics: Actins; Adult; Antigens, Neoplasm; Biomarkers; Extracellular Matrix; Female; Fibronectins; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Lymphangioleiomyomatosis; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Respiratory Mucosa; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the different expression of transforming growth factor beta(1) (TGF-beta(1)) in lung cancer specimens of Gejiu miners, and non-miners in other regions.. Thirty specimens of Gejiu miners' lung cancer and 30 specimens of non-miners' were observed in this experiment. The expression of TGF-beta(1) protein and TGF-beta(1) mRNA were detected by the methods of immunohistochemistry and in situ hybridization. The results were quantitatively analyzed using image analysis system.. The positive rate of TGF-beta(1) protein expression in Gejiu miners and non-miners was 75.39%and 44.78% respectively, and the positive rate of TGF-beta(1) mRNA was 63.96% and 34.07% respectively. There were significant differences between the two groups (P < 0.01).. The expression of TGF-beta(1) in lung cancer of Gejiu miners was significantly higher than that of non-miners. The pathogenesis of lung cancer may be different between Gejiu miner and non-miners. High expression of TGF-beta(1) may be one of the reasons of high incidence of lung cancer in Gejiu miners.

Topics: Humans; Immunohistochemistry; In Situ Hybridization; Lung Neoplasms; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

In dose-escalation studies of radiotherapy (RT) for non-small-cell lung cancer (NSCLC), radiation pneumonitis (RP) is the most important dose-limiting complication. Transforming growth factor-beta1 (TGF-beta1) has been reported to be associated with the incidence of RP. It has been proposed that serial measurements of plasma TGF-beta1 can be valuable to estimate the risk of RP and to decide whether additional dose-escalation can be safely applied. The aim of this study was to evaluate prospectively the time course of TGF-beta1 levels in patients irradiated for NSCLC in relation to the development of RP and dose-volume parameters.. Plasma samples were obtained in 68 patients irradiated for medically inoperable or locally advanced NSCLC (dose range, 60.8-94.5 Gy) before and 4, 6, and 18 weeks after the start of RT. Plasma TGF-beta1 levels were determined using a bioassay on the basis of TGF-beta1-induced plasminogen activator inhibitor-1 expression in mink lung cells. All patients underwent chest computed tomography scans before RT that were repeated at 18 weeks after RT. The computed tomography data were used to calculate the mean lung dose (MLD) and to score the radiation-induced radiologic changes. RP was defined on the basis of the presence of either radiographic changes or clinical symptoms. Symptomatic RP was scored according to the Common Toxicity Criteria (Grade 1 or worse) and the Southwestern Oncology Group criteria (Grade 2 or worse). Multivariate analyses were performed to investigate which factors (pre- or posttreatment TGF-beta1 level, MLD) were associated with the incidence of RP. To improve our understanding of the time course of TGF-beta1 levels, we performed a multivariate analysis to investigate which factors (pre-RT TGF-beta1 level, MLD, RP) were independently associated with the posttreatment TGF-beta1 levels.. The pre-RT TGF-beta1 levels were increased in patients with NSCLC (median 21 ng/mL, range, 5-103 ng/mL) compared with healthy individuals (range, 4-12 ng/mL). On average, the TGF-beta1 levels normalized toward the end of treatment and remained stable until 18 weeks after RT. In 29 patients, however, TGF-beta1 was increased at the end of RT with respect to the pre-RT value. The multivariate analyses revealed that the MLD was the only variable that correlated significantly with the risk of both radiographic RP (p = 0.05) and symptomatic RP, independent of the scoring system used (p = 0.05 and 0.03 for Southwestern Oncology Group and Common Toxicity Criteria systems, respectively). The TGF-beta1 level at the end of RT was significantly associated with the MLD (p <0.001) and pre-RT TGF-beta1 level (p = 0.001).. The MLD correlated significantly with the incidence of both radiographic and symptomatic RP. The results of our study did not confirm the reports that increased levels of TGF-beta1 at the end of RT are an independent additional risk factor for developing symptomatic RP. However, the TGF-beta1 level at the end of a RT was significantly associated with the MLD and the pre-RT level.

Topics: Aged; Aged, 80 and over; Analysis of Variance; Biomarkers; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Radiation Pneumonitis; Radiotherapy Dosage; Reference Values; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

To achieve in situ tumor antigen uptake and presentation, intratumoral administration of ex vivo-generated, gene-modified murine bone marrow-derived dendritic cells (DC) was used in a murine lung cancer model. To attract mature host DC and activated T cells at the tumor site, the DC were transduced with an adenoviral vector expressing secondary lymphoid tissue chemokine (CCL21/SLC). Sixty percent of the mice treated with 10(6) DC-AdCCL21 intratumorally (7-10 ng/ml/10(6) cells/24 h of CCL21) at weekly intervals for 3 weeks showed complete tumor eradication, whereas only 25% of mice had complete resolution of tumors when mice were treated with fibroblasts expressing CCL21. In contrast only 12% of the mice treated with unmodified or control vector modified DC (DC-AdCV) showed complete tumor eradication. DC-AdCCL21 administration led to increases in the CD4(+), CD8(+), and CD3(+)CXCR3(+) T cells, as well as DC expressing CD11c(+) DEC205(+). CD4(+)CD25(+) T-regulatory cells infiltrating the tumors were markedly reduced after DC-AdCCL21 therapy. The tumor site cellular infiltrates were accompanied by the enhanced elaboration of granulocyte macrophage colony-stimulating factor, IFN-gamma, MIG/CXCL9, IP-10/CXCL10, and interleukin 12, but decreases in the immunosuppressive mediators transforming growth factor beta and prostaglandin E(2). DC-AdCCL21-treated tumor-bearing mice showed enhanced frequency of tumor-specific T lymphocytes secreting IFN-gamma, and tumor protective immunity was induced after DC-AdCCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9, or IFN-gamma significantly reduced the antitumor efficacy of DC-AdCCL21. These findings provide a strong rationale for the evaluation of DC-AdCCL21 in cancer immunotherapy.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Cancer Vaccines; CD3 Complex; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CCL21; Chemokines, CC; Cytokines; Dendritic Cells; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Immunoenzyme Techniques; Immunotherapy; Lung Neoplasms; Mice; Mice, Inbred BALB C; T-Lymphocytes; Time Factors; Transforming Growth Factor beta

To investigate the relevance of transforming growth factor-beta (TGF-beta) dynamics in plasma for identification of patients at low risk for developing pneumonitis as a complication of thoracic radiotherapy (RT).. Non-small cell lung cancer patients undergoing conventional RT were included in the prospective study. Concentrations of TGF-beta were measured in the patients' plasma prior to and weekly during 6 weeks of RT. The incidence of symptoms of early post-irradiation lung injury, i.e. symptomatic radiation pneumonitis, was correlated with TGF-beta parameters.. Forty-six patients were included in the study. Eleven patients (24%) developed symptomatic radiation pneumonitis. Absolute TGF-beta plasma levels did not differ between the groups of patients without or with pneumonitis. However, patients who developed pneumonitis tended to show increases in TGF-beta levels in the middle of the RT course relative to their pre-treatment levels while TGF-beta plasma levels of patients who did not develop pneumonitis tended to decrease over the RT treatment. The difference in the relative TGF-beta dynamics between the groups reached marginal significance in the third week of the treatment (P = 0.055) but weakened towards the end of the RT course. The utility of TGF-beta testing was evaluated at each RT week based on the test's ability to yield more accurate estimate of complication probability in an individual patient compared to empirically expected probability in similar group of patients. The ratio of TGF-beta level at week 3/week 0 being <1 showed an ability to improve the prediction of freedom from pneumonitis, yet with a large degree of uncertainty (wide confidence intervals). The accuracy of prediction deteriorated at later time points (weeks 4, 5 and 6) rendering the end-RT ratios without predictive power.. We observed a trend of plasma TGF-beta concentration to decrease below the pre-treatment value during the RT treatment in patients who did not develop pulmonary complications after the RT treatment. However, this trend was not consistent enough to warrant safe decision-making in clinical setting.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cohort Studies; Dose-Response Relationship, Radiation; Female; Humans; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Probability; Prognosis; Radiation Pneumonitis; Radiotherapy Dosage; Risk Assessment; Sensitivity and Specificity; Statistics, Nonparametric; Transforming Growth Factor beta

Rapid senescence was induced into human lung adenocarcinoma A549 cells by transforming growth factor-beta1. Lectin blot analysis of membrane glycoprotein samples showed that the binding of Ricinus communis agglutinin-I to protein bands increased markedly while those of other lectins together with protein components did not change significantly with senescence. This indicates that the beta-1,4-galactosylation of N-linked oligosaccharides is stimulated by rapid senescence. Analysis of the enzymatic background of senescence showed 1.5 times higher beta-1,4-galactosyltransferase (beta-1,4-GalT) activity and 2-5 times higher expression levels of beta-1,4-GalT II, III, V, and VI genes are associated with rapid senescence. Incubation of the cells on RCA-I-coated plates in the absence of fetal calf serum showed that the viability of the senescent cells is half that of the control cells. Therefore, it is hypothesized that galactose residues expressed by rapid senescent can induce a lethal signal in cells if they interact with appropriate receptors.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Survival; Cellular Senescence; Galactose; Humans; Lung Neoplasms; Oligosaccharides; Transforming Growth Factor beta; Transforming Growth Factor beta1

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-beta1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-beta1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-beta1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-beta1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-beta1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-beta1. Impaired NK cytotoxicity by TGF-beta1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-beta1.

Topics: Case-Control Studies; Cells, Cultured; Colorectal Neoplasms; Cytotoxicity, Immunologic; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lung Neoplasms; NK Cell Lectin-Like Receptor Subfamily C; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, Natural Killer Cell; Transforming Growth Factor beta; Transforming Growth Factor beta1

The c-Jun NH2-terminal kinase (JNK) has been implicated in the function of transforming growth factor beta (TGF-beta). To test the role of JNK, we examined the effect of compound disruption of the murine genes that encode the ubiquitously expressed isoforms of JNK (Jnk1 and Jnk2). We report that JNK-deficient fibroblasts isolated from Jnk1-/- Jnk2-/- mice constitutively express TGF-beta1. Complementation studies demonstrate that JNK is a repressor of Tgf-beta1 gene expression. This mechanism of regulation of TGF-beta1 expression by JNK represents an unexpected form of cross-talk between two important signaling pathways. Together, these data demonstrate that the JNK pathway may contribute to the regulation of autocrine TGF-beta1-mediated biological responses in vivo.

Topics: Activin Receptors, Type I; Animals; Autocrine Communication; Bromodeoxyuridine; Cell Cycle; Cell Division; Cell Movement; Electrophoretic Mobility Shift Assay; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Genes, Dominant; Homozygote; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Male; Mice; Mice, Knockout; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

The Nkx2.1 homeobox gene and transforming growth factor-beta1 (TGF-beta1) are essential for organogenesis and differentiation of the mouse lung. NKX2.1 is a marker of human lung carcinomas, but it is not known whether this gene participates in early tumorigenesis. Addition of TGF-beta1 to TGF-beta1-responsive nontumorigenic mouse lung cells cotransfected with a NKX2.1Luc luciferase reporter and either a Sp1 or Sp3 plasmid showed a significant increase or decrease, respectively, in NKX2.1Luc transcription. Cotransfection of Sp3 and dominant-negative TGF-beta type II receptor plasmids negated the effect of Sp1. Cotransfected Sp1 plasmid with either dominant-negative Smad2 or Smad3 or Smad4 plasmids significantly decreased NKX2.1Luc transcription. Electrophoretic mobility shift assays revealed binding of Sp1 and Smad4 to the NKX2.1 promoter. With a TGF-beta1 heterozygous mouse model, Nkx2.1 mRNA and protein in lungs of TGF-beta1 heterozygous mice were significantly lower compared to wildtype (WT) littermates. Competitive reverse transcription (RT)-polymerase chain reaction (PCR) and immunostaining showed that Nkx2.1 mRNA and protein decreased significantly in adenomas and adenocarcinomas compared to normal lung tissue. Our in vitro data showed that regulation of Nkx2.1 by TGF-beta1 occurs through TGF-beta type II receptor and Smad signaling, with Sp1 and Sp3 in lung cells. Our in vivo data showed reduced Nkx2.1 in lungs of TGF-beta1 heterozygous mice compared to WT mice, that is detectable in adenomas, and that is further reduced in carcinogenesis, and that correlates with reduction of Sp1, Sp3, and Smads in lung adenocarcinomas. Our findings suggest that reduced Nkx2.1 and TGF-beta1 signaling components may contribute to tumorigenesis in the lungs of TGF-beta1 heterozygous mice.

Topics: Animals; Blotting, Northern; Cell Transformation, Neoplastic; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Lung; Lung Neoplasms; Mice; Nuclear Proteins; Receptors, Transforming Growth Factor beta; RNA, Messenger; Thymidine; Thyroid Nuclear Factor 1; Transcription Factors; Transforming Growth Factor beta; Tritium

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Gene Expression; Humans; Luciferases; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Neovascularization, Pathologic; Peptide Fragments; Repetitive Sequences, Nucleic Acid; Survival Rate; Thrombospondin 1; Transfection; Transforming Growth Factor beta

The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were investigated in i.v., s.c. and i.p. injection models in mice. Seven groups of female C57BL/6 or nude mice were fed a basal diet (control group) or the basal diet supplemented with KTI or BBI (5, 15, or 50 g/kg). Here we show that, in an in vivo spontaneous metastasis assay, the diet supplementation with KTI (15 and 50 g/kg), but not with BBI, for 28 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21 days after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI (15 or 50 g/kg) treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. Immunoblot analysis revealed that KTI specifically reduced expression of uPA protein as well as phosphorylation of MAP kinase and PI3 kinase proteins in the cells stimulated with agonists (G-CSF for 3LL cells or TGF-beta1 for HRA cells). These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Dietary Supplements; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Models, Animal; Ovarian Neoplasms; Peritoneal Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitor, Kunitz Soybean; Trypsin Inhibitors; Urokinase-Type Plasminogen Activator

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

Topics: Biomarkers; Carcinoma, Non-Small-Cell Lung; Humans; Lung; Lung Neoplasms; Radiation Injuries; 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

Topics: Animals; Antisense Elements (Genetics); DNA Primers; DNA, Complementary; Gene Expression; Immunoenzyme Techniques; In Situ Hybridization; Lung; Lung Neoplasms; Mice; Paraffin Embedding; Polymerase Chain Reaction; Protein Isoforms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Sensitivity and Specificity; Transforming Growth Factor beta

The interaction between Fas and Fas ligand (FasL) is involved in the apoptotic death of a number of cells including lymphocytes. Forced expression of FasL in tumors can induce apoptosis of infiltrating Fas-positive T cells; accordingly, tumors can survive in the milieu of systemic immune responses. However, FasL-expressing murine lung carcinoma (A11) and melanoma (B16) cells did not develop subcutaneous tumors and FasL-expressing A11 (A11/FasL) cells produced few spontaneous lung metastatic foci in syngeneic mice. The mice that rejected A11/FasL cells were resistant to subsequent challenge of parent A11 but not irrelevant B16 cells. Vaccination of mice with UV-treated A11/FasL, but not UV-treated A11 cells, however, augmented the growth rate of A11 but not B16 tumors, both of which were subsequently inoculated. The number of lung metastatic foci of A11 cells was also increased in the mice that received UV-treated A11/FasL but not UV-treated A11 cells. Intraperitoneal injection of UV-treated A11/FasL cells resulted in the production of larger amounts of immunosuppressive TGF-beta in peritoneal exudate than that of UV-treated A11 cells. Expression of the CD80 costimulatory molecule in tissues where UV-treated A11/FasL cells were inoculated was lower than the expression at an untreated A11/FasL-injected site. Our results indicated that apoptotic FasL-expressing tumor cells could impair host immune responses against the tumors, in contrast to potent antitumor immunity generated by viable FasL-expressing tumors.

Topics: Animals; B7-1 Antigen; Cancer Vaccines; Carcinoma; Cell Division; Drug Screening Assays, Antitumor; Fas Ligand Protein; Female; Injections, Intraperitoneal; Lung Neoplasms; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Peritoneal Cavity; Transforming Growth Factor beta; Tumor Cells, Cultured; Ultraviolet Rays; Vaccination

Rapamycin is an effective inhibitor of human renal cancer metastasis.. Human renal cell cancer (RCC) is common and is 10 to 100 times more frequent in patients with end-stage renal disease (ESRD) and candidates for renal transplantation. Treatment of metastatic RCC is largely ineffective and is further undermined by immunosuppressive therapy in transplant recipients. A treatment regimen that prevents transplant rejection while constraining RCC progression would be of high value.. We developed a human RCC pulmonary metastasis model using human RCC 786-O as the tumor challenge and the severe combined immunodeficient (SCID) beige mouse as the host. We explored the effect of rapamycin, cyclosporine, or rapamycin plus cyclosporine on the development of pulmonary metastases and survival. The effects of the drugs on tumor cell growth, apoptosis, and expression of vascular endothelial growth factor (VEGF-A) and transforming growth factor beta1 (TGF-beta1) were also investigated.. Rapamycin reduced, whereas cyclosporine increased, the number of pulmonary metastases. Rapamycin was effective in cyclosporine-treated mice, and rapamycin or rapamycin plus cyclosporine prolonged survival. Rapamycin growth arrested RCC 786-O at the G1 phase and reduced VEGF-A expression. Immunostaining of lung tissues for von Willebrand factor was minimal and circulating levels of VEGF-A and TGF-beta1 were lower in the rapamycin-treated mice compared to untreated or cyclosporine-treated mice.. Our findings support the idea that rapamycin may be of value for patients with RCC and that its antitumor efficacy is realized by cell cycle arrest and targeted reduction of VEGF-A and TGF-beta1. A regimen of rapamycin and cyclosporine, demonstrated to be effective in reducing acute rejection of renal allografts, may prevent RCC progression as well, and has the potential to prevent mortality due to RCC in patients with ESRD who have received renal allografts.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclosporine; Gene Expression; Humans; Immunosuppressive Agents; Kidney Neoplasms; Lung Neoplasms; Mice; Mice, SCID; Sirolimus; Survival Rate; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

The influence of transforming growth factor beta (TGF-beta) signaling on Neu-induced mammary tumorigenesis and metastasis was examined with transgenic mouse models. We generated mice expressing an activated TGF-beta type I receptor or dominant negative TGF-beta type II receptor under control of the mouse mammary tumor virus promoter. When crossed with mice expressing activated forms of the Neu receptor tyrosine kinase that selectively couple to the Grb2 or Shc signaling pathways the activated type I receptor increased the latency of mammary tumor formation but also enhanced the frequency of extravascular lung metastasis. Conversely, expression of the dominant negative type II receptor decreased the latency of Neu-induced mammary tumor formation while significantly reducing the incidence of extravascular lung metastases. These observations argue that TGF-beta can promote the formation of lung metastases while impairing Neu-induced tumor growth and suggest that extravasation of breast cancer cells from pulmonary vessels is a point of action of TGF-beta in the metastatic process.

Topics: Activin Receptors, Type I; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis; Drosophila Proteins; Female; Genes, Dominant; Genes, erbB-2; GRB2 Adaptor Protein; Humans; Lactation; Ligases; Lung Neoplasms; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Mitotic Index; Neoplasm Proteins; Nerve Tissue Proteins; Pregnancy; Protein Serine-Threonine Kinases; Proteins; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Deletion; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

To help identify genes, which may regulate metastasis in lung cancer, we performed representational difference analysis between a patient-derived non-small cell lung carcinoma (NSCLC) and immortalized normal human bronchial epithelial cells. This analysis revealed that bone morphogenetic proteins-2/4 (BMP) mRNA was expressed in the lung carcinoma. BMP-2/4 are known to induce pluripotent cell differentiation, enhance cell migration and stimulate proliferation during embryonic development. Despite being powerful morphogens it is not known whether BMP-2/4 have significant biological activity in human carcinomas. Furthermore, it has not been established whether the mature active BMP-2/4 protein is aberrantly expressed in patient-derived tumors. The purpose of this study was to determine whether the expression of the mature BMP-2/4 protein is disregulated in human lung carcinomas and to establish whether it has adverse biological activity. This study reveals that the mature BMP-2 protein, but not BMP-4, is highly over-expressed in human NCSLC with little to no expression in normal lung tissue or benign lung tumors. The expression of BMP-2 localized specifically to the cancer cells. Recombinant BMP-2 stimulated in vitro, the migration and invasiveness of the A549 and H7249 human lung cancer cell lines. In vivo, recombinant BMP-2 enhanced the growth of tumors formed from A549 cells injected subcutaneously into nude mice. Furthermore, inhibition of BMP-2 activity with either recombinant noggin or anti-BMP-2 antibody resulted in a significant reduction in tumor growth. This study shows that expression of the mature BMP-2 protein is disregulated in the majority of NSCLC. BMP-2 enhancement of tumor cell migration and invasion, as well as stimulating tumor growth in vivo, suggests it has important biological activity in lung carcinomas.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carcinoma, Non-Small-Cell Lung; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

p38 is associated with a macromolecular tRNA synthetase complex. It has an essential role as a scaffold for the complex, and genetic disruption of p38 in mice causes neonatal lethality. Here we investigated the molecular mechanisms underlying lethality of p38-mutant mice. p38-deficient mice showed defects in lung differentiation and respiratory distress syndrome. p38 was found to interact with FUSE-binding protein (FBP), a transcriptional activator of c-myc. Binding of p38 stimulated ubiquitination and degradation of FBP, leading to downregulation of c-myc, which is required for differentiation of functional alveolar type II cells. Transforming growth factor-beta (TGF-beta) induced p38 expression and promoted its translocation to nuclei for the regulation of FBP and c-myc. Thus, this work identified a new activity of p38 as a mediator of TGF-beta signaling and its functional importance in the control of c-myc during lung differentiation.

Topics: Amino Acyl-tRNA Synthetases; Animals; Animals, Newborn; Cell Differentiation; Cell Division; DNA Helicases; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation; Homozygote; Humans; Immunoenzyme Techniques; Lung Neoplasms; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Pulmonary Alveoli; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Saccharomyces cerevisiae; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Two-Hybrid System Techniques; Ubiquitins

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

Most murine lung tumors are composed of differentiated epithelial cells. We have reported previously that surfactant protein (SP)-D is expressed in urethane-induced tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury but have not been measured in mice. In this study, we sought to determine whether SP-D could be detected in murine serum and discovered that it was increased in mice bearing lung tumors. Serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D nulls, and 63.6 +/- 9.0 ng/ml in SP-D-overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three different protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbumin in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors and generally reflects the tumor burden but is also elevated during lung inflammation.

Topics: Adenocarcinoma; Animals; Carcinogens; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, ras; Lung Neoplasms; Male; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Transgenic; Ovalbumin; Pulmonary Surfactant-Associated Protein D; Rats; Transforming Growth Factor beta; Urethane

Claudin-4 has been identified as an integral constituent of tight junctions and has been found to be highly expressed in pancreatic cancer. The aim of the present study was to elucidate the effect of claudin-4 on growth and metastatic potential in pancreatic cancer cells, as well as the regulation of claudin-4 by oncogenic pathways. Claudin-4 was stably overexpressed in SUIT-2 pancreatic cancer cells, and its effect on invasion and growth in vitro was examined by using two-chamber invasion assays, soft agar assays, and fluorescence-activated cell sorter analysis. Claudin-4 localization was characterized by light and electron microscopy, and pulmonary colonization was analyzed in vivo after injection of claudin-4 overexpressing cells into the tail vein of nude mice. Overexpression of claudin-4 was associated with significantly reduced invasive potential in vitro and inhibited colony formation in soft agar assays. In vivo, tail vein-injected claudin-4 overexpressing cells formed significantly less pulmonary metastases in comparison with mock-transfected cells. These effects were not caused by changes in proliferation, cell cycle progression, or matrix metalloproteinase gelatinolytic activity, but were paralleled by increased cell contact formation. Moreover, proinvasive transforming growth factor beta was able to down-regulate claudin-4 in PANC-1 cells. Inhibition of Ras signaling by using dominant-negative Ras and specific inhibitors of both downstream effectors mitogen-activated protein/extracellular signal-regulated kinase kinase and phosphatidylinositol 3'-kinase also decreased claudin-4 expression. Our findings identify claudin-4 as a potent inhibitor of the invasiveness and metastatic phenotype of pancreatic cancer cells, and as a target of the transforming growth factor beta and Ras/Raf/extracellular signal-regulated kinase pathways.

Topics: Carcinoma, Pancreatic Ductal; Cell Adhesion; Cell Cycle; Cell Division; Cell Line, Tumor; Claudin-4; Humans; Immunohistochemistry; Lung Neoplasms; Matrix Metalloproteinase 2; Membrane Proteins; Neoplasm Invasiveness; Pancreatic Neoplasms; ras Proteins; Receptors, Cell Surface; Signal Transduction; Transforming Growth Factor beta

The type V TGF-beta receptor (TbetaR-V)/IGFBP-3 receptor mediates the IGF-independent growth inhibition induced by IGFBP-3. It also mediates the growth inhibitory response to TGF-beta1 in concert with other TGF-beta receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Here we demonstrate that TbetaR-V is identical to LRP-1/alpha2M receptor as shown by MALDI-TOF analysis of tryptic peptides of TbetaR-V purified from bovine liver. In addition, 125I-IGFBP-3 affinity-labeled TbetaR-V in Mv1Lu cells is immunoprecipitated by antibodies to LRP-1 and TbetaR-V. RAP, an LRP-1 antagonist, inhibits binding of 125I-TGF-beta1 and 125I-IGFBP-3 to TbetaR-V and diminishes IGFBP-3-induced growth inhibition in Mv1Lu cells. Absent or low levels of LRP-1, as with TbetaR-V, have been linked to the malignant phenotype of carcinoma cells. Mutagenized Mv1Lu cells selected for reduced expression of LRP-1 have an attenuated growth inhibitory response to TGF-beta1 and IGFBP-3. LRP-1-deficient mouse embryonic fibroblasts lack a growth inhibitory response to TGF-beta1 and IGFBP-3. On the other hand, stable transfection of H1299 human lung carcinoma cells with LRP-1 cDNA restores the growth inhibitory response. These results suggest that the LRP-1/TbetaR-V/IGFBP-3 receptor is required for the growth inhibitory response to IGFBP-3 and TGF-beta1.

Topics: Animals; Carcinoma; Cattle; Cell Division; Cell Line, Tumor; Cells, Cultured; Humans; Insulin-Like Growth Factor Binding Protein 3; Liver; Low Density Lipoprotein Receptor-Related Protein-1; Lung Neoplasms; Mice; Mice, Knockout; Precipitin Tests; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Sequence Homology, Amino Acid; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.

Topics: Adoptive Transfer; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Chemokine CCL19; Chemokine CXCL10; Chemokines; Chemokines, CC; Chemokines, CXC; Cytokines; Dendritic Cells; Dinoprostone; Female; Herpesvirus 4, Human; Injections, Intralesional; Interferon-gamma; Leukocyte Count; Lung Neoplasms; Lymphocyte Count; Macrophage Inflammatory Proteins; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; T-Lymphocyte Subsets; Th1 Cells; Transforming Growth Factor beta

Recent studies have evaluated the cytokine network involved in the local immune response to tumors. In addition to infiltrating inflammatory cells, tumors also produce cytokines and growth factors that may alter tumor growth and tumor immunogenicity. Ninety-one samples of NSCLC were used in this study. We measured the expression of VEGF, TNF-alpha, TGF-beta, IL-6, IL-8, IL-12, INF-gamma, and MCP-1 in NSCLC tissues, by ELISA. The expression of IL-6 and IL-8 were significantly higher in squamous cell carcinoma than in adenocarcinoma (p=0.016 and p<0.001, respectively). The expression of TGF-beta, MCP-1 and IL-8 were significantly higher in pulmonary metastasis positive than negative cases (p=0.002, p=0.001, and p=0.008, respectively). In multivariate logistic regression analysis, the expression of TGF-beta was an independent risk factor for the occurrence of pulmonary metastasis (p=0.008, 95% CI=1.002-1.011). We confirmed that tumor infiltrating stromal cells were major sources of TGF-beta by immunohistochemical analysis. The expression of VEGF and IL-8 were significantly higher in cases with central necrosis (p=0.006 and p=0.011, respectively). We speculated that TGF-beta expression in tumor infiltrating stromal cells may regulate the occurrence of spontaneous pulmonary metastasis in NSCLC. (Ann Thorac Cardiovasc Surg 2003; 9: 295-300)

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Non-Small-Cell Lung; Culture Techniques; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Interferon-gamma; Interleukin-12; Interleukin-6; Interleukin-8; Logistic Models; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Probability; Prognosis; Sampling Studies; Sensitivity and Specificity; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The epithelial alteration in interstitial pneumonias is one of the repair processes at the sites of disease activity. Regenerative epithelial cells may participate in remodeling of the lung. To determine the phenotype of regenerative epithelial cells in usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), the expression of Clara cell 10KD protein (CC10), cytokeratin (CK) 14 and 17, surfactant apoprotein (SP)-A, KL-6/MUC1, transforming growth factor (TGF) beta2 were examined in 25 patients with UIP, 9 patients with NSIP and normal lung tissues from 10 patients with lung cancer. In honeycomb lesions of UIP, non-ciliated columnar cells mainly expressed CC10, cuboidal cells expressed CC10, CK17, CK14 and SP-A in descending order. Fibroblastic foci are covered by CK17, CK14, CC10, and a few SP-A positive flattened or cuboidal cells. Regenerative epithelium in NSIP mainly comprised cuboidal cells expressing SP-A, CC10 and CK17. KL-6 was more remarkably expressed in cuboidal and non-ciliated columnar cells both in UIP and NSIP. Expression of TGFbeta2 was observed in cuboidal and flattened epithelium. In severe fibrotic areas, CC10 expressing cells were more prominent, while SP-A positive cells were more prominent in less fibrotic areas. Regenerative epithelial cells in remodeling area in UIP may be derived from bronchiolar basal cells and Clara cells, while most of those in NSIP may be derived from type II pneumocytes. The different origin of regenerative epithelium may reflect the severity and extent of the injury and the degree of consequent fibrosis in UIP and NSIP.

Topics: Antigens; Antigens, Neoplasm; Apoproteins; Bronchi; Enzyme Inhibitors; Epithelial Cells; Female; Fibroblasts; Fibrosis; Glycoproteins; Humans; Keratins; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Male; Middle Aged; Mucin-1; Mucins; Phenotype; Phospholipases; Proteins; Pulmonary Surfactant-Associated Proteins; Regeneration; Transforming Growth Factor beta; Transforming Growth Factor beta2; Uteroglobin

We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.

Topics: Adenocarcinoma; Animals; Cell Cycle Proteins; Cell Division; DNA Primers; Female; G1 Phase; Gene Expression Profiling; Genes, Regulator; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urethane

Transforming growth factor-beta (TGF-beta) represses surfactant protein B (Sp-B) gene transcription through a mechanism that remains unknown. A homeodomain and a forkhead transcription factor, NKX2.1 and HNF-3, respectively, are known activators of Sp-B transcription. Because SMADs are the effectors of TGF-beta-induced gene activation, we examined the possibility that gene repression by TGF-beta may also occur through interactions of SMADs with NKX2.1 and HNF-3. We found that lung epithelial carcinoma H441 cells contain SMAD2/3 and -4, which localize to the nucleus in response to TGF-beta treatment. The activity of a transfected Sp-B promoter/reporter construct was reduced in a dose-dependent manner by TGF-beta. Cotransfection with a mutant, constitutively activated form of the Tgf-beta type I receptor repressed Sp-B promoter activity in the absence of TGF-beta ligand. Dominant negative mutants of Smads blocked the repressor activity of TGF-beta. SMAD3, but not SMAD2, mediated the repressor activity of TGF-beta on the Sp-B promoter. Mutations within a 70-base pair domain that includes binding sites for NKX2.1, hepatocyte nuclear factor 3 (HNF-3), or cAMP response element-binding protein (CREB) eliminated SMAD3-dependent repression of Sp-B transcription. Electrophoretic mobility shift analysis showed no evidence for direct binding of SMAD3 to the Sp-B promoter, and a DNA binding mutant of SMAD3 also repressed Sp-B, suggesting that direct DNA binding of SMAD3 may not be required. Using a mammalian two hybrid assay, we found physical and functional interactions between SMAD3 and both NKX2.1 and HNF-3. Also, a glutathione S-transferase-fused SMAD3 directly binds to in vitro synthesized NKX2.1 or HNF-3, demonstrating protein-protein interactions between SMAD3 and the two transcriptional factors. The DNA binding of NKX2.1 to Sp-B promoter was reduced in response to TGF-beta treatment, although expression of Nkx2.1 was not affected. We conclude that SMAD3 interactions with the positive regulators NKX2.1 and HNF-3 underlie the molecular basis for TGF-beta-induced repression of Sp-B gene transcription.

Topics: Activin Receptors, Type I; Carcinoma; Cyclic AMP Response Element-Binding Protein; DNA-Binding Proteins; Epithelial Cells; Gene Expression Regulation; Genes, Reporter; Glutathione Transferase; Humans; Lung Neoplasms; Mutagenesis, Site-Directed; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Pulmonary Surfactant-Associated Protein B; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Repressor Proteins; Signal Transduction; Smad3 Protein; Thyroid Nuclear Factor 1; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Two-Hybrid System Techniques

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Topics: Adenoviridae; Animals; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcification, Physiologic; Carcinoma; Cell Line; Cell Transformation, Viral; Cells, Cultured; Coxsackie and Adenovirus Receptor-Like Membrane Protein; DNA, Recombinant; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Green Fluorescent Proteins; Humans; Injections, Intramuscular; Luminescent Proteins; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Osteogenesis; Receptors, Virus; Recombinant Fusion Proteins; Recombinant Proteins; Stem Cells; Stromal Cells; Transduction, Genetic; Transforming Growth Factor beta; Transgenes; Tumor Cells, Cultured

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial beta-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitative metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 microns segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

Topics: Animals; Bone Matrix; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Carcinoma, Ductal, Breast; Computer Systems; Cytokines; DNA, Neoplasm; Female; Genes, Reporter; Growth Substances; Heart; Humans; Injections; Lac Operon; Lung Neoplasms; Mammary Glands, Animal; Mice; Mice, Nude; Neoplasm Proteins; Organ Specificity; Osteoclasts; Osteolysis; Paracrine Communication; Parathyroid Hormone-Related Protein; Polymerase Chain Reaction; Proteins; Radiography; Sensitivity and Specificity; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Although oxygen is required for normal aerobic respiration, hyperoxia (95% O(2)/5% CO(2)) damages DNA, inhibits proliferation in G1, S and G2 phases of the cell cycle, and induces necrosis. The current study examines whether growth arrest in G1 protects pulmonary epithelial cells from oxidative DNA damage and cell death. Mv1Lu pulmonary adenocarcinoma cells were chosen for studies because hyperoxia inhibits their proliferation in S and G2 phase, while they can be induced to arrest in G1 by altering culture conditions. Hyperoxia inhibited proliferation, increased intracellular redox, and rapidly reduced clonogenic survival. In contrast, Mv1Lu cells treated with transforming growth factor (TGF)-beta1, deprived of serum or grown to confluency, arrested and remained predominantly in G1 even during exposure. Growth arrest in G1 significantly enhanced clonogenic survival by 10-50-fold. Enhanced survival was not due to reduction in the intracellular redox-state of the cells, but instead was associated with reduced DNA strand breaks and p53 expression. Our findings suggest that the protective effects of G1 is mediated not simply by a reduction in intracellular ROS, but rather through an enhanced ability to limit or rapidly recognize and repair damaged DNA.

Topics: Adenocarcinoma; Animals; Blotting, Western; Cell Death; Cell Division; Cell Survival; Comet Assay; Culture Media, Serum-Free; DNA Damage; Epithelial Cells; Flow Cytometry; G1 Phase; Lung Neoplasms; Mink; Oxidation-Reduction; Oxygen; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Genetic mechanisms underlying origin and progression of lung cancer are still poorly understood, despite the numerous studies which identified many genomic alterations. Using polymorphic microsatellites, allelic imbalances have been frequently found at loci such as 3p, 5q, 8p, 9p and 9q, 11p and 11q, and 17q without either histologic specificity or prognosis value. We report allelotyping results in 54 cases (50 smokers) of primary lung adenocarcinoma (50 men/4 women) resected at one institution. To perform this study, a panel of seven microsatellites were chosen upon their likely involvement in lung cancer or in the cell cycle. A highly sensitive method was designed using fluorescent PCR coupled with quantification on an automated DNA sequencer. We report that at least one allelic imbalance was observed in 87% of adenocarcinoma. Alterations at 17q23 tended to be associated with early stage tumors (I and II) and longer survivals (P = 0.05 and P = 0.06, respectively). Furthermore, concomitant alterations were found at 9p21 and at either 9q34 or 3p24 loci (P = 0.003 and P = 0.004, respectively). The presence of genes coding for TGF-beta receptors I and II at these loci suggests that the TGF-beta/CDK inhibitor P16/P15 signaling pathway might be involved in lung adenocarcinoma development.

Topics: Adenocarcinoma; Adult; Aged; Alleles; Cells, Cultured; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 9; Cyclin-Dependent Kinase Inhibitor p16; Female; Genotype; Humans; Lung Neoplasms; Male; Mass Spectrometry; Microsatellite Repeats; Middle Aged; Polymerase Chain Reaction; Prognosis; Sequence Analysis, DNA; Signal Transduction; Smoking; Time Factors; Transforming Growth Factor beta

To determine the mechanisms involved in the evasion from TGF-beta growth regulation in the small cell lung carcinoma (SCLC) cell lines and the non-small cell lung carcinoma (NSCLC) cell lines, we studied: (a) production of TGF-beta1 and TGF-beta2; (b) percentage of cells expressing TGF-beta RII; (c) responsiveness of the tumour cell lines to exogenous TGF-beta1 or TGF-beta2; and (d) presence of mRNA transcripts of the three TGF-beta isoforms and of the TGF-beta RII. Our results indicate that the SCLC cell lines do not synthesize the isoforms TGF-beta1 and TGF-beta2 nor the TGF-beta RII, thus avoiding inhibitory autocrine and paracrine TGF-beta actions. However, NSCLC cell lines express not only TGF-beta1, TGF-beta2 and TGF-beta RII mRNA transcripts, but also synthesize both isoforms and the TGF-beta RII. Although approximately 50% of the cells from the studied cell lines expressed the TGF-beta RII, different cell lines varied greatly in the sensitivity to the inhibitory action of TGF-beta. This could result from alterations in: (i) the structure of TGF-beta RII; (ii) the phosphorylation motif of TGF-beta RI; (iii) the molecules involved in the intracellular signalling pathway of TGF-beta; and (iv) cell cycle regulation.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphorylation; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

We have investigated mechanisms of mitochondrial stress-induced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca(2+)](c), and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFbeta1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-X(L) were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFbeta1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondria-to-nucleus signaling pathway operating through increased [Ca(2+)](c) plays an important role in cancer progression and metastasis.

Topics: Adenocarcinoma; Adenosine Triphosphate; Apoptosis; Calcium Signaling; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cathepsin L; Cathepsins; Cysteine Endopeptidases; DNA, Mitochondrial; Electron Transport; Electron Transport Complex IV; Ethidium; Gene Expression Regulation, Neoplastic; Humans; Intracellular Membranes; Lung Neoplasms; MAP Kinase Signaling System; Membrane Potentials; Mitochondria; Neoplasm Invasiveness; Neoplasm Proteins; Oligoribonucleotides, Antisense; Phenotype; Stress, Physiological; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Transforming growth factor B (TGF-beta) is a potent immunosuppressive cytokine that is frequently associated with mechanisms of tumor escape from immunosurveillance. We report that transplantation of murine bone marrow (BM) expressing a dominant-negative TGF-beta type II receptor (TbetaRIIDN) leads to the generation of mature leukocytes capable of a potent antitumor response in vivo. Hematopoietic precursors in murine BM from donor mice were rendered insensitive to TGF-beta via retroviral expression of the TbetaRIIDN construct and were transplanted in C57BL/6 mice before tumor challenge. After i.v. administration of 5 x 10(5) B16-F10 murine melanoma cells into TbetaRIIDN-BM transplanted recipients, survival of challenged mice at 45 days was 70% (7 of 10) versus 0% (0 of 10) for vector-control treated mice, and surviving TbetaRIIDN-BM mice showed a virtual absence of metastatic lesions in the lung. We also investigated the utility of the TGF-beta-targeted approach in a mouse metastatic model of prostate cancer, TRAMP-C2. Treatment of male C57BL/6 mice with TbetaRIIDN-BM resulted in the survival of 80% (4 of 5) of recipients versus 0% (0 of 5) in green fluorescent protein-BM recipients or wild-type controls. Cytolytic T-cell assays indicate that a specific T-cell response against B16-F10 cells was generated in the TbetaRIIDN-BM-treated mice, suggesting that a gene therapy approach to inducing TGF-beta insensitivity in transplanted BM cells may be a potent anticancer therapy.

Topics: Animals; Genetic Therapy; Green Fluorescent Proteins; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Luminescent Proteins; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retroviridae; T-Lymphocytes, Cytotoxic; Transfection; Transforming Growth Factor beta

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Collagen; Culture Media, Conditioned; Drug Combinations; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Hemorrhage; Humans; Laminin; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Paracrine Communication; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Sequence Deletion; Skin Ulcer; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

The cytokines expressed in tumor microenvironments are thought to be important mediators of both the host immune response and tumor survival. The source of these cytokines includes tumor cells, infiltrating leukocytes, fibroblasts, and other stromal elements. We previously reported that tumor-infiltrating lymphocytes (TIL) from human non-small cell lung cancer (NSCLC) express predominantly type 1 cytokines, which are known to enhance cell-mediated immunity. The purpose of this study is to assess the cytokine mRNA expression of human NSCLC primary cell lines and the capacity of the tumor-associated cytokines to modulate the development of TIL cytolytic activity against the autologous tumor. Cytokine mRNA expression was determined by RT-PCR and the capacity of TIL to kill autologous lung tumor cells was measured by the chromium-51 (51Cr) release assay. All NSCLC primary cell lines expressed mRNA for IL-4, IL-6, and transforming growth factor-beta1 (TGFbeta1), whereas IL-10 was expressed in only 1/7 cell lines. When added to TIL cultures stimulated with anti-CD3+IL-2, IL-4 and IL-10 enhanced and TGF-beta1 suppressed the development of TIL cytolytic activity against autologous tumor cells. The effects of IL-6 were inconsistent and for the group, were not statistically significant. These results demonstrate that human NSCLC cells express cytokines with the capacity to regulate the in situ anti-tumor immune response. However, the effects of tumor-derived cytokines varied qualitatively and quantitatively suggesting the balance between specific type 2 cytokines or TGF-beta1 within tumor microenvironments may influence prognosis or response to immunotherapy.

Topics: Carcinoma, Non-Small-Cell Lung; CD3 Complex; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; DNA Primers; Humans; Interleukins; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Plasma transforming growth factor beta1 (TGFbeta1) levels are elevated in patients with lung cancer. As TGFbeta1 is mainly found in platelets and as nonmalignant pulmonary diseases (NMPD) are frequently associated with lung cancer, we investigated the potential contribution of platelet degranulation and/or of a concomitant NMPD to the increased plasma levels of TGFbeta1 reported in patients with lung cancer.. Blood samples were collected in duplicate from 30 healthy subjects, 14 patients suffering from NMPD and 37 patients with lung cancer. The platelet count was determined and the samples were processed to obtain plasma. One sample was collected in EDTA (EDTA plasma) and the other in a mixture inhibiting platelet degranulation (PIM plasma). TGFbeta1 concentrations and beta-thromboglobulin (betaTG) levels, an index of platelet degranulation, were measured in both plasma samples.. TGFbeta1 and betaTG plasma levels measured in PIM plasma were lower than those obtained in EDTA plasma. With respect to PIM plasma, both TGFbeta1 and betaTG levels were higher in patients with lung cancer than those with NMPD and in healthy individuals. In patients with NMPD, only TGFbeta1 levels were increased as compared to healthy controls, betaTG levels being similar.. Methods for collecting and processing blood samples are critical in determining reliable circulating TGFbeta1 levels. Increased TGFbeta1 plasma levels observed in patients with lung cancer are related, at least partly, to concomitant NMPD and also to platelet degranulation as proved by increased betaTG levels.

Topics: Adult; Aged; beta-Thromboglobulin; Blood Platelets; Cell Degranulation; Female; Humans; Lung Neoplasms; Male; Middle Aged; Pulmonary Disease, Chronic Obstructive; Transforming Growth Factor beta; Transforming Growth Factor beta1

We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1beta), transforming growth factor beta 1 (TGF-beta1), fibronectin, and prostaglandin E2 (PGE2) by pure fibroblasts (fourth passage). A fibroblast colony-forming units (CFU-F) assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta, TGF-beta1, and fibronectin in conditioned mediums (CM) of fibroblast cultures were measured by enzyme-linked immunosorbent assay (ELISA) kit and PGE(2) by radioimmunoassay (RIA) kit. Confluence was achieved in the 60% of LCP and 78% of BCP primary cultures compared with 100% of NC, and only fibroblasts from seven LCP and six BCP cultures had the capacity to proliferate following four subcultures. Levels of IL-1beta were below 10 pg/ml in both patient groups, while NC had a mean value of 5882.57+/-221.61 pg/ml. Levels of TGF-beta1 in BCP were lower than NC values ( P<0.05). LCP and BCP had significantly decreased levels of fibronectin when compared to NC values ( P<0.05 and P<0.01, respectively). Levels of PGE2 in LCP were higher compared to NC ( P<0.01). In conclusion, BM fibroblasts from LCP and BCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta, TGF-beta1, fibronectin, and PGE2 production.

Topics: Bone Marrow Cells; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Culture Media, Conditioned; Dinoprostone; Female; Fibroblasts; Fibronectins; Humans; Interleukin-1; Lung Neoplasms; Transforming Growth Factor beta

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-alpha5, a trace amount of laminin-alpha3, laminins-beta1 and -gamma1, type IV collagen, and perlecan. Transforming growth factor-beta1 significantly induces expression of laminin-alpha1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-alpha5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.

Topics: Animals; Basement Membrane; Cell Line, Transformed; Extracellular Matrix; Fibroblasts; Gene Expression; Laminin; Lung Neoplasms; Mice; Pulmonary Alveoli; Respiratory Mucosa; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. beta IGH3, TGF-beta inducible gene, was originally isolated from lung adenocarcinoma cell line (A549). We have hypothesized that beta IGH3 mRNA levels could be predictors of the development and invasion of lung cancer.. The study included 71 lung cancer cases. The beta IGH3 mRNA levels were quantified by real time reverse transcription polymerase chain reaction (RT-PCR) using LightCycler.. The beta IGH3 mRNA levels were elevated in tumor tissues from lung cancer (1.058 +/- 1.212) compared with non-malignant lung tissues (0.304 +/- 0.159) (p = 0.0001). No significant difference in beta IGH3 mRNA levels was found among gender, age, pathological subtype and lymph node metastasis. The beta IGH3 mRNA levels were elevated in tumor tissues from T4 lung cancer (1.425 +/- 1.470) compared with those from T1 lung cancer (0.702 +/- 0.655) (p = 0.05) and T2 lung cancer (0.736 +/- 0.734) (p = 0.044).. Using the LightCycler RT-PCR assay, the determination of beta IGH3 mRNA level might provide a potential marker for the aggressiveness of lung cancer. However, further studies and a longer follow-up are needed to confirm the impact of beta IGH3 on the biological behavior of the tumor.

Topics: Adult; Aged; Aged, 80 and over; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.

Topics: Animals; Cell Division; Cell Line; Homeostasis; Humans; Integrins; Lung Neoplasms; Matrix Metalloproteinase 14; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Oligopeptides; Protein Binding; Protein Structure, Tertiary; Proteins; TNF Receptor-Associated Factor 2; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Active suppression by T regulatory cells plays an important role in the down-regulation of T cell responses to foreign and self-Ags. Thus far, the potential role of CD4(+)CD25(+) T cells in human tumors has not been reported. In this work we show that lung tumors contain large numbers of these cells and that they have constitutive high-level expression of CD152 (CTLA-4). Furthermore, the CD4(+)CD25(+) T cells mediate potent inhibition of autologous T cell proliferation. Finally, regulatory T cells from patient tumors failed to inhibit the proliferation of allogeneic T cells. Together these results suggest that the CD4(+)CD25(+) T cells found in lung tumors selectively inhibit the host immune response and therefore could contribute to the progression of lung cancer.

Topics: Abatacept; Antigens, CD; Antigens, Differentiation; Carcinoma, Non-Small-Cell Lung; CD4-Positive T-Lymphocytes; Cells, Cultured; CTLA-4 Antigen; Humans; Immunoconjugates; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Receptors, Interleukin-2; Transforming Growth Factor beta

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

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

Transforming growth factor-beta1 (TGF- beta1) is a multifunctional factor and is known to affect tumor growth in malignant tumors. The effects of TGF-beta1 on angiogenesis, stromal formation, and immune function suggest its possible involvement in tumor progression. The authors examined whether TGF-beta1 levels may be correlated with angiogenesis, clinicopathologic factors, and survival in patients with surgically resected lung carcinoma.. TGF-beta1 protein was extracted from 53 nonsmall cell lung carcinoma tissue samples (19 squamous cell carcinomas, 33 adenocarcinomas, and 1 adenosquamous cell carcinoma), and its level was measured by enzyme-linked immunosorbent assay. To assess tumor angiogenesis, microvessel density (MVD) was determined by CD31 immunostaining.. The protein level of TGF-beta1 was 289 picograms per milligram of protein (pg/mg protein), ranging from 94 pg/mg protein to 584 pg/mg protein. The TGF-beta1 protein level was significantly higher in patients with lymph node metastasis compared with patients who were without lymph node metastasis (P = 0.02), and the TGF-beta1 protein level was significantly higher in patients with Stage III disease (TNM classification) compared with patients who had Stage I and II disease (P = 0.03). There was no significant correlation between the TGF-beta1 protein level and any of the other clinicopathologic factors that were considered. A significant positive correlation between TGF-beta1 protein level and MVD was noted (P < 0.01). Furthermore, in patients with adenocarcinoma, a significant correlation between TGF-beta1 protein level and prognosis was detected by multivariate analysis (P = 0.028).. TGF-beta1 seems to affect tumor angiogenesis and to play an important role in tumor progression in patients with nonsmall cell lung carcinoma. Furthermore, the TGF-beta1 protein level may be an independent predictor of survival in patients with adenocarcinoma of the lung.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Disease Progression; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Predictive Value of Tests; Prognosis; Prospective Studies; Regression Analysis; Transforming Growth Factor beta

Previous studies showed that TGF-beta down-regulates aryl hydrocarbon (AhR) expression in human lung carcinoma cells A549. Here we analyzed the molecular mechanisms by which TGF-beta modulates AhR expression. A 5799-nucleotide 5'-flanking region of human AhR gene was isolated. Transient transfection studies of full-length (hAhRP) and deletion promoter constructs indicate the requirement of a cis-regulatory element encompassing -1980 to -1892 for full constitutive activity. Basal hAhRP activity occurs in a cell-specific manner; human hepatoma HepG2 cells possess a 10-fold higher activity compared with A549 cells. TGF-beta exerts cell-specific effects on hAhRP activity. Treatment of cells with 100 pM TGF-beta leads to a 50% inhibition in A549 and a 3-fold induction in HepG2 cells. Deletion mutagenesis identified a TGF-beta-responsive sequence containing a functional conserved Smad-binding element. Transient overexpression of Smad 2, 3, and 4 indicates that these signal transducers modulate hAhRP activity. The down-regulation of AhR by TGF-beta is modulated by 5'-TG-3'-interacting factor (TGIF). Transient overexpression of TGIF in MDA-MB231 and HepG2 cells led to inhibition of hAhRP activity and a similar decrease of AhR mRNA expression. Our findings indicate that Smad proteins are involved in the cell-specific regulation of AhR expression by TGF-beta.

Topics: 5' Untranslated Regions; Activin Receptors, Type I; Binding Sites; Carcinoma; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Homeodomain Proteins; Humans; Liver Neoplasms; Lung Neoplasms; Mutagenesis, Site-Directed; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Aryl Hydrocarbon; Receptors, Transforming Growth Factor beta; Regulatory Sequences, Nucleic Acid; Repressor Proteins; RNA, Messenger; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Resistance to apoptosis and ability to promote angiogenesis are integral features of the metastatic phenotype. Human gene CC3 is a metastasis suppressor for variant small cell lung carcinoma and a mouse melanoma in vivo. We have shown previously that metastasis-suppressing function of CC3 might be due at least in part to the ability of CC3 protein to predispose tumor cells to apoptosis. Here we demonstrate that CC3 has a previously unidentified effect on the ability of tumor cells to induce angiogenesis in vitro. Expression of CC3 in three different tumor cell lines significantly diminished their angiogenic character as manifested in the in vitro proliferation and migration assays with endothelial cells of both macro- and microvascular origin. Expression of CC3 induced changes in RNA levels of several angiogenic modulators consistent with the overall reduction in angiogenic properties. These results indicate that expression of CC3 has a dual effect on phenotype of tumor cells ultimately inhibiting their metastatic potential.

Topics: Acetyltransferases; Carcinoma, Small Cell; Cell Line; Cell Movement; Culture Media, Conditioned; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Lung Neoplasms; Lymphokines; Neovascularization, Pathologic; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The transforming growth factor-beta1 (TGF-beta1) responsive epithelial non-small-cell lung cancer (NSCLC) cell line NCI-H727 was used to identify potential target genes involved in TGF-beta1-mediated responses. Comparative cDNA expression patterns between cells treated with TGF-beta1 and those treated with vehicle were generated by differential mRNA display. One 496-bp fragment, differentially increased threefold by TGF-beta1 and hybridizing to a 2.7-kb mRNA species in NCI-H727 cells by Northern analysis, revealed no significant match to any known gene sequence. The mRNA transcript of this novel gene that we named differentially expressed nucleolar TGF-beta1 target (DENTT) is expressed in several normal human tissues, with the highest level of expression in brain. Human brain cDNA library screening and 5' rapid amplification of cDNA ends yielded full-length DENTT cDNA containing an 1899-bp open reading frame encoding a predicted 633-amino-acid protein with four potential nuclear localization signals (NLSs) and two coiled-coil regions. DENTT contains a conserved 191-residue domain that shows significant identity to, and defines, the TSPY/TSPY-like/SET/NAP-1 superfamily. Enhanced green fluorescent protein (EGFP)-tagged full-length DENTT transfected into COS-7 cells showed nucleolar and cytoplasmic localization. Transfection of EGFP-tagged DENTT NLS deletion constructs lacking the bipartite NLS-1 were excluded from the nucleolus. While NLS-1 is necessary for nucleolar localization of DENTT, it is not sufficient for sole nucleolar localization. Our data show that DENTT mRNA induction by TGF-beta1 correlates with induction of TGF-beta1 mRNA, induction of extracellular matrix gene expression, and inhibition of colony formation in soft agarose in TGF-beta1 responsive NSCLC cells when exposed to TGF-beta1. TGF-beta1 does not induce DENTT mRNA expression in TGF-beta1 nonresponsive NSCLC cells. Our data suggest that this novel TGF-beta1 target gene has distinct domains for direction to different subnuclear locations.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Brain; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Cloning, Molecular; COS Cells; DNA-Binding Proteins; DNA, Complementary; Gene Expression Profiling; Gene Expression Regulation; Histone Chaperones; Humans; Lung Neoplasms; Molecular Sequence Data; Nuclear Proteins; Nucleosome Assembly Protein 1; Proteins; RNA, Messenger; Sex-Determining Region Y Protein; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells. However, increased percentages of CD4(+)CD25(+) T cells were observed in the non-small cell lung cancer tumor-infiltrating lymphocytes and ovarian cancer tumor-associated lymphocytes. Furthermore, these CD4(+)CD25(+) T cells were found to secrete transforming growth factor-beta, consistent with the phenotype of regulatory T cells. Despite a generalized expression of lymphocyte activation markers in the tumor-associated T-cell populations, the CD8(+) T cells expressed low levels of CD25. To determine whether expression of CD25 could be restored on the CD8 cells, tumor-associated T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After stimulation, nearly all of the CD8 T cells expressed CD25. Furthermore, despite the low levels of interleukin 2, IFN-gamma, and tumor necrosis factor-alpha secretion by the tumor-associated and peripheral blood T cells at baseline, stimulation with anti-CD3 and anti-CD28 monoclonal antibodies significantly increased the fraction of cells producing these cytokines. Thus, tumor-associated T cells from patients with early and late-stage epithelial tumors contain increased proportions of CD4(+)CD25(+) T cells that secrete the immunosuppressive cytokine transforming growth factor-beta. Furthermore, our results are consistent with previous reports showing impaired expression of CD25 on CD8(+) T cells in cancer patients. Finally, increased lymphocyte costimulation provided by triggering the CD28 receptor is able to increase CD25 expression and cytokine secretion in tumor-associated T cells. These observations provide evidence for the contribution of regulatory T cells to immune dysfunction in cancer patients.

Topics: Carcinoma, Non-Small-Cell Lung; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Down-Regulation; Female; Humans; Interferon-gamma; Interleukin-2; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Neoplasm Staging; Ovarian Neoplasms; Receptors, Antigen, T-Cell; Receptors, Interleukin-2; Th1 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To correlate the volume of lung irradiated with changes in plasma levels of the fibrogenic cytokine transforming growth factor beta (TGFbeta) during radiotherapy (RT), such that this information might be used to predict the development of symptomatic radiation-induced lung injury (SRILI).. The records of all patients with lung cancer treated with RT with curative intent from 1991 to 1997 on a series of prospective normal tissue injury studies were reviewed. A total of 103 patients were identified who met the following inclusion criteria: (1) newly diagnosed lung cancer of any histology treated with RT +/- chemotherapy with curative intent; (2) no evidence of distant metastases or malignant pleural effusion; (3) no thoracic surgery after lung RT; (4) no endobronchial brachytherapy; (5) follow-up time more than 6 months; (6) plasma TGFbeta1 measurements obtained before and at the end of RT. The concentration of plasma TGFbeta1 was measured by an enzyme-linked immunosorbent assay. Seventy-eight of the 103 patients were treated with computed tomography based 3-dimensional planning and had dose-volume histogram data available. The endpoint of the study was the development of SRILI (modified NCI [National Cancer Institute] common toxicity criteria).. The 1-year and 2-year actuarial incidence of SRILI for all 103 patients was 17% and 21%, respectively. In those patients whose TGFbeta level at the end of RT was higher than the pre-RT baseline, SRILI occurred more frequently (2-year incidence = 39%) than in patients whose TGFbeta1 level at the end of RT was less than the baseline value (2-year incidence = 11%, p = 0.007). On multivariate analysis, a persistent elevation of plasma TGFbeta1 above the baseline concentration at the end of RT was an independent risk factor for the occurrence of SRILI (p = 0.004). The subgroup of 78 patients treated with 3-dimensional conformal radiotherapy, who consequently had dose-volume histogram data, were divided into groups according to their TGFbeta1 kinetics and whether their V(30) level was above or below the median of 30%. Group I (n = 29), with both a TGFbeta1 level at the end of RT that was below the pre-RT baseline and V(30) < 30%; Group II (n = 35), with a TGFbeta1 level at the end of irradiation that was below the baseline but a V(30) > or = 30% or with a TGFbeta1 level at the end of RT that was above the pre-RT baseline but V(30) < 30%; Group III (n = 14), with both a TGFbeta1 level at the end of RT that was above the baseline and V(30) > or = 30%. A significant difference was found in the incidence of SRILI among these three groups (6.9%, 22.8%, 42.9%, respectively, p = 0.02).. (1) An elevated plasma TGFbeta1 level at the end of RT is an independent risk factor for SRILI; (2) The combination of plasma TGFbeta1 level and V(30) appears to facilitate stratification of patients into low, intermediate, and high risk groups. Thus, combining both physical and biologic risk factors may allow for better identification of patients at risk for the development of symptomatic radiation-induced lung injury.

Topics: Aged; Analysis of Variance; Biomarkers; Dose Fractionation, Radiation; Female; Follow-Up Studies; Humans; Lung; Lung Neoplasms; Male; Radiation Injuries; Radionuclide Imaging; Radiotherapy, Conformal; Risk Factors; Transforming Growth Factor beta

The antitumor efficiency of secondary lymphoid organ chemokine (SLC), a CC chemokine that chemoattracts both dendritic cells (DCs) and T lymphocytes,was evaluated in SV40 large T-antigen transgenic mice that develop bilateral multifocal pulmonary adenocarcinomas. Injection of recombinant SLC in the axillary lymph node region led to a marked reduction in tumor burden with extensive lymphocytic and DC infiltration of the tumors and enhanced survival. SLC injection led to significant increases in CD4 and CD8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen. The cellular infiltrates were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN-gamma inducible protein 10, and monokine induced by IFN-gamma (MIG). In contrast, lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor beta was decreased in response to SLC treatment. In vitro, after stimulation with irradiated autologous tumor, splenocytes from SLC-treated mice secreted significantly more IFN-gamma and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Angiogenesis Inhibitors; Animals; Antigens, Polyomavirus Transforming; Chemokine CCL21; Chemokines; Chemokines, CC; Cytokines; Dendritic Cells; Disease Models, Animal; Endothelial Growth Factors; Lung Neoplasms; Lymph Nodes; Lymphokines; Mice; Mice, Transgenic; Neovascularization, Pathologic; Recombinant Proteins; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim was to investigate the influence of pulmonary metastases of the rhabdomyosarcoma R1H on the radiation response of the lung of the WAG/Rij rat.. Three groups of animals were investigated: metastases-free animals treated with fractionated irradiation of the lungs; metastases-bearing animals receiving no irradiation; and metastases-bearing animals treated with fractionated irradiation initiated 14, 21 or 28 days after induction of pulmonary metastases of the R1H-tumour by i.v. injection of viable tumour cells. Metastases were thus treated at various well-defined sizes. Total doses of 20-60Gy were applied in fractions of 2 Gy within 11 days. Complication rate and survival time were used as endpoints.. About 2 months after onset of irradiation treatment, animals had to be sacrificed because of severe respiratory distress either caused by irradiation-induced lung damage (median 57 days, range 36-77 days), or because of development of lung metastases (65, 20-160 days). A decrease of the ED(50) (dose required to induce lethal lung damage in 50% of irradiated animals) was determined for metastases-bearing animals. This effect increased with metastatic volume.. The results suggest that the presence of tumours in the lung decreased the lung tolerance to radiation. This effect can hardly be explained by a reduction in functional lung volume by metastatic volume.

Topics: Animals; Female; Lung; Lung Neoplasms; Male; Radiation Tolerance; Rats; Rhabdomyosarcoma; Transforming Growth Factor beta

Mice heterozygous for deletion of the transforming growth factor beta1 (TGF-beta1) gene show an enhanced rate of lung tumorigenesis following carcinogen treatment. Since the growth inhibitory activity of TGF-beta1 in epithelial cells is associated with K-ras p21, and K-ras mutations commonly occur in chemically-induced mouse lung tumors, we postulated that tumors in heterozygous TGF-beta1 mice might be more likely to have K-ras mutations compared with tumors in wildtype TGF-beta1 mice. Urethane-induced lung tumors in AJBL6 TGF-beta1 +/- and +/+ mice were examined for K-ras mutations by polymerase chain reaction/single strand conformation polymorphism analysis and sequencing. Mutation frequencies were similar in both genotypes: 12/18 +/- tumors (67%) and 10/16 +/+ tumors (62%). Mutations occurred in 80% +/- and 75% +/+ carcinomas, but in only 50% of the adenomas of both TGF-beta1 genotypes. Codon 61 A-->G transition mutations were predominant, occurring in 61% +/- and 44% +/+ tumors. Three +/- (17%) and three +/+ (19%) tumors showed codon 12 mutations, mostly G-->A transitions. Two +/- tumors had both codon 61 and codon 12 mutations. Interestingly, carcinomas with mutations in codon 61 were larger than those with codon 12 changes. It appears that the mechanism of enhanced susceptibility of TGF-beta1+/- mice to urethane-induced lung carcinogenesis does not involve selective development of tumors with K-ras mutations.

Topics: Adenoma; Animals; Carcinogenicity Tests; Carcinogens; Carcinoma; Crosses, Genetic; DNA Mutational Analysis; DNA, Neoplasm; Female; Genes, ras; Genetic Predisposition to Disease; Genotype; Heterozygote; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mutagenicity Tests; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urethane

Expression of transforming growth factor betas (TGF betas), tumor necrosis factor (TNF) family members, interferons (IFNs), macrophage migration inhibitory factor (MIF) and granulocyte macrophage colony stimulating factor (GM-CSF) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks of age, were given 2,000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until killed at week 25. Total RNAs were extracted from 15 adenocarcinomas. Four samples of normal lung tissue from untreated rats served as controls. The expression of TGFbeta1, TGFbeta2, TGFbeta3, TNFalpha, TNFbeta and lymphotoxin beta (Ltbeta) was significantly higher in adenocarcinomas than in normal lung tissues. In contrast, MIF was expressed at the same level in neoplasms and normal tissue and no expression of IFNbeta, IFNgamma and GM-CSF was apparent in either adenocarcinomas or normal lung tissues. These results suggest that elevated expression of TGFbetas and TNF family members may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.

Topics: Adenocarcinoma; Administration, Oral; Animals; Carcinogens; Disease Models, Animal; Drinking; Lung Neoplasms; Male; Nitrosamines; Rats; Rats, Wistar; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor (TGF)-beta strongly inhibits epithelial cell proliferation. Alterations of TGF-beta signaling are thought to play a role in tumorigenesis. We show in the present study that most lung cancer cell lines have lost the growth-inhibitory response to TGF-beta signal, and that those with TGF-beta unresponsiveness can be divided into two major groups, TGF-beta type II receptor (TGFbetaRII)(+)/Smad7(+) and TGFbetaRII(-)/Smad7(-), suggesting the heterogeneous mechanisms underlying the TGF-beta responsiveness. The mechanism of the loss of TGFbetaRII expression of the latter group was further studied, identifying aberrant DNA methylation of the promoter region in a limited fraction of cell lines. Interestingly, we found that the alteration of chromatin structure because of histone deacetylation may also be involved, showing a good correlation with loss of TGFbetaRII expression. This notion was supported by the findings of a restriction enzyme accessibility assay, of a chromatin immunoprecipitation assay with anti-acetyl histone antibodies, and of an in vivo induction of TGFbetaRII expression by histone deacetylase inhibitors including trichostatin A (TSA) and sodium butyrate. In vitro induction of TGFbetaRII promoter reporter activity by TSA was also detected and found to require the CCAAT box within the -127/-75 region. A positive regulatory mechanism for TGFbetaRII expression in a TGF-beta-expressing cell line was also investigated, and a TPA-responsive element (TRE)-like motif, TRE2, was detected in addition to the previously reported TRE-like motif Y element in the positive regulatory region. Alterations in two discrete proteins interacting with these two TRE-like motifs were also suspected of being involved in the loss of TGFbetaRII expression. This is the first study to demonstrate that, in addition to the TSA-responsive region and TRE2 motif in the TGFbetaRII promoter, the alteration of histone deacetylation may be involved in the loss of TGFbetaRII expression in lung cancer cell lines.

Topics: Acylation; Chromatin; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Silencing; Histones; Humans; Lung Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

The invasive transformation of A-459 lung epithelial carcinoma cells has been linked to the autocrine regulation of malignant phenotypic changes by transforming growth factor beta (TGF-beta). Here we demonstrate, using stable 13C glucose isotopes, that the transformed phenotype is characterized by decreased CO2 production via direct glucose oxidation but increased nucleic acid ribose synthesis through the nonoxidative reactions of the pentose cycle. Increased nucleic acid synthesis through the nonoxidative pentose cycle imparts the metabolic adaptation of nontransformed cells to the invasive phenotype that potentially explains the fundamental metabolic disturbance in tumor cells: highly increased nucleic acid synthesis despite hypoxia and decreased glucose oxidation.

Topics: Adenocarcinoma; Cell Transformation, Neoplastic; Glucose; Humans; Lung Neoplasms; Oxidation-Reduction; Pentose Phosphate Pathway; Ribose; Transforming Growth Factor beta; Tumor Cells, Cultured

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.

Topics: Carcinoma; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glycosylation; Humans; Hyaluronan Receptors; Hyaluronic Acid; Lung Neoplasms; Oncostatin M; Peptides; Protein Processing, Post-Translational; Sulfates; Transforming Growth Factor beta; Tumor Cells, Cultured

TSC-36 (TGF-beta1-stimulated clone 36) is a TGF-beta1 inducible gene whose product is an extracellular glycoprotein that contains a single follistatin module. TSC-36 is highly expressed in the lung, but its physiological function is unknown. In an attempt to elucidate it, we investigated the effect of TSC-36 on proliferation of human lung cancer cell lines. We found a correlation between expression of TSC-36 and cell growth: TSC-36 mRNA was not detected in cells derived from small cell lung cancer (SCLC) cells, a highly aggressive neoplasm, but was detected in some non-small cell lung cancer (NSCLC) cells, a moderately aggressive neoplasm. This suggested an antiproliferative function for TSC-36. To address this question, NSCLC PC-14 cells, which express very low level of TSC-36 protein, were transfected with TSC-36 cDNA and the proliferative capacity of stable transfectants was determined by measuring the doubling time, colony forming activity in soft agar and the level of incorporation of (3)H-thymidine into DNA. Under normal culture conditions, the transfected cells showed a longer doubling time, lower plating efficiency and lower rate of DNA synthesis than the parental cells and the control neo transfectant cells. These findings suggested that expression of TSC-36 caused growth inhibition in human lung cancer cells.

Topics: Blotting, Northern; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Flow Cytometry; Follistatin-Related Proteins; Glycoproteins; Humans; Lung Neoplasms; Plasmids; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

To determine whether changes in TGF-beta plasma levels during radiation therapy may be useful in predicting radiation-induced pulmonary injury and tumour response in non-small-cell lung cancer (NSCLC) patients.. Plasma TGF-beta was investigated in 27 patients with stage III NSCLC, who were treated with 60 Gy (2Gy/day) radiotherapy with or without carboplatin. TGF-beta was measured prior to beginning radiotherapy and weekly during treatment; evaluated as a ratio between TGF-beta levels obtained during treatment and the pretreatment TGF-beta level. The endpoints of the study were development of symptomatic radiation pneumonitis and tumour response.. Nine of the 27 patients developed pneumonitis. The patients who developed pneumonitis had high persistent TGF-beta levels throughout the course of treatment (TGF-beta ratio>1), whereas the TGF-beta levels in patients who did not develop pneumonitis were unchanged or declined towards normal (TGF-beta ratio < 1). Patients who responded to treatment had low or normal TGF-beta levels during treatment compared with patients who failed to respond. Other parameters such as pretreatment TGF-beta values, carboplatin treatment or field size did not appear to have a significant effect, which is probably due to the small number of patients entered in the study.. This pilot study, with a limited number of patients, suggests the hypothesis that elevated TGF-beta levels during radiotherapy may not only indicate patients with a higher risk of developing pulmonary toxicity but also patients with a higher risk of treatment failure. This remains to be tested in a larger clinical study.

Topics: Adult; Aged; Humans; Lung; Lung Neoplasms; Middle Aged; Pilot Projects; Radiation Injuries; Radiotherapy; Transforming Growth Factor beta; Treatment Failure

Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-beta) superfamily involved in a range of biological activities. The loss of sensitivity to TGF-beta is frequent in human lung cancers and inactivation of Smad family members are thought to play important roles in disruption of TGF-beta signaling. In the study presented here, we characterized the biological and biochemical functions of six Smad2 and Smad4 mutants, which we previously identified in human lung cancers. All mutant Smad2 and Smad4 were in fact found to be defective in transmitting growth inhibitory signals originating from TGF-beta and incapable of activating Smad/hFAST-1-mediated transcription. Transcriptional activation of plasminogen activator inhibitor type 1 (PAI-1) was impaired in four of the six mutants due to the defects in homo- and/or hetero-oligomerization with wild-type Smads. In contrast, the remaining two Smad mutants showed a modest reduction in the PAI-1 transcriptional activation and apparently retained the ability to oligomerize with wild-type Smads. Significant loss of growth inhibition and Smad/hFAST-1-mediated transcriptional activation by all of the six mutants suggested that Smad mutants are indeed functionally impaired Smad mutations and may play a role in lung tumorigenesis. Moreover, the present findings suggest that in addition to the impairment in the homo- and/or hetero-oligomerization, there may be an alternative mechanism producing disruption of TGF-beta signaling, involving hFAST-1-or possibly other transcriptional cofactor(s)-mediated transcriptional activation.

Topics: DNA-Binding Proteins; Forkhead Transcription Factors; Gene Silencing; Genes, Tumor Suppressor; Genetic Heterogeneity; Humans; Lung Neoplasms; Nerve Growth Factors; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Smad Proteins; Smad2 Protein; Smad4 Protein; Trans-Activators; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenopus Proteins

The cathepsins B, L, and H are expressed ubiquitously and represent the major proportion of lysosomal enzymes. They are involved in bulk proteolysis in the lysosomes, processing of proteins and matrix degradation. Under pathological conditions the participation of cathepsins, especially their secreted forms, was observed in inflammation, tumor progression and metastasis. The enzymatic activity of cathepsins is regulated by posttranslational modification, localization, maturation, changes in pH, and their interaction with inhibitors. Regulation at the level of transcription is not well elucidated. The aim of this study was to investigate the effect of IL-1 beta, IL-6, IL-10, TGF-beta 1, and HGF on mRNA expression and protein level in human lung epithelial cell lines A-549 and BEAS-2B. IL-6 leads to a twofold increase in cathepsin L mRNA expression, whereas TGF-beta 1 decreases the amount of cathepsin L mRNA. At protein level, using enzyme immunoassay, it was shown that IL-6 induced increased amounts of cathepsin L but not cathepsin B. In contrast, after incubation of bronchial epithelial cells with TGF-beta 1 the cathepsin L concentration was decreased. In conclusion, gene expression of cathepsins B and L is variable. The cytokines IL-6 and TGF-beta 1 modulate cathepsin gene expression.

Topics: Carcinoma; Cathepsin B; Cathepsin L; Cathepsins; Cell Line; Cell Line, Transformed; Cysteine Endopeptidases; Endopeptidases; Enzyme Induction; Epithelial Cells; Hepatocyte Growth Factor; Humans; Interleukin-1; Interleukin-10; Interleukin-6; Lung; Lung Neoplasms; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta (TGF-beta) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF-beta in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF-beta inducible reporter construct, p3TP-Lux. Previous work from this laboratory using pharmacologic agents suggested that a phosphatidylcholine-specific phospholipase C and protein kinase C may be involved in early aspects of TGF-beta signaling. Here we provide evidence that TGF-beta induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP-Lux reporter plasmid are simultaneously treated with TGF-beta and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF-beta alone. We also find elevated levels of phosphocholine in cells following TGF-beta treatment. Further, exogenously added bacterial phosphatidylcholine phospholipase C (PC-PLC) is capable of inducing expression of the p3TP-Lux reporter to the same extent as TGF-beta indicating that the bacterial PC-PLC can mimic the TGF-beta effect. In contrast, neither hexanoyl sphingosine (a ceramide analogue) nor arachadonic acid induce expression of the p3TP-Lux reporter. Measurements with the fluorescent, calcium-sensitive dye, FURA2, indicated that there was no change in intracellular calcium in response to TGF-beta. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF-beta induction of the p3TP-Lux reporter. Thus the TGF-beta signaling pathway appears to involve the production of diacylglycerol but is independent of calcium.

Topics: Acetophenones; Arachidonic Acid; Bridged-Ring Compounds; Calcium; Chelating Agents; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix; Humans; Hydrolysis; Lipids; Lung Neoplasms; Norbornanes; Phosphatidylcholines; Phosphodiesterase Inhibitors; Plasmids; Protein Isoforms; Second Messenger Systems; Sphingosine; Thiocarbamates; Thiones; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Type C Phospholipases

We previously reported elevated levels of TGF-beta1 in patients with renal carcinoma. Certain aspects led us to ask whether they might be caused by chronic damage to the kidney(s). Here we report on an extended set of patients with various renal diseases, lung cancer, humoral immunodeficiency and controls. For latent TGF-beta1 in plasma, we find that the control, immunodeficiency, lung cancer and kidney transplant groups do not differ significantly (means, 7.0-8.8 ng/ml). Also, acute short-term renal stress (extracorporal lithotrypsy) does not lead to an increase of TGF-beta1. However, the pyelonephritis patients present with levels of 19.0 ng/ml, chronic extracorporal dialysis patients with 15.5 ng/ml, and renal cell carcinoma patients with 22.8 ng/ml. For active TGF-beta1 these findings are exactly recovered. For serum levels, only the renal carcinoma group presents with significantly elevated levels of TGF-beta1. Kidney transplantation seems to normalize TGF-beta1 levels, while in the kidney cancer patients surgery has an effect only in part of the group. We conclude that elevated plasma TGF-beta1 levels are common in at least two chronic renal disease conditions, and that it normalizes with restoration of renal function. It is tempting to speculate that chronic elevation of TGF-beta1 in these patients may be critically involved in these conditions predisposing to renal cancer.

Topics: Carcinoma, Renal Cell; Common Variable Immunodeficiency; Humans; Kidney Diseases; Kidney Failure, Chronic; Kidney Neoplasms; Kidney Transplantation; Lithotripsy; Lung Neoplasms; Pyelonephritis; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) signaling requires the functional interaction of two distinct receptors, type I (RI) and type II (RII), at the cell surface. Exposure of cells to TGF-beta results in receptor internalization and down-regulation (Zwaagstra et al., 1999, Exp. Cell Res. 252, 352362); however, little is known about the subsequent fate of RI or RII. In this study the cellular distribution of RI was examined in cells before and after treatment with ligand. RI was localized by immunocytochemistry and confocal microscopy using two polyclonal antisera directed against two different epitopes, one in the C-terminal region and one in the N-terminal region of the cytoplasmic domain. The majority of RI molecules in untreated MvlLu and A549 cells were found to be intracellular. Treatment of MvlLu and A549 cells with 100 pM TGF-beta1 for 24 h at 37 degrees C caused a redistribution of surface RI on MvlLu cells, as evidenced by surface RI aggregation. Unexpectedly, this TGF-beta1 treatment also caused redistribution and accumulation of intracellular RI in and around the nucleus for both MvlLu and A549 cells. Nuclear accumulation of RI was also promoted independently of ligand receptor activation by treatment of MvlLu cells with olomoucine, an agent that results in growth arrest. The capacity of RI to localize in the nucleus was confirmed by microscopic examination of 293 cells transiently expressing RI fused to green fluorescent protein (RI-GFP). Olomoucine treatment of these cells resulted in the movement of RI-GFP into the nucleus. Our results indicate that growth arrest alters intracellular transport/routing of RI and may indicate that RI functions not only at the cell surface but inside the cell as well.

Topics: Activin Receptors, Type I; Animals; Cell Cycle; Cell Line; Cell Membrane; Cell Nucleus; Cyclin-Dependent Kinases; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation; Golgi Apparatus; Green Fluorescent Proteins; Humans; Kinetin; Luminescent Proteins; Lung Neoplasms; Mink; Protein Serine-Threonine Kinases; Purines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Respiratory Mucosa; Signal Transduction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Fibrosis in normal tissues is a common and dose-limiting late complication of radiotherapy at many cancer sites, but its pathogenesis is poorly understood. We undertook a controlled study of the effect of irradiation on the collagen production of fibroblasts cultured from skin biopsies taken from patients undergoing radiotherapy treatment. Eight weeks after a single 8 Gy fraction using 300 kV X-rays, five patients treated at the Royal Marsden Hospital underwent biopsy of the irradiated site and of the contralateral, unirradiated body site. Fibroblasts from irradiated and control, unirradiated sites were cultured in vitro, and collagen production rates were measured during a 48-hour incubation under standardized conditions and in the presence and absence of transforming growth factor beta(1)(TGF beta(1)), 1 ng/ml, using HPLC. Collagen production was elevated in cells cultured from irradiated skin; median collagen production rates 61.16 pmoles hydroxyproline/10(5)cells/hour in irradiated cells, 39.78 pmoles hydroxyproline/10(5)cells/hour in unirradiated cells, P = 0.016 (Mann-Whitney U-test). In fibroblasts from unirradiated sites, collagen production rates were increased by the addition of TGF beta(1); however, in three of the cell lines cultured from irradiated sites this effect of TGF beta(1)on collagen production was not observed.

Topics: Aged; Aged, 80 and over; Biopsy; Breast Neoplasms; Cells, Cultured; Chromatography, High Pressure Liquid; Collagen; Colonic Neoplasms; Female; Fibroblasts; Flow Cytometry; Humans; Lung Neoplasms; Male; Middle Aged; Prostatic Neoplasms; Skin; Time Factors; Transforming Growth Factor beta

Transforming Growth Factor-beta 1 (TGF-beta 1) regulates the proliferation of normal epithelial cells, and resistance to TGF-beta 1 growth inhibition is a common feature of human cancers including lung cancer. In order to understand the mechanism of resistance to growth inhibition by TGF-beta 1 and to reverse the regulation of proliferation in lung cancer, we determined the genomic structure of the genes involved in the signal transduction pathway of TGF-beta 1 and performed an initial mutation survey of the complete coding region of the genes in lung cancer and cell lines with the resistance to growth inhibition by TGF-beta 1. First, a mutation analysis of the TGF-beta type II receptor (TGF-beta RII) was performed. Point mutations of the gene were detected in several colon cancers and an adenocarcinoma of the lung in the poly-A sequence. No mutations of Smad 2, 3, 4, 5 and TGF-beta type I receptor (TGF-beta IR) genes were detected in a series of the tumors we tested, although several mutations of Smad 2 and 4 were previously reported. Frequent alterations of the p15 gene and reduced expression of p21 we already reported from our previous studies. We also determined the genomic structure of the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), which is involved in activating TGF-beta 1, and performed an initial mutation survey of the complete coding sequences of the gene. A point mutation at exon 40 was found in one lung adenocarcinoma cell line. In summary, alterations in the many genes involved in the signal transduction of TGF-beta 1 were found and may mediate the loss of TGF-beta 1 responsiveness in lung cancer. The molecular targets for the regulation of the proliferation of lung cancer are thought to be p15, p21 and the transcriptional regulators.

Topics: Cell Division; Colonic Neoplasms; Drug Resistance, Neoplasm; Gene Deletion; Humans; Lung Neoplasms; Point Mutation; Polymerase Chain Reaction; Receptor, IGF Type 2; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Recent in vivo and in vitro studies have demonstrated a wide spectrum of biologic activities of cytokines in the pathogenesis and progression of malignancy. Tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) have emerged as two of the many host-derived mediators that seem to interfere with both antiproliferative and tumorigenic effects in malignant tumours including lung cancer. However, their association with tumour prognosis or prognostic factors has not yet been completely clarified. In this study, we assessed TNF-alpha and TGF-beta mRNA expression by RT-PCR technique in 61 NSCLC samples, demonstrating the presence of TNF-alpha and TGF-beta mRNA in 55.74% and 45.9% of cases, respectively. We also evaluated the expression of the two distinct transmembrane TNF receptors. TNFR-I and TNFR-II, with a PCR-positive signal in 70.49% and 65.57% of cases, respectively. In 49 of the 61 cases, we evaluated the prognostic impact of the two growth-inhibiting factors using the Kaplan-Meier analysis. In the univariate analysis patients without nodal metastatic involvement (P = 0.02), less advanced tumour stage (P = 0.02) or TNF-alpha and TGF-beta positive cancers (P = 0.01 and P = 0.03) showed a favourable prognosis in terms of overall survival. Since our previous studies demonstrated a significant association between NSCLC behaviour, neoangiogenesis and bcl-2 expression, we investigated the putative relation between TNF-alpha and TGF-beta on the one hand, and vascular count (as a measure of tumour angiogenesis) and bcl-2 protein expression, on the other hand. Our results showed a significant direct association between TNF-alpha and bcl-2 (P = 0.05) and an inverse association between TNF-alpha and microvessel count (P = 0.03). Moreover, as previously demonstrated, we observed a significant inverse correlation between bcl-2 protein expression and vascular count (P = 0.05), suggesting that the favourable effect of TNF-alpha on clinical outcome may be related to a bcl-2-mediated low neovascular development.

Topics: Adult; Aged; Antigens, CD; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Female; Gene Expression; Humans; Lung Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The interaction of cytokines and their net balance with regard to macrophage activation (or deactivation) and immune stimulation (or suppression), ultimately determines the success of host-immune response at sites of active infection. A regulatory role for interleukin (IL)-12 in production of transforming growth factor (TGF)-beta1 has been suggested, however, remains controversial. In this study, we analyzed the effect of IL-12 on TGF-beta1 expression in the human lines, K562 and A549, and in primary human monocytes and macrophages. We found that IL-12 down-regulates TGF-beta1 mRNA expression in K562, monocytes and bone marrow cells, and to a lesser extent in the A549 cells. Using constructs containing different regions of the first promoter of the TGF-beta1 gene and a reporter gene, we also demonstrate that this effect is mediated through the TGF-beta1 gene promoter in the K562 and monocytic cell types. In conclusion, the critical role of IL-12 in the early activation of the immune response to pathogens may include down-modulation of TGF-beta1 gene activity.

Topics: Autocrine Communication; Bone Marrow Cells; Carcinoma; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Interleukin-12; K562 Cells; Lung Neoplasms; Macrophages; Macrophages, Alveolar; Monocytes; Neoplasm Proteins; Promoter Regions, Genetic; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

To elucidate the role of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta type II receptor (TGF-beta RII) as tumor-suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF-beta1 gene with A/J mice to produce AJBL6 TGF-beta1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF-beta1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription-polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-beta1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF-beta1 HT mouse provides an excellent model system to examine carcinogen-induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF-beta type I receptor (TGF-beta RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF-beta RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-beta RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-beta RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-beta RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF-beta RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-beta1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-beta RII mRNA e

Topics: Animals; Blotting, Northern; Carcinogens; Crosses, Genetic; Female; Gene Amplification; Gene Dosage; Genes, Tumor Suppressor; Immunohistochemistry; In Situ Hybridization; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urethane

Mutations and expression of the transforming growth factor-beta receptor type II (TGF-beta RII) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-beta RII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-beta RII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-beta RII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.

Topics: Adenocarcinoma; Animals; Carcinogens; Gene Expression; Lung Neoplasms; Male; Mutation; Nitrosamines; Polymorphism, Single-Stranded Conformational; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the angiogenic factors produced by tumor cells, vascular endothelial growth factor is considered to be the most potent and pathologically important. The synthesis of this growth factor has been shown to be modulated through Sp1 function following stimulation by tumor necrosis factor alpha (TNF-alpha). Oligodeoxynucleotides (ODNs) were synthesized with either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a mutated form of this sequence (mt-Sp1 decoy ODNs). Using the hemagglutinating virus of Japan (HVJ)-liposome method, we transferred these ODNs into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and transforming growth factor beta1 and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, and cell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy ODNs. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness.

Topics: Adenocarcinoma; Binding Sites; Cell Division; Cell Movement; Endothelial Growth Factors; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glioblastoma; Humans; Liposomes; Lung Neoplasms; Lymphokines; Neoplasm Invasiveness; Oligonucleotides; Respirovirus; RNA, Messenger; Sp1 Transcription Factor; Thromboplastin; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim of this study was to compare the function of lung fibroblasts obtained from surgically biopsied specimens of patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (UIP; n = 5), nonspecific interstitial pneumonia (NSIP; n = 5), and normal parts of surgically resected lungs (control; n = 5). The results showed that (1) fibroblasts obtained from UIP showed increased contractility compared with those obtained from NSIP or controls (UIP, 72.7 +/- 6.21%; NSIP, 32.8 +/- 5.46; controls, 28.5 +/- 3.51, p < 0.01 in UIP versus NSIP or control); (2) this increase in contractility was consistent with enhanced F-actin content in fibroblasts; (3) conditioned media from UIP fibroblast cultures enhanced control fibroblast contractility, whereas those obtained from NSIP or controls did not; (4) the 180 and 25 kD products representing the contractility in conditioned media were identified as fibronectin (ED-A domain) and TGF-beta1 by immunoblots, respectively; (5) the UIP-conditioned media contained higher amounts of fibronectin or TGF-beta 1 (fibronectin: UIP 289 +/- 47.1 ng/ml, NSIP 121 +/- 23.0, control 118 +/- 16.0; TGF-beta1: UIP 798 +/- 119 pg/ml, NSIP 246 +/- 69.1, control 247 +/- 53.6, p < 0.01 in UIP versus NSIP or control); () the contractility positively correlated with the amount of either fibronectin (r = 0.867, p < 0.001, n = 15) or TGF-beta 1 (r = 0.939, p < 0.001, n = 15), respectively. Thus, UIP fibroblasts showed greater contractility than did NSIP fibroblasts and up-regulated control fibroblasts.

Topics: Actins; Adenocarcinoma; Analysis of Variance; Biopsy; Cells, Cultured; Culture Media, Serum-Free; Female; Fibroblasts; Gels; Hamartoma; Humans; Lung; Lung Diseases; Lung Diseases, Interstitial; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific (125)I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 microM t-RA decreased specific (125)I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC(1) receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 microM t-RA and 3 microM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-beta2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-beta 1 mRNA was unaffected and TGF-beta 3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC(1) receptor and TGF-beta 3 mRNA but up-regulating TGF-beta 2 mRNA.

Topics: Binding Sites; Cell Division; Cyclic AMP; Dose-Response Relationship, Drug; Down-Regulation; Humans; Kinetics; Lung Neoplasms; Protein Binding; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the p

Topics: Adenoma; Animals; Blotting, Western; Bronchi; Carcinogens; Carcinoma, Non-Small-Cell Lung; Disease Models, Animal; DNA Primers; Female; Humans; Immunohistochemistry; In Situ Hybridization; Lung Neoplasms; Mice; Mice, Inbred A; Mice, Inbred C57BL; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; 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

TGF beta 1 commonly produced by normal and neoplastic human cells, has capacity to regulate new blood vessel formation, to establish and maintain the vessel wall integrity; was found to have some significance in the lung cancer prognosis. Tumour angiogenesis is an important factor for tumour growth and metastasis. The purpose of this study was to find, if the immunoexpression of TGF beta 1 has any significance in determination of the histologic subtypes of carcinoids?; to find, if TGF beta 1 has any role and relation to carcinoids angiogenesis?; and to explore TGF beta 1 expression and angiogenesis with respect to metastatic potential of carcinoids. The study was performed on 48 resected broncho-pulmonary carcinoids: 35 typical (TC) and 13 atypical (AC), classified according to the WHO. Semiquantitative analysis for TGF beta 1 was performed. Sections stained using monoclonal antibody against TGF beta 1 were scored in scale from 0 to 4, according to the percentage of positively stained cells (pc) plus percentage of positively stained stroma (ps). The microvessels stained with CD34 monoclonal antibody, were counted in 0.75 mm2 field (microvessel density--MD), using the computerised image analysis system SAMBA 2005 (the morphometric software). The histologic subtype of carcinoids was related to age of the pts (AC occurred in older pts than TC, p = 0.027), to the tumour size (AC were larger than TC: respectively--3.25 cm and 2.4 cm, p = 0.009). Lymph node metastases were significantly more frequent in AC than in TC (38% vs 13%, p = 0.025). 85% carcinoids showed TGF beta 1 expression with various intensity, mainly in the stroma. There was no significant correlation between TGF beta 1 expression and tumour size, the histologic subtype nor the lymph node metastases. The angiogenesis expressed as MD, was not related to histology, nor to the presence of lymph node metastases. There was no correlation between TGF beta 1 expression and angiogenesis. Shown in our study, lack of relation between TGF beta 1 expression and angiogenesis, could support some of the published data indicating indirect action of TGF beta 1 on the angiogenesis. The rich vascularity found in carcinoids morphology could result from TGF beta 1, commonly expressed by the tumoural stroma. The angiogenesis nor TGF beta 1 expression do not determinate the carcinoids histology.

Topics: Adult; Biomarkers, Tumor; Carcinoid Tumor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neovascularization, Pathologic; Retrospective Studies; Transforming Growth Factor beta; Transforming Growth Factor beta1

Bryostatin 1 and phorbol esters reduce the intracellular melanin level in high metastatic overexpressing nPKCdelta BL6 (BL6T) cells, thereby inducing white experimental metastasis in syngeneic mice. We evaluate here the possible differences between white and black metastases induced by both treatments on the proliferative and metastatic potential as well as on the expression of some cytokines involved in the metastatic process such as TGFbeta, IL-10 and IFNgamma. The level of expression of gelatinase A is also considered. White and black metastases induced after the injection of bryostatin 1- or phorbol ester-treated cells into the tail vein of syngenic mice were isolated and analysed for the levels of LDH usually used as markers of cytotoxicity, for the levels of cytokines and gelatinase A or dissociated and cultured in vitro for a few passages. The cultured cells were analysed in vitro for the proliferative capacity and the melanin synthesis. The same cells were also re-injected into syngeneic mice and the number of experimental metastases were counted after 17 days or injected with matrigel in order to quantify the proliferative capacity in vivo. The results show only one significant difference between bryostatin I and phorbol ester, namely the cells obtained from white bryostatin 1-treated cells return to a black phenotype after a few passages in culture. This suggests that PKC mediates many of the biological effects of bryostatin 1 but that its effect is lost in vitro. On the other hand, white and black metastases (at least for metastases induced by BL6T cells treated with phorbol ester) do appear significantly different. In vivo white metastases show lower levels of LDH, lower levels of proliferative capacity into matrigel, higher levels of TGFbeta and IFNgamma and, when re-injected into syngeneic mice, give big black metastases. Therefore, in murine melanoma cells, the treatment with bryostatin I induces the appearance of a white population expressing different levels of TGFbeta, IFNgamma, IL-10 and gelatinase A. Such a white population is more difficult to diagnose and is capable of turning into a more aggressive phenotype under suitable environmental conditions.

Topics: Animals; Antineoplastic Agents; Bryostatins; Interferon-gamma; Interleukin-10; L-Lactate Dehydrogenase; Lactones; Lung Neoplasms; Macrolides; Matrix Metalloproteinase 2; Melanoma; Mice; Mice, Inbred C57BL; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) is known as the growth factor that stimulates the synthesis of extracellular matrix. Recently, TGF-beta has been found to control the growth of cancer cells. Small chondroitin-dermatan sulfate (decorin) is an abundant extracellular matrix component. TGF-beta1 stimulates the synthesis of decorin, and decorin is considered to bind TGF-beta1. The activity of decorin in neutralizing TGF-beta1 activity suggests that decorin serves as a negative-feedback regulator of TGF-beta1 activity. To investigate the role and relationship of TGF-beta1 and decorin in the formation of central fibrosis in pulmonary adenocarcinoma, we performed an immunohistochemical study of TGF-beta1 and decorin in 61 cases of T1 pulmonary adenocarcinoma. Positive stainings for TGF-beta1 were shown in 40 cases and negative in 21 cases. Twenty-seven of 32 cases with central fibrosis were positive for TGF-beta1. Positive staining for TGF-beta1 was significantly related to the appearance of central fibrosis in pulmonary adenocarcinoma. When central fibrosis was composed of proliferative connective tissue with loose staining for decorin, cancer cells showed intense staining for TGF-beta1. When central fibrosis was composed of old fibrotic tissue with dense staining for decorin, cancer cells showed weak staining for TGF-beta1. Our results suggest that TGF-beta1 has an important role in the formation of central fibrosis in pulmonary adenocarcinoma, and decorin may play a role as a negative feedback regulator in the production of TGF-beta1 in pulmonary adenocarcinoma.

Topics: Adenocarcinoma; Decorin; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Lung Neoplasms; Proteoglycans; Pulmonary Fibrosis; Survival Rate; Transforming Growth Factor beta

We and others have reported that cyclosporine (CsA) induces increased expression of transforming growth factor-beta1 (TGF-beta1) in vitro as well as in vivo. In view of similarities between tacrolimus and CsA with respect to immunosuppressive mechanisms, we determined whether tacrolimus, in a fashion similar to CsA, induces TGF-beta1 hyperexpression in mammalian cells.. We studied the induction of TGF-beta1 mRNA by tacrolimus using reverse transcription-polymerase chain reaction and Northern blot analysis in normal human T cells and A-549 cells (human lung adenocarcinoma cell line), a cell line used to study the biology of TGF-beta and the induction of TGF-beta1 by CsA. We also measured the induction of TGF-beta1 protein by tacrolimus in activated human T cells, peripheral blood mononuclear cells, and A-549 cells, using sandwich enzyme-linked immunosorbent assay.. A significant increase in the TGF-beta1 mRNA expression was observed after treatment of T cells or A-549 cells. Tacrolimus treatment resulted also in heightened production of TGF-beta1 protein by activated T cells, A-549 cells, or peripheral blood mononuclear cells activated with anti-CD3, phytohemagglutinin, and concanavalin A.. Our observations that tacrolimus stimulates TGF-beta1 hyperexpression in mammalian cells suggest a unifying mechanism for the immunosuppressive as well as nephrotoxic properties of tacrolimus, as the multifunctional TGF-beta1 is a potent immunosuppressive and fibrogenic cytokine.

Topics: DNA; Gene Expression Regulation; Humans; Immunosuppressive Agents; Lung Neoplasms; RNA, Messenger; T-Lymphocytes; Tacrolimus; Transforming Growth Factor beta; Tumor Cells, Cultured

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

Topics: Adenocarcinoma; Animals; Coculture Techniques; Enzyme Induction; Enzyme Precursors; Gene Expression Regulation, Neoplastic; Humans; Hydrazines; Image Processing, Computer-Assisted; Interferon-gamma; Lipopolysaccharides; Lung Neoplasms; Macrophage Activation; Macrophages; Mice; Neoplasm Proteins; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Oxidative Stress; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Proteins; Recombinant Fusion Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Current immunosuppression strategies involve inhibition of T cell activation and/or lymphocyte proliferation. During T cell cycle progression/activation, the expression of cyclins and cyclin dependent kinases is increased. In this study, we examined whether cyclosporine A (CsA) suppresses the cell cycle progression through the induction of the cell cycle inhibitor p21. Because CsA induces the expression of transforming growth factor (TGF)-beta, and TGF-beta induces p21 expression, we also determined whether CsA's induction of p21 is dependent on or independent of TGF-beta.. Using reverse transcription assisted polymerase chain reaction and western blot analysis, we studied the induction of p21 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by CsA. The stimulation of p21 promoter activity was studied by luciferase assay using p21-luc, chimeric plasmid DNA containing a p21 promoter segment, and luciferase reporter gene. The dependence of CsA's induction of p21 was studied using anti-TGF-beta antibody and TGF-beta altered A-549 cells.. CsA induced p21 mRNA protein expression and stimulated its promoter activity in lymphoid (T cells) and nonlymphoid (human lung adenocarcinoma, A-549 cells).CsA's induction of p21 was inhibited both by a neutralizing anti-TGF-beta antibody and in TGF-beta-altered A-549 cells, consistent with its effects on p21 requiring TGF-beta.. These data support the hypothesis that at least one component of CsA's antiproliferative effects may occur through the induction of p21 and that this induction is dependent on TGF-beta. Should p21 induction be a viable immunosuppressive strategy, inducing this molecule independent from the fibrogenic cytokine TGF-beta might reduce the toxicity associated with current immunosuppression.

Topics: Adenocarcinoma; Antibodies; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cyclosporine; Enzyme Inhibitors; Humans; Immunosuppressive Agents; Lung Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Recent studies have suggested that transforming growth factor(TGF)-beta1 acts as a multifunctional regulator of cell growth, and also modifies tumor progression and metastasis. In the present study, we investigated the effects of TGF-beta1 on the proliferation and experimental pulmonary metastasis of MCS-1. MCS-1 are undifferentiated type cloned tumor cells established from a mesenchymal chondrosarcoma which spontaneously occurred in the soft tissue of a female Chinese hamster. MCS-1 cells were pretreated with TGF-beta1 (0, 0.05, 0.5, 2, 10 ng/ml) for 72 hours in a medium containing 1% fetal bovine serum, then tested for in vitro growth by the MTT method, in vivo growth by subcutaneous inoculation into athymic nude mice (1 x 10(6) cells/mouse) and experimental pulmonary metastasis by injection into the lateral tail vein of athymic nude mice (5 x 10(4) cells/mouse). TGF-beta1 significantly inhibited in vitro growth of MCS-1, depending on its concentrations, and also experimental metastasis with maximal inhibition at 0.5 or 2 ng/ml treatment compared to untreated controls. TGF-beta1, however, was ineffective for in vivo subcutaneous growth of MCS-1. These results indicated that TGF-beta1 might be an inhibitor of metastasis of mesenchymal chondrosarcomas including other types of non-epitherial cartilage or bone formation tumors.

Topics: Animals; Bone Neoplasms; Cattle; Chondrosarcoma, Mesenchymal; Cricetinae; Female; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Transforming Growth Factor beta; Transplantation, Heterologous

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

To determine the identity of a major membrane/ extracellular matrix (ECM)-associated 66-kDa protein in human corneal endothelial cells and its phosphorylation in vivo.. A membrane/ECM-associated 66 kDa protein was purified from human corneal endothelial cells of 50-80 year-old donors by a three-step procedure that included preparative SDS-PAGE, preparative isoelectric focusing (IEF) and HPLC using a C-18 column. The phosphorylation of the 66-kDa protein was determined by both endogenous kinases and exogenous protein kinase A in the presence of endogenous or added cAMP, or added Walsh inhibitor (a specific inhibitor of protein kinase A). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. The phosphoamino acids were identified following hydrolysis of the purified phosphorylated 66-kDa protein and thin layer chromatography with standard phosphoamino acids.. Following purification, the 66-kDa protein showed a single band on SDS-PAGE, a single species on two dimensional (2D)-gel electrophoresis and a single peak during C-18 HPLC. The partial N-terminal sequence of the 66-kDa protein matched with that of the 68-kDa beta ig-h3 protein (minus the signal peptide) of lung adenocarcinoma cells. Furthermore, on Western blot analysis, the 66-kDa protein immunoreacted with anti-beta ig-h3 past signal peptide (residue nos.24-32) antibody but not with the anti-beta ig-3 signal peptide (residue nos.1-9) antibody. During the phosphorylation of endothelial proteins by endogenous kinases or added protein kinase A in the presence of endogenous or added cAMP, the 66 kDa protein was the major substrate with its Ser residues phosphorylated in both cytosolic- and membrane/ECM-fractions.. The human corneal endothelial 66-kDa protein is identical to the 68-kDa beta ig-h3 protein (minus signal peptide) from lung adenocarcinoma cells. The corneal protein exists in a phosphorylated state in vivo with its Ser residues phosphorylated by a cAMP-dependent protein kinase A.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Amino Acid Sequence; Blotting, Western; Cell Membrane; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Endothelium, Corneal; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Lung Neoplasms; Middle Aged; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins; Phosphorylation; Transforming Growth Factor beta

This study examined the metastatic capacity of clonal populations of 4NQO-induced rat malignant oral keratinocytes following orthotopic transplantation to athymic mice. Polygonal and spindle cells formed well-differentiated squamous cell carcinomas (keratin positive and vimentin negative) and undifferentiated spindle cell tumours (keratin negative and vimentin positive), respectively, in almost 100% of animals at the site of inoculation (floor of mouth). Transplantation of 5x 10(6) cells of either cell type at high cell density resulted in approximately 50% of animals forming pulmonary metastases. By contrast, inoculation of 2x 10(6) differentiated polygonal cells resulted in the formation of significantly fewer pulmonary metastases than the undifferentiated spindle cells. A single well-differentiated clone of polygonal cells and 3 of 4 of the undifferentiated spindle cell lines produced comparable levels of TGF-beta1. One undifferentiated spindle cell line expressed significantly more TGF-beta1 and, following transplantation orthotopically, fewer animals formed pulmonary metastases despite the formation of primary tumours in almost all grafted animals, suggesting that TGF-beta1 can act as a tumour suppressor in this cell type. All cell lines produced comparable amounts of TGF-beta2. The clones of polygonal cells were markedly inhibited and the spindle cells were only partially inhibited by exogenous TGF-beta1. Both cell types expressed high-affinity TGF-beta cell surface receptors; the ratio of type I to type II TGF-beta receptors was 1.0:<3.0 in the spindle cells and 1.0:17.9 in the polygonal clone. The results suggest that differentiated rat malignant oral keratinocytes are less aggressive and have a decreased potential to metastasise than their undifferentiated spindle cell counterparts. This may be attributable, in part, to a change in TGF-beta receptor profile leading to the partial loss of response to exogenous TGF-beta1.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transplantation; Clone Cells; Keratinocytes; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Nude; Mouth Neoplasms; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Plasma transforming growth factor-beta1 (TGFbeta1) levels are increased in many malignancies at the time of diagnosis, including all forms of lung carcinoma. Therefore, the potential use of TGFbeta1 as a plasma marker to predict the long term outcome of lung carcinoma patients treated with radiotherapy (RT) was evaluated.. Plasma samples for 59 newly diagnosed lung carcinoma patients were assayed for TGFbeta1 before RT (pre RT), at the end of RT (end RT), and during follow-up after RT. TGFbeta1 was extracted from plasma using an acid-ethanol method. An enzyme-linked immunoadsorbent assay was used to quantify the plasma TGFbeta1 levels. The normal value for this assay is < or =7.5 ng/mL. Disease status at last follow-up was without knowledge of TGFbeta1 levels. Comparisons within groups and between groups were estimated using analysis of variance and the Student t test for unpaired data, respectively.. The 59 patients were divided into 2 groups according to their disease status at last follow-up: those with no evidence of disease (NED) (n = 13) and those with disease (WD) (n = 46). The median follow up was 26.8 months and 12.4 months, respectively, for the NED and WD groups. No significant differences were found in the clinical characteristics between the two groups. The plasma TGFbeta1 level before RT was significantly higher in the WD group (mean +/- standard error of the mean [SEM] = 12.5+/-1.7 ng/mL; median = 8.6 ng/mL) compared with the NED group (mean +/- SEM = 6.0+/-1.0 ng/mL; median = 6.0 ng/mL) (P = 0.037). At the time of last follow-up, WD patients had a significantly higher plasma TGFbeta1 level (mean +/- SEM = 11.6+/-1.3 ng/mL; median = 9.6 ng/mL) compared with NED patients (mean +/- SEM = 3.7+/-0.5 ng/mL; median = 3.6 ng/mL) (P = 0.002).. These data demonstrate that plasma TGFbeta1 may be a useful tumor marker in patients with lung carcinoma.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Case-Control Studies; Disease-Free Survival; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Prognosis; Transforming Growth Factor beta

High concentrations of O(2) inhibit epithelial cell proliferation that resumes on recovery in room air. To determine whether growth arrest is mediated by transforming growth factor-beta (TGF-beta), changes in cell proliferation during exposure to hyperoxia were assessed in the mink lung epithelial cell line Mv1Lu and the clonal variant R1B, which is deficient for the type I TGF-beta receptor. Mv1Lu cells treated with TGF-beta accumulated in the G(1) phase of the cell cycle as determined by propidium iodide staining, whereas proliferation of R1B cells was unaffected by TGF-beta. In contrast, hyperoxia inhibited proliferation of both cell lines within 24 h of exposure through an accumulation in the S phase. Mv1Lu cells treated with TGF-beta and exposed to hyperoxia accumulated in the G(1) phase, suggesting that TGF-beta can inhibit the S phase accumulation observed with hyperoxia alone. Cyclin A was detected in cultures exposed to room air or growth arrested by hyperoxia while decreasing in cells growth arrested in the G(1) phase by TGF-beta. Finally, hyperoxia failed to activate a TGF-beta-dependent transcriptional reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF-beta signaling.

Topics: Adenocarcinoma; Animals; Cell Division; Epithelial Cells; Flow Cytometry; G1 Phase; Gene Expression; Genes, Reporter; Luciferases; Lung; Lung Neoplasms; Mink; Oxygen; S Phase; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.

Topics: Animals; Carcinoma; Collagenases; Female; Gelatinases; Lung Neoplasms; Male; Matrix Metalloproteinase 9; Mice; Mice, Nude; Prostatic Neoplasms; RNA, Catalytic; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Transforming Growth Factor beta

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.

Topics: Adenocarcinoma; Adoptive Transfer; Animals; Bone Marrow Transplantation; Colonic Neoplasms; Graft vs Host Disease; Immunosuppressive Agents; Interleukin-2; Intestines; Killer Cells, Natural; Liver; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, SCID; Skin; Time Factors; Transforming Growth Factor beta; Transplantation, Homologous

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

Transforming growth factor-beta (TGF-beta) is a growth modulator that inhibits the proliferation of many epithelial cells through interaction with its receptors, the type I and type II receptors (TGF-beta RI and RII) by activating their serine/threonine kinase activities. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. To examine the roles of TGF-beta1, -beta2, and -beta3 and TGF-beta RI and RII in chemically induced mouse lung tumorigenesis, we used immunohistochemical and in situ hybridization analyses to measure the expression of their proteins and mRNAs in A/J mice treated with the carcinogen urethane to induce lung adenomas. Immunostaining for the TGF-beta ligands and receptors was detected in the epithelia of the bronchioles of untreated and treated A/J mice at similar levels. Immunostaining for the TGF-beta ligands and receptors was also detected in adenomas by 2 mo. While immunostaining for TGF-beta1, -beta2, and -beta3 and TGF-beta RI in adenomas was detected at levels comparable to those in bronchioles, immunostaining for TGF-beta RII was less intense in adenomas than in bronchioles. Decreased immunostaining for TGF-beta RII in adenomas persisted for at least 8 mo after exposure to urethane, whereas immunostaining for TGF-beta1, -beta2, and -beta3 and TGF-beta RI persisted at levels comparable to those in normal bronchioles. In situ hybridization studies conducted with TGF-beta receptor riboprobes showed a corresponding reduction in expression of TGF-beta RII mRNA but not of TGF-beta RI mRNA in adenomas compared with expression in bronchioles. Expression of TGF-beta RII mRNA was also examined in non-tumorigenic and tumorigenic mouse lung cells; expression of TGF-beta RII mRNA was lower in the tumorigenic cells derived from urethane-induced lung tumors. These data suggest that a decrease in expression of TGF-beta RII may contribute to autonomous cell growth and may play an important role in mouse lung carcinogenesis induced by urethane.

Topics: Activin Receptors, Type I; Animals; Carcinogens; Cell Division; Cell Line; Cell Transformation, Neoplastic; Down-Regulation; Epithelial Cells; Female; Lung; Lung Neoplasms; Mice; Mice, Inbred A; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Urethane

p21waf1/cip1 encodes a cyclin-dependent kinase inhibitor that is transcriptionally activated by the p53 tumor suppressor gene, transforming growth factor beta 1 (TGF-beta 1), AP2, and other pathways. Because p21waf1/cip1, p53, and TGF-beta 1 all regulate apoptosis and the cell cycle, we tested the hypothesis that their relative protein levels would correlate with biological features including the survival of non-small cell lung cancer (NSCLC) patients. We conducted an immunohistochemical analysis of p21waf1/cip1 and TGF-beta 1 and identified four patient groups with distinct survival outcomes. Concordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and high TGF-beta 1 expression or low p21waf1/cip1 and low TGF-beta 1 expression) predicted 70% disease-free survival at 2000 days of follow-up. Discordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and low TGF-beta 1 expression or low p21waf1/cip1 and high TGF-beta 1 expression) predicted 35% disease-free survival (P = 0.0003; log-rank test). These survival relationships were not attributable to differences in grade, stage, or p53 status. Although current models do not fully explain these complex interactions, most of these data fit a paradigm whereby TGF-beta 1 regulation determines NSCLC survival. In addition to the survival correlation, we found that high p21waf1/cip1 protein expression correlated with high tumor grade (P = 0.014). There is little evidence that p21waf1/cip1 protein levels accurately predict p53 mutation status in NSCLC; specifically, 20 of 48 (42%) tumors with p53 mutations contained high levels of p21waf1/cip1 protein. These findings indicate that p21waf1/cip1 immunohistochemical analysis may provide useful information concerning the biological properties of NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease-Free Survival; Enzyme Inhibitors; Female; Follow-Up Studies; Genes, p53; Humans; Immunohistochemistry; Lung Neoplasms; Male; Mutation; Neoplasm Staging; Prognosis; Sex Characteristics; Survival Analysis; Time Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

For understanding the local immune response in human non-small cell lung cancer (NSCLC), we investigated both Th1 and Th2-type as well as TGF-beta1 cytokine mRNA expression in 10 fresh tumor biopsies, the corresponding tumor and short term TIL cell lines as well as patient PBMC. A methodology based on a highly sensitive quantitative RT-PCR was used. We found that IL-6 mRNA was highly expressed in all tumor biopsy samples analyzed (4 LLC, 3 ADC and 3 SCC). IL-10 mRNA was expressed in 7 of 10 biopsies whereas IL-4 mRNA expression was moderate. Analysis of type I cytokines revealed a low expression level of IL-2 mRNA, while IFNgamma and GM-CSF expression was high in the majority of the tumor lesions studied. Quantitatively, high amounts of Th2-type cytokine mRNA were detected at the tumor site with IL-6 as the predominant lymphokine. A high mRNA expression level of the immunosuppressive cytokine TGF-beta1 was observed in all NSCLC. To identify the cell types responsible for the production of TGF-beta1, IL-6, IL-10 and GM-CSF at the tumor site, tumor and TIL cell lines were derived from the corresponding biopsies. All the 3 tumor cell lines analysed were found to express high amount of TGF-beta1 but not IL-10 mRNA, 2 expressing IL-6 and GM-CSF. Five short term TIL cell lines established in the presence of IL-2 expressed high level of IL-10, IL-4 and IFNgamma but not IL-2 mRNA. Strikingly, high expression of IL-10 mRNA was also observed in all 6 patient PBMC analyzed as compared to controls. Together, our results indicate the existence of a local and peripheral Th-2-type cytokine pattern in patients bearing NSCLC.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cytokines; Female; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; RNA, Messenger; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

Topics: Adult; Aged; Animals; Dogs; Female; Humans; Lung Neoplasms; Male; Middle Aged; Minimally Invasive Surgical Procedures; Plastic Surgery Procedures; Pneumonectomy; Surgical Wound Dehiscence; Survival Rate; Transforming Growth Factor beta; Wound Healing

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

Topics: Animals; Apoptosis; Carcinogenicity Tests; Cell Cycle Proteins; Cell Division; Gene Targeting; Genes, Tumor Suppressor; Liver; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Transforming Growth Factor beta

Surgical excision of liver tumors represents the only curative treatment for primary and metastatic liver malignancies. It has been suspected that hepatectomy may stimulate growth of microscopic tumors. To determine whether local or systemic factors after hepatectomy are responsible for enhancement of tumor growth, the effects of hepatectomy on the experimental growth of liver or pulmonary tumors were examined.. One hour after injection of 10(6) Morris hepatoma cells into either the portal or femoral vein, which produces isolated liver and lung tumors, respectively, animals were randomized to undergo 0%, 30%, or 70% partial hepatectomy (PH).. Animals that underwent portal injection of tumor had significantly increased liver tumor burden after PH (sham, 25 +/- 7 vs PH, 94 +/- 17; p < 0.01), whereas animals that underwent femoral injection had no change in lung tumor burden after PH. PH was associated with significantly increased levels of transforming growth factor-alpha, transforming growth factor-beta, and basic fibroblast growth factor in the liver but not in the lung.. Changes in liver cytokine-growth factor activation may contribute to enhanced tumor growth in the liver after hepatectomy.

Topics: Animals; Carcinoma, Hepatocellular; Fibroblast Growth Factor 2; Hepatectomy; Injections, Intravenous; Liver; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred BUF; Transforming Growth Factor alpha; Transforming Growth Factor beta

Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.

Topics: Activins; Animals; Bone Morphogenetic Proteins; Cell Line; DNA-Binding Proteins; Drug Synergism; Epidermal Growth Factor; Epithelial Cells; Gene Expression; Humans; Inhibins; Keratinocytes; Lung; Lung Neoplasms; Mink; RNA, Messenger; Signal Transduction; Smad6 Protein; Smad7 Protein; Tetradecanoylphorbol Acetate; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate prospectively the utility of plasma transforming growth factor beta1 (TGFbeta1) as a marker for the development of symptomatic radiation pneumonitis.. Seventy-three patients with lung cancer treated with curative intent are reported herein. Plasma TGFbeta1 samples were obtained before, weekly during, and at each follow-up after radiation therapy (RT). TGFbeta1 was extracted using an acid/ethanol method. An enzyme-linked immunosorbent assay was used to quantify plasma TGFbeta1 concentrations. The TGFbeta1 level at the end of RT was considered "normal" if it was both < or = 7.5 ng/ml and less than the pretreatment value. All patients were followed for at least 6 months, unless symptomatic pneumonitis developed sooner. Pneumonitis was defined by National Cancer Institute (NCI) common toxicity criteria.. Fifteen of the 73 patients (21%) developed symptomatic pneumonitis and the remaining 58 (79%) did not. A normal plasma TGFbeta1 by the end of RT, as defined above, was more common in patients who did not develop pneumonitis. A return of the plasma TGFbeta1 to normal accurately identified patients who would not develop pneumonitis with both a sensitivity and positive predictive value of 90%.. Plasma TGFbeta1 levels appear to be a useful means to identify patients at low risk for the development of pneumonitis from thoracic RT. Thus, monitoring of plasma TGFbeta1 levels may identify candidates for dose escalation studies in the treatment of lung cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prospective Studies; Radiation Pneumonitis; Transforming Growth Factor beta

Previously we reported the malignant progression of QR-32, a regressor-type tumor clone, following co-implantation with foreign bodies (gelatin sponge or plastic plate) in normal syngeneic C57BL/6 mice. We also reported that the progression of QR-32 cells by a gelatin sponge was significantly inhibited in the mice administered polysaccharide K (PSK) and that PSK induced an increase of radical scavengers, especially manganese superoxide dismutase (Mn-SOD), locally at the site of tumor tissues. In this study, to reveal the possible mechanism by which PSK induced Mn-SOD in the tumor tissues, we examined the mRNA expression and protein levels of inflammatory cytokines in the tissues. We found that mRNAs of tumor necrosis factor alpha (TNFalpha) and interleukin-1alpha (IL-1alpha) were considerably expressed in both PSK-treated and phosphate-buffered-saline-treated tumors, and that the mRNA expression and protein level of interferon gamma (IFNgamma) increased in the tumor tissues treated with PSK. In vitro treatment of QR-32 cells with IFNgamma did not significantly increase the production of Mn-SOD; however, the combination of IFNgamma with TNFalpha increased the Mn-SOD production more effectively than did any of the cytokines used singly. Furthermore, we observed the down-regulation of the mRNA expression and protein level of transforming growth factor beta (TGFbeta) in the tumor tissues treated with PSK, and that in vitro treatment of QR-32 cells with TGFbeta decreased the production of Mn-SOD. These results suggest that PSK suppresses the progression of QR-32 cells by increasing Mn-SOD via the modulation of inflammatory cytokines; that is, by decreasing TGF-beta and increasing IFN-gamma.

Topics: Adjuvants, Immunologic; Animals; Enzyme Induction; Female; Fibrosarcoma; Foreign-Body Reaction; Gelatin; Gene Expression Regulation, Neoplastic; Interferon-alpha; Interferon-gamma; Interleukin-1; Lung Neoplasms; Manganese; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Transplantation; Proteoglycans; RNA, Messenger; RNA, Neoplasm; Superoxide Dismutase; Surgical Sponges; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-betas (TGFbetas) play a prominent role in tumour growth and metastasis by enhancing angiogenesis and suppressing immune surveillance. Despite the increased interest in the effect of TGFbetas on tumour progression, little is known about the importance of TGFbeta3 and its receptor CD105 in breast cancer. In the present study, we measured the plasma levels of TGFbeta3, CD105-TGFbeta3 complexes and TGFbeta1 in 80 patients with untreated early-stage breast cancer using an enhanced chemiluminescence ELISA method. Of the 80 patients, 14 were histologically confirmed as having axillary lymph node metastases, while the remainder had no evidence of lymph node involvement. The results showed that levels of both TGFbeta3 and CD105-TGFbeta3 complex were significantly elevated in patients with positive lymph nodes compared to those without node metastasis. Furthermore, the levels of both TGFbeta3 and CD105-TGFbeta3 complex correlated with lymph node status. The only patient who died of the disease had very high plasma levels of TGFbeta3 and CD105-TGFbeta3 complex and positive lymph nodes; this patient developed lung metastases within 2 years of diagnosis. No significant correlation was seen between either TGFbeta3 or CD105-TGFbeta3 complex levels and tumour stage, size or histological grade. Plasma TGFbeta1 levels were not correlated with node metastasis, tumour stage, grade or size. Our data suggest that plasma levels of TGFbeta3 and CD105-TGFbeta3 complex may be of prognostic value in the early detection of metastasis of breast cancer.

Topics: Adult; Antigens, CD; Axilla; Breast Neoplasms; Endoglin; Enzyme-Linked Immunosorbent Assay; Female; Humans; Luminescent Measurements; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Receptors, Cell Surface; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

The distribution of the three isoforms of transforming growth factor (TGF)-beta (TGF-beta 1, -beta 2, and -beta 3) as well as their signaling receptor, TGF-beta type I receptor (T beta R-I), in bronchiolo-alveolar cell hyperplasia (BAH) and bronchiolo-alveolar carcinoma (BAC) was examined by immunohistochemistry using specific antibodies. The results showed that the expression of one or more TGF-beta subtypes occurred in a large proportion of BAC and BAH tissues. The expression of TGF-beta 1 and TGF-beta 3 was increased in tumor cells compared with hyperplastic cells. BAC with expression of two or three ligands was associated with a higher incidence of lymph node metastasis. These results suggest that hyperplastic cells and cancer cells derived from bronchiolo-alveolar cell can synthesize and secrete TGF-beta isoforms and its receptor T beta R-I which may contribute to the progression of BAC in part.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Adult; Aged; Bronchi; Female; Humans; Hyperplasia; Lung Neoplasms; Male; Middle Aged; Pulmonary Alveoli; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

We tested 20 human carcinoma samples for the production of transforming growth factor beta (TGFbeta) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFbeta. According to this assay, the supernatants contained both active and latent TGFbeta. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFbeta. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFbeta-specific mAb. It is important to note that, when TGFbeta-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFbeta may help the survival of potentially immunogenic tumour cells in immunocompetent patients.

Topics: Carcinoma; Female; Humans; Lung Neoplasms; Lymphocyte Activation; Male; Melanoma; Ovarian Neoplasms; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

To determine whether changes in the plasma Transforming Growth Factor beta1 (TGF beta1) concentration during radiotherapy could identify patients at risk for developing symptomatic radiation pneumonitis.. Thirty-six patients who received radiation therapy with curative intent for lung cancer (n = 31), Hodgkin's disease (n = 4), or thymoma (n = 1) were evaluated prospectively. All patients had serial plasma TGF beta1 measurements obtained before, during, and after treatment. Plasma TGF beta1 was quantified using an enzyme-linked immunosorbent assay. Pneumonitis was defined clinically. Plasma TGF beta1 levels were considered to have normalized if the following occurred: the last on-treatment TGF beta1 level was both <7.5 ng/ml and lower than the pretreatment level.. Thirteen of these 36 patients developed pneumonitis. Significant changes in plasma TGF beta1 levels during treatment were seen only in the subset of patients whose TGF beta1 levels were >7.5 ng/ml at baseline (n = 22). Failure of plasma TGF beta1 to normalize by the end of treatment, as defined above, much more accurately identified patients at risk for symptomatic pneumonitis if their baseline TGF beta1 was >7.5 ng/ml than if it was <7.5 ng/ml.. Changes in plasma TGF beta1 levels during radiotherapy appears to be a useful means by which to identify patients at risk for the development of symptomatic radiation pneumonitis, particularly in the subset of patients whose pretreatment TGF beta1 levels are >7.5 ng/ml.

Topics: Biomarkers; Hodgkin Disease; Humans; Lung Neoplasms; Prospective Studies; Radiation Pneumonitis; Sensitivity and Specificity; Thymoma; Transforming Growth Factor beta

In the United States, pulmonary adenocarcinomas have recently replaced squamous cell carcinomas as the most frequent type of lung cancer encountered. The incidence of pulmonary adenocarcinoma continued to increase worldwide.. To determine the roles of the transforming growth factor-beta 1 (TGF-beta 1), and TGF-beta type I receptor (T beta R-I), and the TGF-beta type II receptor (T beta R-II) in the progression of a pulmonary adenocarcinoma, their respective expressions have been immunohistologically studied in specimens from 120 pulmonary adenocarcinoma patients.. The overall prognosis was significantly poorer for patients showing positive TGF-beta 1, T beta R-I, T beta R-II expressions than for patients who were negative to all three immunostainings (P < 0.01). Our multivariate analysis also revealed that a positive TGF-beta 1 response significantly affect prognosis (P < 0.05).. TGF-beta 1, T beta R-I, and T beta R-II play important roles in tumor progression, and a positive TGF-beta 1 expression can serve as a pulmonary adenocarcinoma marker. T beta R-I and T beta R-II expressions are necessary for TGF-beta signal transduction.

Topics: Activin Receptors, Type I; Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Survival Rate; Transforming Growth Factor beta

In tissue culture conditions, exogeneous active transforming growth factor-beta1 (TGF-beta1) enhances the lethal effect of DNA-damaging agents (UV-C, gamma rays, cisplatin, methotrexate and 5-fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild-type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF-beta1 and UV-C was studied on mink and human cell lines, and the biomodulation by TGF-beta1 of cell killing by cisplatin, gamma rays, 5-fluorouracil or methotrexate was tested only on human cells. As investigated with UV-C-irradiated human A549 cells, TGF-beta1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF-beta1 might affect the curative and/or undesirable secondary side effects of cancer therapy.

Topics: Animals; Antineoplastic Agents; Cell Death; Cisplatin; DNA Damage; Drug Synergism; Fluorouracil; Humans; Lung Neoplasms; Methotrexate; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-beta and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-beta in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-beta involves PKC. TGF-beta 1 and PMA increased expression of TGF-beta 1 mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-beta 2 and TGF-beta 3 were more complex; while TGF-beta 2 and TGF-beta 3 mRNAs increased transiently in response to TGF-beta 1 in NHBE cells and TGF-beta 3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCI-H727 where TGF-beta 2 mRNA increased dramatically, TGF-beta 1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contrast, while TGF-beta 1 also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-beta 1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-beta 1, PAI-1 and PA by TGF-beta 1 and PMA in NSCLC, but differential regulation of the genes for PAI-1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-beta 1, PAI-1 and PA to TGF-beta 1 and PMA were inhibited by the serine/ threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-beta 1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-beta responses by inhibiting a protein serin

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Blotting, Northern; Bronchi; Carcinoma, Non-Small-Cell Lung; Cells, Cultured; DNA Probes; Enzyme Inhibitors; Extracellular Matrix; Fibronectins; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Plasminogen Activator Inhibitor 1; Plasminogen Activators; Protein Serine-Threonine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured

In addition to autoregulating its own expression, transforming growth factor-beta 1 (TGF-beta1) also regulates the production of proteases, protease inhibitors and extracellular matrix proteins. To investigate the relationship between plasminogen activator (PA), plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix in malignant and normal lung epithelial cells and to determine whether malignant lung epithelial cells may be more invasive than normal lung epithelial cells because of differences in expression of these proteins in response to TGF-beta, the regulation of PA, PAI-1, fibronectin, laminin and thrombospondin by TGF-beta1 in human non-small cell lung cancer (NSCLC) cells was examined and compared with normal human bronchial epithelial (NHBE) cells. TGF-beta1 caused a persistent increase in expression of the mRNAs for both PA and PAI-1 in NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. By immunoprecipitation analysis, it was shown that TGF-beta1 also induced a corresponding increase in the amount of PAI-1 protein in these NSCLC cells as well. In contrast, while TGF-beta1 also increased expression of PAI-1 mRNA in NHBE cells, expression of PA mRNA decreased simultaneously. Treatment of NSCLC cells with TGF-beta1 resulted in a persistent increase in expression of the mRNAs for fibronectin, laminin and thrombospondin; expression of fibronectin protein also increased after treatment with TGF-beta1 in these cells. When NHBE cells were similarly cultured in the presence of TGF-beta1, expression of fibronectin mRNA also increased in a persistent manner; however, only an early transient increase in the level of the mRNAs for laminin and thrombospondin was detected in these cells. These data show that there is differential regulation of the genes for PA and PAI-1 and the extracellular matrix protein fibronectin in response to TGF-beta1 not only when NSCLC and NHBE cells are compared, but also when different NSCLC cells are compared with each other.

Topics: Bronchi; Carcinoma, Non-Small-Cell Lung; Cells, Cultured; Culture Media, Conditioned; Endopeptidases; Epithelium; Extracellular Matrix Proteins; Fibronectins; Gene Expression; Humans; Lung Neoplasms; Plasminogen Activators; Precipitin Tests; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

A role in tumor progression has been proposed for transforming growth factor-beta 1 (TGF beta 1) and interleukin (IL)-8 as well as for IL-1, which itself induces the production of TGF beta 1 and IL-8 in many cell types. TGF beta 1 and IL-8 production and their regulation by IL-1 in five non-small-cell (NSC) lung tumor cell lines were evaluated. Moreover, their levels were evaluated in 29 NSC lung tumors. All cell lines constitutively produced TGF beta 1, and three produced IL-8. After IL-1 beta treatment, TGF beta 1 production was upregulated in two cell lines, whereas IL-8 production was markedly upregulated in two, induced in one, and unmodified in two. In tumors, the levels of TGF beta 1, IL-8, and IL-1 beta were higher than in normal counterparts (p < 0.001), and a positive correlation between IL-8 and IL-1 beta levels (p < 0.001) was found. TGF beta 1, IL-8, and IL-1 beta mRNA expression was examined in 12 tumors. TGF beta 1 mRNA was detected in all cases, IL-8 mRNA in 7, and IL-1 beta MRNA was undetectable. TGF beta 1, IL-8, and IL-1 beta immunoreactivity was then studied by immunohistochemistry. TGF beta 1 and IL-8 immunoreactivity was observed in neoplastic cells; IL-1 beta immunoreactivity was observed in mononuclear cells. In conclusion, in tumors IL-1 beta levels positively correlated with those of IL-8, and IL-1 beta as well as TGF beta 1 and IL-8 levels were significantly higher than in normal tissues.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Humans; Immunohistochemistry; Interleukin-1; Interleukin-8; Lung Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

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

Transforming growth factor beta1 (TGFbeta1) is a potent growth inhibitor for most cells, including neoplastic cells. However, there are several types of malignant cells that are resistant to its growth-inhibitory effect. LMC19, a highly malignant rat urothelial cell line, lacks TGFbeta1 receptor (TbetaRI) and is insensitive to the growth-suppresive effect of TGFbeta1. We transfected an expression vector containing human TbetaRI into this cell line. In control cells transfected with the neo gene alone, no inhibitory effect on growth was observed in vitro by the addition of anti-TGFbeta1 antibody or recombinant TGFbeta1 into serum-free medium. In contrast, the growth of all transfectants tested was inhibited significantly under serum-free conditions because of their endogenous TGFbeta synthesis. The growth was reduced further by the addition of recombinant TGFbeta1. This response pattern is consistent with TGFbeta1 mediating its effects by an autocrine and paracrine mechanism. The tumorigenicity of the cells was tested in a heterotopically transplanted urinary bladder system, which was generated as an orthotopic test site in athymic nude mice. All nine mice tested receiving control cells formed deeply invasive, undifferentiated-cell carcinomas and multiple metastatic foci in the lungs. In contrast, none of the mice receiving transfectants of TbetaRI formed bladder tumors or metastases. Taken together, these observations indicate that TbetaRI exhibits a potent tumor suppressor effect in bladder carcinoma.

Topics: Animals; Culture Media, Serum-Free; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Rats; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The level of hyaluronic acid (HA) was determined in the pleural fluid of 99 patients, including 19 with malignant mesothelioma, 27 with lung cancer, 1 with breast cancer, 1 with mediastinal tumor and 51 with non-malignant diseases. With a cut-off level at 100 micrograms/ml, the pleural fluid concentration of HA was high in 36.8% of patients (7 of 19) with malignant mesothelioma and 1.3% of patients (1 of 80) with lung cancer and other malignant and non-malignant diseases. The mean concentration of pleural fluid HA was significantly higher in patients with mesothelioma than in those with lung cancer and other malignant and non-malignant diseases. The pre-test probability of MM was 5.9% in this series. The LRs for > or = 100, 50-99 and < or = 49 micrograms/ml are 28.3, 3.3 and 0.5, respectively; these put the post-test probabilities at 64, 17 and 3%, respectively. Indeed, in cases of uncommon disease such as MM, the post-test probability is low even if the cut-off level of HA is > or = 100 micrograms/ml. The discrimination between malignant mesothelioma and lung cancer needs special attention. In these two diseases, the LRs of MM for pleural fluid CEA > 30, 10-30 and < 10 ng/ml were 0.2, 1.9 and 2.4, respectively. The pre-test probability of MM for HA > or = or 100 micrograms/ml is 64%. Furthermore, because the LR for CEA is < 10 ng/ml, the post-test probability is 81%. When the combination of two markers is considered, the high level of HA and the low level of CEA may be useful for the differential diagnosis of MM from pleuritis carcinomatosa.

Topics: Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Heart Failure; Humans; Hyaluronic Acid; Lung Neoplasms; Mesothelioma; Pleural Effusion; Pleural Effusion, Malignant; Prospective Studies; Transforming Growth Factor beta; Tuberculosis, Pleural

Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases.

Topics: Administration, Inhalation; Aged; Asbestos, Serpentine; Asbestosis; Carcinogens; Extracellular Matrix; Humans; Immunohistochemistry; Isomerism; Lung Neoplasms; Mesothelioma; Pleura; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 down-regulates mRNA expression of the aryl hydrocarbon receptor (AhR) and of AhR-inducible genes in A549 cells. Here, we describe a dose-dependent inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytochrome P450 (CYP) 1A1, CYP1B1 and NADPH-quinone-oxidoreductase (NMO-1) mRNA expression as well as TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity in MDA-MB 231 cells. The AhR mRNA expression was not affected by TGF-beta1. TGF-beta-responsiveness was investigated by examining the effect on the expression of responsive genes. While TGF-beta1 up-regulates mRNA expression of TGF-beta1 and TIEG (TGF-beta-inducible early gene) as well as luciferase activity of a responsive reporter plasmid in both cell lines, a down-regulation of c-myc and cyclin A mRNA expression was only found in A549 cells. Furthermore, TGF-beta1 inhibits only cell proliferation of A549 but not of MDA-MB 231 cells. The results show a coregulation of mRNA expression of AhR and cell-cycle regulating genes, and further indicate that the AhR may be involved in regulation of cell proliferation.

Topics: Aryl Hydrocarbon Hydroxylases; Breast Neoplasms; Cell Cycle; Cyclin A; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Female; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Recombinant Fusion Proteins; RNA, Messenger; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

To test the hypothesis that the transforming growth factor-beta (TGF-beta) system has tumor suppressor activity in the mammary gland, we have generated transgenic mice overexpressing a dominant-negative mutant form of the type II TGF-beta receptor, under the control of the mouse mammary tumor virus-long terminal repeat. High-level expression of the transgene was observed in the mammary and salivary glands, with lower expression in the lung, spleen, and testis. Older nulliparous transgenic mice (9-17 months) showed a marked increase in the incidence and degree of lobulo-alveolar side-branching in the mammary glands when compared to wild-type littermates (24.8% of glands examined histologically versus 14.4%; P = 0.004), suggesting a role for endogenous TGF-betas in regulating development or maintenance of mammary alveoli. Spontaneous tumorigenesis was unchanged in the transgenic mice. However, following initiation with the carcinogen 7,12-dimethylbenz[a]anthracene, the transgenic group showed a significant increase in the incidence and multiplicity of mammary tumors when compared with wild-type littermates (40% incidence in transgenic mice versus 22% for wild-type, with 4 of 25 transgenics developing multiple mammary tumors versus 0 of 27 wild-type; P = 0.03). An early increase in the incidence of lung tumors was also observed in transgenic mice, but no difference between genotype groups was seen in the incidence of tumors in tissues in which the transgene is not expressed. The data show that the endogenous TGF-beta system has tumor suppressor activity in the mammary gland and lung.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cocarcinogenesis; Female; Gene Expression; Humans; Lung; Lung Neoplasms; Male; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Transgenes

Exposure of the human lung carcinoma cell line, A549 cells, to transforming growth factor beta1 (TGFbeta1) resulted in an alteration in the morphology from epithelial-like to fibroblastic flat cells. Immunofluorescent study revealed that microfilament organization was partially restored in the TGFbeta1-treated cells. We also investigated the effects of TGFbeta1 on the transformed phenotypes including the anchorage-independent growth and invasive ability of A549 cells and found that the phenotypes were strikingly suppressed in TGFbeta1-treated A549 cells. Accompanying these changes, the levels of the expression of high molecular weight tropomyosin (TM) isoforms, especially TM1 and TM2, were significantly increased in the TGFbeta1-treated cells. The expression level of vinculin was also increased in these cells. These results suggest that the increased tropomyosin and vinculin syntheses may be involved in the suppression of the transformed phenotypes of A549 cells.

Topics: Actins; Animals; Blotting, Western; Carcinoma; Cell Adhesion; Cell Size; Cell Transformation, Neoplastic; Chlorocebus aethiops; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Microscopy, Fluorescence; Neoplasm Invasiveness; Transforming Growth Factor beta; Tropomyosin; Tumor Cells, Cultured; Vero Cells; Vinculin

To investigate the effect of human sense- and antisense-transforming growth factor beta 1 (TGF beta 1) gene on human pulmonary giant cell carcinoma cell (PLA-801D) proliferation.. PLA-801D cells were transfected with an expression vector containing a full length human sense- or antisense-TGF beta 1 gene by gene transfer. The changes of cell proliferation, expressions of nm23 mRNA and c-myc mRNA and the feature of apoptosis were studied on transfected cells and untransfected cells.. The cells transfected by sense-TGF beta 1 gene showed higher TGF beta 1 activity in the conditioned media, growth inhibition in vitro and decreased coloning formation in anchorage-independent assay. The tumorigenic rate was reduced about 47%, the metastasis was inhibited in athymic nude mice as compared with untransfected cells group. The mechanism of such changes described may be associated with the higher expression of nm23 mRNA, and the lower expression of c-myc mRNA as well as the apoptosis in sense-TGF beta 1 gene transfected cells. However, opposing changes were observed in the antisense-TGF beta 1 gene transfected cells.. TGF beta 1 acts as a negative growth regulator of the PLA-801D in vitro as well as in vivo.

Topics: Animals; Antisense Elements (Genetics); Apoptosis; Carcinoma, Giant Cell; Cell Division; Gene Expression; Genes, myc; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Monomeric GTP-Binding Proteins; Neoplasm Transplantation; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate TGF beta 1 gene expression and its effect on murine tumor growth following direct intratumoral injection of naked plasmid DNA encoding human TGF beta 1.. LM3 murine lung adenocarcinoma cells, were injected subcutaneously to T739 mice and grew to tumor nodules in 2 weeks, Multiple direct intratumoral injections of plasmid DNA, PMAM-neo-TGF beta 1, were given and were compared with saline or vector plasmid administration groups. The growth of tumor was observed till the 8th week when the mice were killed for Northern blot analysis and histopathological study of tumoral tissue.. The growth of tumor was boosted in the TGF beta 1 gene treated mice as compared with the control groups, whereas there was no significant difference in the metastatic behavior. Northern blot showed efficient expression of TGF beta 1 mRNA in the treated group.. TGF beta 1 may stimulate tumor growth in vivo through certain mechanisms. Direct intratumoral injection of nude plasmid DNA may be a promising gene transfer strategy in vivo.

Topics: Adenocarcinoma; Animals; DNA; Gene Transfer Techniques; Genetic Therapy; Humans; Lung Neoplasms; Male; Mice; Neoplasm Transplantation; Transforming Growth Factor beta; Tumor Cells, Cultured

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII.

Topics: Activin Receptors, Type I; Blotting, Northern; Carcinoma, Small Cell; Cell Line; Cycloheximide; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Although it is recognized that three isoforms of transforming growth factor-beta (TGF-beta) exist in mammals, their expression, distribution, and function in injury and repair are not well characterized. Using immunohistochemistry and antibodies to synthetic peptides of TGF-beta 1, TGF-beta 2, and TGF-beta 3, we determined the distribution of TGF-beta isoforms in lung sections with acute and chronic lesions of idiopathic pulmonary fibrosis (IPF), chronic asbestosis and hypersensitivity pneumonitis, as well as non-specific pneumonitis. In lung sections with advanced pulmonary fibrosis and honeycombing, irrespective of the diagnosis, TGF-beta 1 was prominently expressed in epithelial cells and macrophages and was found to be associated with the extracellular matrix. In lungs with early lesions of IPF and only inflammatory changes, TGF-beta 1 was present in alveolar macrophages but TGF-beta 1 was not present in epithelial cells. Small amounts of matrix-associated TGF-beta 1 were present subepithelially in areas of lung sections from patients with IPF with minimal inflammation and no fibrosis. In normal lungs with no evidence of inflammation or fibrosis TGF-beta 1 was not seen in alveolar macrophages, epithelial cells, or extracellularly. TGF-beta 2 and TGF-beta 3 were expressed in alveolar macrophages, epithelial cells, and smooth muscle cells of vessels and bronchi of normal lungs and lungs with both inflammatory and fibrotic changes. Our findings suggest that while TGF-beta 2 and TGF-beta 3 are ubiquitously expressed in the lung, TGF-beta 1 is expressed in epithelial cells of fibrotic lungs where the presence of TGF-beta 1 is not disease-specific but an indication of the chronicity of the injury.

Topics: Adult; Aged; Antibody Specificity; Asbestosis; Biopsy; Cysts; Epithelium; Humans; Immunohistochemistry; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Macrophages; Middle Aged; Muscle, Smooth, Vascular; Pulmonary Fibrosis; Transforming Growth Factor beta

Three small cell lung cancer cell lines established from a single patient during longitudinal follow-up were examined for in vitro expression of TGF beta and TGF beta receptors, i.e. the components of an autocrine loop. GLC 14 was established prior to treatment, GLC 16 on relapse after chemotherapy and GLC 19 on recurrence after radiotherapy. TGF beta was detected by ELISA and TGF beta receptors by chemical crosslinking to radiolabelled TGF beta 1. Furthermore, TGF beta and TGF beta receptor mRNAs were detected by northern blot analysis. Expression of type II TGF beta receptor mRNA and protein was found in GLC 16 and GLC 19. These cell lines were also growth inhibited by exogenously administrated TGF beta 1. TGF beta 1 mRNA and protein in its latent form was only expressed in the radiotherapy-resistant cell line, GLC 19. The results indicate that disease progression in this patient was paralleled by a gain in sensitivity to the growth inhibition by TGF beta 1 due to type II TGF beta receptor, and a gain of latent TGF beta 1 protein. Lack of type II receptor expression in GLC 14, which was also resistant to growth inhibition by exogenous TGF beta 1, was not due to gross structural changes in the type II receptor gene, as examined by Southern blotting. Also, the type I receptor could not be detected by ligand binding assay in this cell line, despite expression of mRNA for this receptor. This agrees with previous findings that type I receptor cannot bind TGF beta 1 without co-expression of the type II receptor.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Small Cell; Cell Division; Disease Progression; DNA, Neoplasm; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Lung Neoplasms; Middle Aged; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

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

Retinoids show promise for prevention and treatment of cancers. In most cases, the mechanisms of their anticancer effects are poorly defined, but interactions with cytokine genes have been postulated in several systems. The effects of trans-retinoic acid (RA) on proliferation and cytokine gene expression in the human lung carcinoma Lu-CSF-1 are reported. RA exhibited cell-cycle independent inhibition of Lu-CSF-1 growth while stimulating endogenous interleukin-1 beta and suppressing granulocyte-macrophage colony-stimulating factor and IL-6 mRNAs. Reduction in granulocyte-macrophage colony-stimulating factor and IL-6 message was associated with reduced RNA stability and was translated into reduced protein levels. IL-1 beta mRNA stability was not decreased, and elevation in IL-1 beta protein levels was of a comparable magnitude to the increased amounts of its RNA. Growth inhibition similar to that following RA treatment could be reproduced by exposing cells to exogeneous IL-1 beta alone. These data suggest that changes in autologous cytokine gene expression might contribute to growth inhibition of lung cancer cells by RA.

Topics: Antineoplastic Agents; Cell Division; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Humans; Interleukin-1; Interleukin-6; Lung Neoplasms; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In vivo and in vitro studies have linked small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells along a differentiation continuum. The transition of a SCLC toward a NSCLC phenotype is modeled in culture by the simultaneous overexpression of myc and ras genes in cultured SCLC cells. A major phenotypic distinction between SCLC and NSCLC in culture is that SCLC cells usually grow in floating aggregates, whereas NSCLC cells and myc- plus ras-expressing SCLC cells grow as adherent spreading monolayers like other epithelial cells. The present studies examine how myc, ras, cell aggregation, and attachment to laminin may interact to modulate transitions between the SCLC and NSCLC phenotypes. We find that myc-expressing SCLC cells, which normally grow as anchorage-independent cells in plastic flasks, will adhere to laminin and exhibit an epithelial morphology. In this setting, the cells express both NSCLC and SCLC markers, thus resembling a tumor type previously termed NSCLC with neuroendocrine features. Anchorage-dependent SCLC cells simultaneously expressing the myc family and an exogenous ras oncogene move further toward the NSCLC phenotype than the above myc-expressing cells. However, forced suspension of such cells restores the expression of neuroendocrine SCLC features. These studies indicate that cell environment, as much as gene expression events, profoundly affects aspects of the SCLC cell phenotype.

Topics: Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Adhesion; Cell Aggregation; DNA-Binding Proteins; Epithelium; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, ras; Humans; Hydrogels; Laminin; Lung Neoplasms; Neurosecretory Systems; Phenotype; Polyhydroxyethyl Methacrylate; Protein Kinase C; Protein Kinase C beta; RNA, Messenger; RNA, Neoplasm; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The mechanism causing finger clubbing in patients with lung cancer (LC) is still unclear. We compared age, cigarette consumption, data on blood gas analysis and pulmonary function tests among patients with LC with clubbing (n = 30) and without clubbing (n = 28) and among patients with pulmonary emphysema (PE) with (n = 11) and without clubbing (n = 17). We also examined serum concentrations of transforming growth factor beta 1 (TGF beta 1) and insulin-like growth factor-I (IGF-I) in the patients and healthy volunteers (n = 21). There were no differences in age or cigarette consumption. LC groups showed normal levels of Pao2 and Paco2, suggesting that neither hypoxaemia nor hypercapnia caused clubbing in these patients. The level of serum TGF beta 1 in patients with LC with clubbing was significantly higher than in other groups (P < 0.005), whereas levels of IGF-I did not differ among the groups. Our data suggest that TGF beta 1 may play a role in the mechanism of clubbing in patients with LC.

Topics: Age Factors; Aged; Blood Gas Analysis; Carcinoma; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Middle Aged; Osteoarthropathy, Secondary Hypertrophic; Pulmonary Emphysema; Respiratory Function Tests; Smoking; Transforming Growth Factor beta

To determine the frequency with which elevated plasma transforming growth factor-beta 1 (TGF beta 1) concentrations occur in lung cancer patients, to determine the kinetics of TGF beta 1 expression during and after radiotherapy and to correlate plasma TGF beta 1 levels with disease status after treatment.. Plasma samples were obtained before, during and after radiotherapy in 54 patients with lung cancer and 20 normal controls. Plasma TGF beta 1 levels were measured using an enzyme-linked immunosorbent assay.. Baseline TGF beta 1 levels in lung cancer patients and normal controls were 13.0 +/- 2.5 and 4.4 +/- 0.3 ng/ml, respectively. Elevated TGF beta 1 were found in 50% (27/54) of lung cancer patients. During radiation therapy plasma TGF beta 1 levels declined, however, by the completion of treatment the mean TGF beta 1 level had not normalized in patients with lung cancer. The TGF beta 1 level at last follow-up correlated with disease status in those patients with an increased pretreatment plasma level. Three of four patients with no evidence of cancer had normal follow-up TGF beta 1 levels, compared to 2/16 patients with residual or recurrent tumor (P = 0.02).. Elevated plasma TGF beta 1 levels occur frequently in patients with lung cancer. In those patients with an elevated plasma TGF beta 1 level at diagnosis, monitoring this level may be useful in detecting both disease persistence and recurrence after therapy.

Topics: Aged; Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor beta

Effects of the expression of the platelet-derived growth factor (PDGF), insulin-like growth factor-II (IGF-II), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 were studied individually and in combinations with the clinicopathologic features and prognosis of pulmonary adenocarcinoma using paraffin embedded tissue specimens. Tumor sections from 90 patients with pulmonary adenocarcinoma were stained immunohistochemically for PDGF, IGF-II, bFGF and TGF-beta 1 by the ABC method. The survival rate was worse in patients in whom each of the four growth factors was expressed than in those where growth factors were not expressed. The reduced expression of the four growth factors correlated with less tumor aggressiveness and better prognosis of pulmonary adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunohistochemistry; Insulin-Like Growth Factor II; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Platelet-Derived Growth Factor; Prognosis; Staining and Labeling; Survival Analysis; Transforming Growth Factor beta

In addition to infiltrating inflammatory cells, tumors also produce cytokines and growth factors that may alter tumor growth, tumor immunogenicity, and the host immune response. To characterize the expression profile of human non-small cell lung cancer (NSCLC)-derived cytokines, the mRNA expression of type 1 and type 2 cytokines in five human NSCLC lines was analyzed by reverse transcriptase-PCR. Expression of interleukin 5 (IL-5) and IL-10 was demonstrated in all tumor lines evaluated, whereas IL-4 was present in three of five lines and IL-13 was present in two of five lines. In contrast, none of the tumor lines expressed IL-2 and IFN-gamma. Type 2 cytokine protein production by NSCLC lines was confirmed by immunoprecipitation and cytokine specific ELISA. Tumor-derived IL-10 secretion was significantly augmented by exogenous recombinant cytokines including IL-4 and tumor necrosis factor-alpha. To evaluate whether fresh NSCLC nodules also express a type 2 cytokine pattern, the content of type 1 and type 2 cytokines in tissue homogenates from 13 fresh NSCLC nodules and normal lung surgical specimens was assessed. Human NSCLC nodules contain significantly more type 2 cytokines than does normal lung tissue when corrected for total protein concentration. To identify the cellular source of type 2 cytokine production in tumor nodules, immunohistology was performed on sections from 5 lung squamous cell carcinomas and 5 adenocarcinomas. All of the specimens revealed positive staining for type 2 cytokines within tumor cells. In summary, we report that human NSCLC cells produce type 2 cytokines both in situ and in vitro, which may play an active immunoregulatory role in the lung cancer microenvironment.

Topics: Base Sequence; Carcinoma, Non-Small-Cell Lung; Humans; Immunity, Cellular; Interleukin-10; Interleukin-13; Interleukin-4; Interleukin-5; Lung Neoplasms; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of this study was to examine whether changes in growth factor or cytokine expression could be responsible for the growth inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the human breast cancer MCF-7 cell line. Treatment of MCF-7 cells with 10 nM TCDD for 7 days reduced the cell growth to 60% of control; this effect was partly abolished by cotreatment of the cells with 100 nM 17 beta-estradiol (E2). The inhibition of cell growth by TCDD was accompanied by an enhanced secretion of transforming growth factor-beta (TGF-beta) and the TGF-beta content in cell culture supernatants was 2-fold higher than in controls. Using reverse transcription polymerase chain reaction (RT-PCR), the effect of TCDD on the expression of TGF-beta isoforms, transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) was investigated. It was demonstrated that incubation with 1, 10 and 100 nM TCDD for 24 h increased mRNA levels of TGF-alpha, TNF-alpha and IL-1 beta. The strongest effect was found on IL-1 beta, the mRNA level of which was dose-dependently increased. TCDD had a minor effect on TGF-alpha and TNF-alpha mRNA. The mRNA levels were significantly increased after treatment with 10 and 100 nM TCDD. The mRNA expression of TGF-beta 1 and TGF-beta 2 was unchanged, whereas the TGF-beta 3 mRNA level was enhanced 2 to 3-fold after TCDD treatment. From the results, we suggest that TCDD-induced growth inhibition in MCF-7 cells is related to the growth inhibitory action of a set of growth factors and cytokines which have a contextual action on MCF-7 cell proliferation.

Topics: Analysis of Variance; Breast Neoplasms; Cytokines; Growth Substances; Humans; Interleukin-1; Lung Neoplasms; Polychlorinated Dibenzodioxins; Polymerase Chain Reaction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The in vitro expression level of interleukin-8 (IL-8) correlates with the metastatic potential of human melanoma cells. The purpose of this study was to determine whether the expression level of IL-8 in human melanoma cells is influenced by the organ microenvironment. A375P cells, a low metastatic human melanoma, and A375SM cells, a highly metastatic variant, were injected into the subcutis (s.c.), spleen (to produce liver metastases), and lateral tail vein (to produce lung metastases) of athymic nude mice. Northern blot and immunohistochemical analyses determined that s.c. tumors, lung lesions, and liver lesions expressed high, intermediate, and low IL-8, mRNA, and protein, respectively. This differential regulation of IL-8 was not due to the size or density of the lesions or to selection of subpopulations of cells. We based this conclusion on the results of three experiments: (a) melanoma cell lines established in culture from in vivo-growing tumors exhibited similar levels of IL-8 mRNA transcripts; (b) in a crossover experiment, the level of IL-8 mRNA was always high in A375 tumors reestablished in the skin and low in the tumors reestablished in the liver, regardless of whether the melanoma cells had been first harvested from s.c. or liver tumors; and (c) A375 melanoma cells cocultured with human keratinocytes produced high levels of IL-8 protein, whereas A375 cells cocultured with highly differentiated human hepatoma cells produced decreased levels. When A375P cells were then incubated with cytokines associated with keratinocytes (IL-1 and interferon beta) or hepatocytes (transforming growth factor alpha or beta), IL-1 enhanced the production of IL-8 protein, whereas TGF-beta decreased its production. These data show that IL-8 expression in melanoma cells is modulated by local host factors.

Topics: Adaptation, Psychological; Animals; Cell Communication; Cell Division; Female; Humans; Interleukin-1; Interleukin-8; Keratinocytes; Liver Neoplasms, Experimental; Lung; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Organ Specificity; RNA, Messenger; Skin Neoplasms; Skin Physiological Phenomena; Transforming Growth Factor beta

We immunohistochemically examined the expression of transforming growth factor-beta (TGF-beta) on tissue specimens from primary 124 human lung adenocarcinoma, using a polyclonal antibody. The overall mean immunoreactivity of TGF-beta was 25.7 +/- 22.9, therefore we separated patients into two groups according to their mean immunoreactivity. There were 59 (48%) with a high TGF-beta and 65 (52%) with a low TGF-beta. No correlation was observed between the expression of TGF-beta and clinicopathological factors except for degree of differentiation. The 5-year survival rates of patients with high and low TGF-beta were 71% and 37%, respectively (P < 0.05). A multivariate analysis using the Cox life table regression model showed TGF-beta to be a significantly independent factor. We thus concluded, based on our findings, that the expression of TGF-beta was found to be related to a better prognosis. Therefore, estimating the negative cell proliferation activity induced by TGF-beta on immunohistochemical technique is considered to be useful for determining the patients' prognosis in cases of lung adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Prognosis; Regression Analysis; Sex Factors; Survival Rate; Transforming Growth Factor beta

Specific cDNA probes for transforming growth factor beta s (TGF-beta s) 1, 2, and 3 and TGF-beta types I, II, and III receptors were used to study expression of the mRNAs of the different TGF-beta ligand and TGF-beta receptor isoforms in cultured non-small cell lung cancer (NSCLC) cells and small cell lung cancer (SCLC) cells. Expression of TGF-beta 1 mRNA was detected in both cell types using Northern blot hybridization, with the level of expression of this mRNA being higher in several NSCLC cell lines. In addition, expression of TGF-beta 2 and TGF-beta 3 mRNAs was also detected in NSCLC and SCLC cells but at levels that were lower than that of TGF-beta 1 mRNA. Besides expression of a 3.4-kilobase (kb) TGF-beta 3 transcript, a smaller 2.8-kb TGF-beta 3 transcript was detected in some NSCLC and SCLC cells. TGF-beta 1 and TGF-beta 2 proteins were detected in the conditioned media of NSCLC and SCLC cells, with the levels being higher in several NSCLC cells than in SCLC cells. Expression of TGF-beta types I and II receptor mRNAs was also detected in most NSCLC and SCLC cells, with expression of a 5.5-kb type I receptor mRNA being higher than that of a 5.5-kb type II receptor mRNA in both cell types. In contrast, a 6-kb TGF-beta type III receptor mRNA was detected in only some NSCLC cells and could not be detected in the SCLC cells examined. Also, there was an inverse relationship between the level of expression of the 5.5-kb TGF-beta type I receptor mRNA and that of the 6-kb TGF-beta type III receptor mRNA. Addition of TGF-beta 1 and TGF-beta 2 proteins resulted in an increase in the mRNAs for TGF-beta s 1 and 2 and an increase in the amount of TGF-beta 1 protein in some NSCLC cells, indicating that these cells are responsive to TGF-beta and its effects. At the same time, a differential change in expression of the 2.8- and 3.4-kb TGF-beta 3 transcripts was detected in some lung cancer cells following the addition of TGF-beta 1 and TGF-beta 2. Also, addition of TGF-beta 1 to NSCLC cells inhibited colony formation of some of these cells in soft agarose in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Cell Division; Cell Line; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is known as the growth factor that stimulates the multiplication and accumulation of extracellular matrix. Recently, TGF-beta also has been found to have the ability to control the growth and metastatic potential of cancer cells. It is known that central fibrosis frequently occurs in pulmonary adenocarcinoma and the prognosis becomes poorer as fibrosis become more hyalinized. To estimate the role of TGF-beta in the formation of central fibrosis in pulmonary adenocarcinoma and its influence on the prognosis of patients with pulmonary adenocarcinoma, we performed an immunohistochemical study of TGF-beta in 51 cases of T1 pulmonary adenocarcinoma. Positive stain for TGF-beta was shown in 31 cases, and negative stain was shown in 20 cases. In patients with Stage I, T1 pulmonary adenocarcinoma, the post operative survival curve was compared between positive and negative cases of TGF-beta, and the result showed a tendency toward poorer prognosis in positive cases of TGF-beta. Twenty-four of 51 cases of T1 pulmonary adenocarcinoma had central fibrosis. Twenty of 24 cases with central fibrosis showed positive stain for TGF-beta. It was proven that the appearance of central fibrosis was significantly related to positive stain for TGF-beta in T1 pulmonary adenocarcinoma. According to these results, it is suggested that TGF-beta plays some role in the formation of central fibrosis in pulmonary adenocarcinoma and TGF-beta is possibly a prognostic factor for patients with pulmonary adenocarcinoma.

Topics: Adenocarcinoma; Fibrosis; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Prognosis; Survival Rate; Transforming Growth Factor beta

Whether or not cytokine secretion is impaired in patients with small-cell lung cancer (SCLC), is unknown. We therefore investigated whether cytokine secretion by immunocompetent cells may be suppressed in patients with SCLC.. We determined cytokine secretion by lymphocytes and monocytes in whole blood cell cultures from 58 patients with SCLC, 95 patients with non-small-cell lung cancer (NSCLC), 10 patients with nonmalignant lung disease and from 44 normal healthy individuals by using an enzyme-linked immunosorbent assay (ELISA) specific for the different cytokines measured.. Compares to normal controls, immunocompetent cells from patients with SCLC secreted significantly lower amounts of IL-2, IFN alpha, and IFN gamma upon mitogen stimulation. TNF alpha-secretion was significantly reduced in SCLC extensive disease but not in SCLC limited disease. In contrast, secretion of IL-1 alpha and IL-1 beta was not reduced. In patients with NSCLC, secretion of IL-2 and IFN alpha was significantly reduced. Reduction of IFN gamma secretion was significant in metastasized NSCLC and marginally significant in localized NSCLC. Secretion of TNF alpha, IL-1 alpha and IL-1 beta was not impaired. In addition, cytokine secretion in SCLC patients substantially improved upon successful reduction of tumor load by chemotherapy but not upon ineffective chemotherapy. Furthermore, TGF beta 1 suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha but not of IL-1 alpha and IL-1 beta in whole blood cell cultures from healthy individuals.. Suppression of cytokine secretion in patients with SCLC was selective, dependent on tumor load, different from immunosuppression in NSCLC and seemed to be reconstituted upon reduction of tumor load. These results may suggest interactions between tumor cells and the immune system. TGF beta 1 secreted by SCLC cell lines induced the same selective cytokine suppression as that found in SCLC patients. However, whether or not tumor-derived TGF beta 1 is a factor inducing selective immunosuppression in SCLC patients is presently unclear.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cytokines; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-alpha; Interferon-gamma; Interleukin-1; Interleukin-2; Lung Neoplasms; Middle Aged; Reference Values; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The pathogenesis of mesenchymal and epithelial lung reactions was studied after a single intratracheal instillation of quartz into rats. Relationships between transforming growth factor beta1 (TGF-beta1) and the ras and p53 genes were investigated in silicosis and associated lung cancer. Immunohistochemical reactivity to mature TGF-beta1 was localized intracellularly in fibroblasts and macrophages at the periphery of silicotic granulomas and in stroma adjacent to hyperplastic alveolar type II cells and extracellularly in connective tissue matrix adjacent to hyperplastic alveolar type II cells. TGF-beta1 precursor was localized intracellularly in hyperplastic alveolar type II cells adjacent to granulomas and in the cells of adenomas, but not in carcinomas. Hematite-treated controls showed no reactivity to TGF-beta1. Immunohistochemical localization of pan-reactive p21 ras protein in quartz-treated rat lungs was increased in hyperplastic alveolar type II cells adjacent to granulomas, but not in adenomas and carcinomas. Foci of nuclear immunoreactivity to p53 protein were observed in 25% of the carcinomas.

Topics: Animals; Carcinogens; Culture Techniques; Female; Genes, p53; Immunohistochemistry; Injections, Spinal; Lung Neoplasms; Male; Proto-Oncogene Proteins p21(ras); Rats; Rats, Inbred F344; Silicon Dioxide; Silicosis; Transforming Growth Factor beta

Growth inhibition assays using radioisotope or dye are used to detect transforming growth factor-beta (TGF-beta). Here, we describe a modified bioassay using crystal violet for the quantitative detection of TGF-beta 1 and TGF-beta 2. The procedure is based on staining Mv1Lu mink lung epithelial cells with crystal violet, followed by measurement of the absorbance at 570 nm in individual wells of a 96-well microtiter plate. The number of Mv1Lu cells correlated with the eluted dye intensity. The sensitivity of the bioassay to recombinant TGF-beta 1 and TGF-beta 2 increased approximately twofold by using only 500 Mv1Lu cells in microtiter wells. The bioassay was used to measure TGF-beta activity in the culture supernatant from glioblastoma cells. Culture supernatants were untreated or acid-activated to quantify the active or total TGF-beta, and neutralized with anti-TGF-beta 1 and/or anti-TGF-beta 2 antibody to measure the activity. Both TGF-beta 1 and TGF-beta 2 were detected in the untreated and acid-activated supernatants, and the amounts were calculated by extrapolating from the known recombinant TGF-beta 1 or TGF-beta 2 dilution curve. Our results show that the modified bioassay using crystal violet can measure the levels of TGF-beta 1 and TGF-beta 2 in culture supernatants from malignant glioma cells.

Topics: Biological Assay; Cell Count; Culture Techniques; Gentian Violet; Glioma; Growth Inhibitors; Humans; Lung; Lung Neoplasms; Transforming Growth Factor beta

We investigated effects of soluble mediators secreted by small cell lung cancer (SCLC) cell lines on modulation of cytokine-induced growth of lymphocytes. We found that interleukin-2 (IL-2)-mediated T-cell growth was inhibited by a cytokine constitutively secreted by the SCLC cell line, NCI-N417. Of several cytokines tested, only transforming growth factor beta 1 (TGF beta 1) severely suppressed IL-2-dependent T-cell growth. Using a specific anti-TGF beta 1 antibody, we found that this antibody blocked the immunosuppressive activity secreted by NCI-N417. Thus, the NCI-N417-derived immunosuppressive molecule was serologically identified as TGF beta 1. Further experiments showed that TGF beta 1 was secreted by four of eight SCLC lines tested. mRNA for TGF beta 1 was expressed in NCI-N417 and in SCLC-22H. Constitutive secretion of biologically active TGF beta 1 by SCLC lines suggests that tumour-derived immunosuppression may have clinical relevance.

Topics: Carcinoma, Small Cell; Dose-Response Relationship, Drug; Humans; Interleukin-2; Lung Neoplasms; Mitosis; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

To evaluate the efficacy of transforming growth factor beta (TGF-beta) for the prognosis of pulmonary adenocarcinoma.. TGF-beta was detected immunohistochemically using the avidin-biotin-peroxidase complex technique in resected pulmonary adenocarcinomas from 88 patients.. Of the 88 patients, 39 were TGF-beta negative and 45 TGF-beta positive. The five year survival rate was 56% for the TGF-beta negative and 16% for the TGF-beta positive group.. TGF-beta can be used as a prognostic factor in pulmonary adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cytoplasm; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Paraffin Embedding; Prognosis; Transforming Growth Factor beta

To determine whether plasma transforming growth factor-beta (TGF-beta) levels measured before and during radical radiotherapy for lung cancer could be used to predict patients at risk for the development of radiation pneumonitis.. The first eight patients with lung cancer (nonsmall cell: seven, small cell: one) enrolled in a prospective study designed to evaluate physiological and molecular biologic correlates of radiation induced normal tissue injury are described. The study began in June 1991. All patients were treated with radiotherapy with curative intent. Plasma transforming growth factor-beta levels were obtained before, weekly during, and at each follow-up after treatment. Pretreatment pulmonary function tests and single photon emission computed tomography scans were obtained to assess baseline lung function and were repeated at follow-up visits. Dose-volume histogram analyses were performed to determine the volume of lung which received > or = 30 Gy. Patients were assessed at each follow-up visit for signs and symptoms of pneumonitis.. Five patients developed signs and/or symptoms of pulmonary injury consistent with pneumonitis and three patients did not. In all three patients not developing pneumonitis, plasma TGF-beta levels normalized by the end of radiotherapy. In contrast, four out of five patients who suffered pneumonitis had persistently elevated plasma TGF-beta levels by the end of therapy. This finding appeared to be independent of the volume of irradiated lung.. These results suggest that plasma TGF-beta levels during treatment may be useful to determine which patients are at high risk of developing symptomatic pneumonitis following thoracic radiotherapy. This finding may have implications when planning additional therapy (either chemotherapy or radiotherapy) which may have potentially adverse consequences on the lung.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Middle Aged; Prospective Studies; Radiation Pneumonitis; Time Factors; Transforming Growth Factor beta

We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO.

Topics: Carcinoma, Small Cell; Carcinoma, Squamous Cell; Humans; Lung Neoplasms; Mesothelioma; Platelet Factor 4; Pleural Effusion, Malignant; Transforming Growth Factor beta; Tuberculosis, Pleural

The effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of cytochrome P450IA1 (CYPIA1) was examined in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated human lung cancer A549 cells. Using the reverse transcription-polymerase chain reaction (RT-PCR) it was demonstrated that TGF-beta 1 inhibits CYPIA1 expression in a dose dependent manner. Based on the inhibitory concentration 50 (IC50) of about 5 pM it is suggested that TGF-beta 1 has a physiological function in downregulation of this cytochrome. In the presence of cycloheximide, the effect of TGF-beta 1 on CYPIA1 mRNA disappeared. This finding indicates that protein synthesis may be required for the TGF-beta 1 mediated response of CYPIA1. The possible mechanisms by which TGF-beta 1 interacts with TCDD-responsive drug metabolizing enzymes are discussed.

Topics: Cycloheximide; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Enzyme Induction; Humans; Lung Neoplasms; Oxidoreductases; Polychlorinated Dibenzodioxins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

A growing body of evidence has recently implicated TSP and TGF-beta in the process of malignancy, such as tumor cell proliferation, tumor angiogenesis, and metastasis. The purpose of the present study was to evaluate potential mechanisms of TSP and TGF-beta in tumor cell attachment and invasion. Our results indicate that both TSP and TGF-beta promoted tumor cell attachment and spreading in the presence of plasminogen. The mechanism for these effects appeared to be due, in part, to the capacity of TSP and TGF-beta to induce tumor cell production of (PAI-1). PAI-1, which is a natural inhibitor of tumor-cell associated urokinase-type plasminogen activator (uPA) activity, inhibited activation of plasminogen to plasmin in the growth media, thereby preventing plasmin-induced detachment of cells. The TSP-promoted production of PAI-1 could be inhibited not only by anti-TSP antibodies but also by a neutralizing antibody against TGF-beta. These results suggest that TSP by a mechanism involving TGF-beta can promote cell adhesion through stimulation of tumor cell secretion of PAI-1. These data provide evidence that TSP not only has the capacity of functioning as a matrix protein to directly promote cell-substratum adhesion but that TSP can also stimulate cell adhesion and spreading by modulating cell surface protease expression through stimulation of tumor-associated production of PAI-1.

Topics: Adenocarcinoma; Antibodies; Cell Adhesion; Fibrinolysin; Humans; Lung Neoplasms; Membrane Glycoproteins; Plasminogen Activator Inhibitor 1; Thrombospondins; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The regulation of parathyroid hormone-related protein (PTH-rP) mRNA levels and immunoreactive (ir)PTH-rP formation by peptide growth factors, particularly transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), in squamous cell carcinomas of gynecologic origin is largely unknown.. PTH-rP mRNA levels were evaluated by Northern analysis in A431 cells (derived from a human vulvar epidermoid carcinoma) and ME-180 cells (derived from a human papillomavirus-infected squamous cell carcinoma of the cervix). PTH-rP protein levels in cell culture media were evaluated using both radioimmunoassay and immunoradiometric assay techniques. These results were compared with those from a lung carcinoid cell line known to produce PTH-rP, namely, NCI-H727 cells.. TGF-beta 1 or EGF treatment caused an increase in the levels of PTH-rP mRNA in A431 cells; these increases in PTH-rP mRNA were detectable after 60 minutes of treatment, were maximal at approximately 4-8 hours, and were approximately additive. Immunoreactive PTH-rP was not detectable (using two different PTH-rP immunoassays) in the culture medium or cell sonicates of A431 cells before or after treatment with TGF-beta 1, EGF, or TGF-beta 1 plus EGF. ME-180 cells responded to EGF (but not to TGF-beta 1) with an increase in the level of PTH-rP mRNA as early as 2 hours; irPTH-rP was present (by use of either immunoassay) in the medium of these cells at 8 and 24 hours. In NCI-H727 (human lung carcinoid) cells, TGF-beta 1 and EGF acted alone and synergistically to effect increases in PTH-rP mRNA and the accumulation of irPTH-rP.. TGF-beta 1 and EGF regulation of PTH-rP gene expression in squamous cell carcinomas of gynecologic origin is unique for each cell line studied and different from that in human lung carcinoid cells.

Topics: Base Sequence; Carcinoid Tumor; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genital Neoplasms, Female; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

Transforming growth factor beta (TGF-beta) is a multifunctional polypeptide that regulates the proliferation and differentiation of various cells and has an angiogenic effect in vivo, although it inhibits the growth of cultured endothelial cells. We report here that TGF-beta treatment of quiescent cultures of mouse embryo-derived AKR-2B cells, which are growth-stimulated by TGF-beta, and human lung adenocarcinoma A549 cells, which are growth-inhibited by TGF-beta, results in the induction of vascular endothelial growth factor (VEGF) mRNA and protein. Maximal VEGF mRNA levels occurred 4-8 h after stimulation with a decline to background levels in 24 h. In contrast, the related placenta growth factor mRNA was not induced by TGF-beta in these cells. No VEGF receptor mRNA was seen in AKR-2B cells. Also, TGF-beta treatment of endothelial cells, which express the FLT1 and KDR/FLK-1 receptors for VEGF, did not cause VEGF induction. Because VEGF is known to be a strong angiogenic factor for endothelial cells, the results suggest that the angiogenic effect of TGF-beta on endothelial cells in blood vessels may be mediated at least partly by a paracrine induction of VEGF in other surrounding cell types.

Topics: Animals; Cattle; Cell Line; Cells, Cultured; Endothelial Growth Factors; Endothelium, Vascular; Epithelium; Fibroblasts; Humans; Hybrid Cells; Immunoblotting; Lung Neoplasms; Lymphokines; Mice; Mice, Inbred AKR; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression.

Topics: Carcinoma, Small Cell; Cell Division; Cell Nucleus; Humans; Lung Neoplasms; Receptors, Transforming Growth Factor beta; Retinoblastoma Protein; 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

To determine the biological role of transforming growth factor-beta (TGF-beta) in mammary carcinomas in vivo, estrogen-dependent MCF-7 cells were transfected with a mouse TGF-beta 1 cDNA. Growth characteristics in culture were not altered in the transfected cells. However, the MCF-7/TGF-beta 1 cells formed tumors in ovariectomized athymic mice in the absence of estrogen supplementation. Daily injections of human recombinant TGF-beta 1 supported tumor formation by wild-type MCF-7 cells in castrated nude mice in the absence of exogenous estradiol. In another approach to the same question, the effect of anti-TGF-beta antibodies on tumor formation by estrogen-independent MDA-231 cells was examined. The 2G7 IgG2b (2G7) antibody, which neutralizes TGF-beta 1, -beta 2, and -beta 3, blocked the formation of MDA-231 tumors at the injection site and lung metastases in nude mice. Inoculation of MDA-231 cells inhibited, while injection of 2G7 increased mouse spleen natural killer (NK) activity. 2G7 did not inhibit MDA-231 tumors and metastases in NK-deficient animals. Finally, medium conditioned by MDA-231 cells inhibited lymphocyte-mediated NK activity; this inhibition was abrogated by 2G7, but not by a control IgG2. These data support a positive role for tumor cell TGF-beta in the maintenance and/or progression of mammary carcinoma cells in an intact host.

Topics: Animals; Breast Neoplasms; Estrogens; Female; Humans; Immunocompetence; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Nude; Transforming Growth Factor beta; Tumor Cells, Cultured

It has been considered that growth of human lung cancer cells, like other malignant cells, is positively and negatively regulated by a variety of growth factors via autocrine as well as paracrine mechanisms. The autocrine mechanism is considered to be important in the autonomy of proliferation of cancer cells. Recently, the role of autocrine growth-inhibiting factors such as transforming growth factor beta attracts special attention for better understanding of growth regulation of malignant cells. Here, we have demonstrated that a multifunctional cytokine interleukin 6 (IL-6) had an inhibitory effect on the proliferation of human non-small cell lung cancer cell lines, as shown by the growth accelerating effect of the specific anti-IL-6 antibody as well as the effect of exogenously added IL-6. Moreover, IL-6 can be expressed and released by human lung cancer cells, and these cells had specific IL-6 receptors on their cell surfaces, suggesting an autocrine mechanism. The growth-inhibitory effect of IL-6 was additive to that of transforming growth factor beta, and could not be neutralized by the addition of anti-transforming growth factor beta antibody. These results suggested that IL-6 may function as another class of autocrine growth-inhibiting factor in the growth regulation of human lung cancer. Relatively lower IL-6 sensitivity of these cells than noncarcinogenic human bronchial epithelial cells also suggested that escape from growth regulation by inhibitory factors such as IL-6 could be involved in lung cancer oncogenesis.

Topics: Adenocarcinoma; Cell Division; Humans; Interleukin-6; Lung Neoplasms; Receptors, Immunologic; Receptors, Interleukin-6; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGF beta-receptors and also produce TGF beta mRNAs.

Topics: Blotting, Northern; Carcinoma, Small Cell; Gene Expression; Humans; In Vitro Techniques; Lung Neoplasms; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Immunohistochemical localization of transforming growth factor-beta 1 (TGF-beta 1) was studied in the lungs of rats given crystalline silica or ferric oxide by single intratracheal instillation. Ferric oxide elicited no progressive granulomatous reaction, no epithelial hyperplasia, and no lung tumors; no demonstrable reactivity to TGF-beta 1 was observed. Silica induced a granulomatous reaction with progressive fibrosis, adjacent alveolar type II hyperplasia, and alveolar carcinomas. Rabbit polyclonal antibodies to synthetic peptides corresponding to the first 30 amino acids of mature TGF-beta 1, anti-LC (1-30), and anti-CC (1-30) were used for the localization of intracellular and extracellular TGF-beta 1. An antibody to a peptide corresponding to amino acids 266-278 of the TGF-beta 1 precursor sequence, anti-Pre (266-278), was used to detect the TGF-beta precursor and the latency-associated peptide. Intracellular mature TGF-beta (anti-LC) was demonstrated in fibroblasts and macrophages located at the periphery of silicotic granulomas and in fibroblasts adjacent to hyperplastic type II cells. Extracellular mature TGF-beta 1 was localized in the connective tissue matrix of the granulomas and in the stroma of both hyperplastic type II cells and well-differentiated adenocarcinomas. Immunoreactivity to anti-Pre was localized, intracellularly, in hyperplastic alveolar type II cells and their proliferative lesions adjacent to granulomas, in adenomas, but not in adenocarcinomas. The hyperplastic type II cells appear to be the sites of production and secretion of TGF-beta 1, which may regulate their own growth and differentiation and mediate the production of extracellular TGF-beta 1-associated matrix. The lack of reactivity to TGF-beta 1 precursor in the adenocarcinomas is consistent with the loss of normal cellular differentiation and function. TGF-beta 1 appears to have a pathogenetic role in silica-induced mesenchymal and epithelial lesions. The role of TGF-beta 1 and other cytokines in silica-induced carcinogenesis requires further investigation.

Topics: Adenocarcinoma; Adenoma; Animals; Disease Models, Animal; Female; Ferric Compounds; Hyperplasia; Lung; Lung Neoplasms; Male; Protein Precursors; Pulmonary Alveoli; Rats; Rats, Inbred F344; Silicon Dioxide; Silicosis; Transforming Growth Factor beta

The immunosuppressive state of a tumor-bearing patient is possibly mediated by tumor-derived factor. In this study, the authors characterized lung squamous cell carcinoma-derived immunosuppressive factor (LSCF).. The immunosuppressive activity of QG56 (a lung squamous carcinoma cell line)-derived LSCF was evaluated by the effect of culture supernatant of QG56 on anti-CD3 monoclonal antibody-induced T-cell, response such as proliferation (3H-thymidine uptake), cytotoxicity (51Cr-releasing assay), and expression of cytokine mRNA (polymerase chain reaction). The LSCF was partially purified with an ion-exchange high-performance liquid chromatography (HPLC) and a gel-filtration HPLC.. The LSCF inhibited proliferation, cytotoxicity, and expression of cytokine mRNA of T-cells in a dose-dependent manner. It has a molecular weight of approximately 22 kd, and was sensitive to proteinase K, heating at 60 degrees C, and resistant to treatment with trypsin and pH 3 and 9. These properties appear to be similar to those of transforming growth factor-beta (TGF-beta). However, the activity of the LSCF was not abrogated by anti-TGF-beta sera, and the LSCF did not suppress the proliferation of TGF-beta-sensitive mink lung cells (Mv1Lu).. These data suggest that LSCF may be a novel tumor-derived immunosuppressive protein factor.

Topics: Base Sequence; Carcinoma, Squamous Cell; CD3 Complex; Cytokines; Cytotoxicity, Immunologic; DNA Primers; Humans; Interleukin-2; Lung Neoplasms; Lymphocyte Activation; Molecular Sequence Data; Molecular Weight; Receptors, Interleukin-2; RNA, Messenger; Suppressor Factors, Immunologic; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

We examined factors promoting malignant progression using a weakly malignant variant cell line, ER-1, derived from c-SST-2, a rat mammary carcinoma. ER-1 cells were converted to a highly malignant phenotype (highly tumorigenic, metastatic, invasive in vitro) by the in vitro/in vivo interaction with host cells reactive to foreign body. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) produced by host reactive cells, transiently enhanced the tumorigenicity and in vitro invasiveness of ER-1 cells into an endothelial cell monolayer. The host reactive cells also produced oxygen radicals and induced mutations in ER-1 cells. It is speculated that mutations induced by host reactive cells cause cellular diversification, including the emergence of highly malignant variant cells whose growth is selectively promoted by growth factors such as EGF and TGF-beta.

Topics: Animals; Cell Communication; Cell Division; Cell Movement; Epidermal Growth Factor; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Adenocarcinoma; Breast Neoplasms; Cell Division; Culture Techniques; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Humans; Lung Neoplasms; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We previously reported that murine bone marrow cells activated by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) had potent nonspecific natural suppressor (NS) cell activity. In the present study, we demonstrated that these activated NS cells released a soluble factor (or factors) capable of nonspecifically inhibiting T cell mitogenic responses. Consistent with the properties of transforming growth factor-beta (TGF-beta), treatment of the NS supernates with heat failed to denature the factor, and in fact significantly increased its suppressive activity. The NS suppressor factor strongly inhibited proliferation of the TGF-beta-sensitive tumor cell line, A549. Cytokine activation of suppressive activity correlated with the production of a 10- to 13-kDa protein, consistent with the size of TGF-beta and rIL-3 induced a sevenfold increase in TGF-beta transcription. Finally, neutralizing anti-TGF-beta antibody inhibited the suppressive activity of the supernates, indicating that TGF-beta was responsible for most, if not all, of the suppression expressed by these bone marrow NS cells.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Division; Electrophoresis, Polyacrylamide Gel; Female; Humans; Interferon-gamma; Interleukin-3; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neutralization Tests; Recombinant Proteins; RNA; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Humans; Lung Neoplasms; Plasminogen Inactivators; Tissue Plasminogen Activator; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.

Topics: Blotting, Northern; Carcinoma, Small Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lung Neoplasms; Molecular Weight; Radioligand Assay; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

In cultured, undifferentiated normal human bronchial epithelial (HBE) cells, transglutaminase activity was localized predominantly in the cytosolic fraction of cell lysates. Upon squamous differentiation, this cytosolic activity declined and was replaced by a 40-fold increase in the activity of particulate (membrane-associated) transglutaminase. Immunoblot analysis demonstrated that the cytosolic transglutaminase was Type II (tissue) transglutaminase and that squamous differentiation shifted gene expression to the Type I (epidermal) transglutaminase. Retinoic acid, an inhibitor of squamous cell differentiation, suppressed the increase in Type I transglutaminase. The decrease in Type II transglutaminase activity was unaffected by retinoic acid. Transforming growth factor-beta 1 (TGF-beta 1) enhanced Type II transglutaminase activity about 10-fold in the undifferentiated cells but did not increase Type I transglutaminase or cholesterol sulfate, two early markers of squamous differentiation. TGF-beta 2 was equivalent to TGF-beta 1 in inducing Type II transglutaminase and in inhibiting the growth of HBE cells. The differentiation-related and TGF-beta-induced changes in transglutaminase activity were reflected in the level of transglutaminase Type I and Type II protein and mRNA. Expression of transglutaminases in lung carcinoma cell lines was variable. No correlation was observed between the expression of Type I transglutaminase and the classification of the cells as squamous cell carcinoma. Several lung carcinoma cell lines exhibited high levels of Type II transglutaminase activity that were increased several-fold by TGF-beta 1 treatment. Retinoic acid was ineffective in altering transglutaminase expression in most cell lines but induced Type II transglutaminase in a time- and dose-dependent manner in NCI-HUT-460 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blotting, Northern; Blotting, Western; Bronchi; Cells, Cultured; Epithelium; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Lung Neoplasms; RNA, Messenger; Transforming Growth Factor beta; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Transforming growth factor beta is a strong growth inhibitor for many types of normal and transformed cells, although little is known on the mechanism of this anti-proliferative effect. The human lung adenocarcinoma cell line A549 is growth arrested by TGF-beta 1 and serves as a model for studying this effect. We describe that, concurrent with the inhibition of A549 cell growth, TGF-beta 1 treatment causes a dramatic reduction in the level of expression of the amphiregulin (AR) gene, a recently identified member of the EGF/TGF alpha family. Similar results were also observed with TGF-beta 2. Peak inhibition occurred at 24 hr of treatment and was reversible upon removal of TGF-beta 1. The level of AR protein secreted by A549 cells was also decreased by TGF-beta 1. In contrast, TGF-alpha mRNA was not detected in these cells regardless of TGF-beta 1 treatment. Another TGF-beta inhibited cell line, PC-3 (human prostatic adenocarcinoma) also exhibited a decrease in AR message levels following exposure to TGF-beta 1. The growth inhibitory effects of TGF-beta may in part be mediated by modulation of AR expression.

Topics: Adenocarcinoma; Amphiregulin; Blotting, Northern; Cell Line; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Molecular Weight; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

The response to growth factor stimulation was evaluated in clonally derived rat bladder carcinoma cell lines, ranging from nontumorigenic to tumorigenic and metastatic, in athymic nude mice. In the nontumorigenic cell line D44c, epidermal growth factor (EGF)/transforming growth factor (TGF) alpha weakly stimulated anchorage-dependent, but not -independent, growth. In tumorigenic/nonmetastatic cells (G1-200 Cl-17), EGF/TGF-alpha stimulated markedly anchorage-independent, but marginally anchorage-dependent growth, whereas TGF-beta 1 inhibited anchorage-independent growth and DNA synthesis. In the highly tumorigenic/metastatic cell line LMC19, EGF/TGF-alpha stimulated anchorage-dependent growth weakly and anchorage-independent growth strongly. In these cells, TGF-beta 1 did not inhibit anchorage-independent growth and DNA synthesis but increased the size of colonies irrespective of the presence of EGF, and some cells were scattered around colonies in soft agar. None of the cell lines showed evidence of TGF-alpha-specific mRNA transcription. Expression of TGF-beta 1 mRNA increased in parallel to the biological aggressiveness of the cell lines. Highly tumorigenic and metastatic cells also demonstrated gelatinase activity involving 72 kilodalton and 92 kilodalton types. Our data suggest that the growth-stimulatory effect of EGF/TGF-alpha in soft agar may be limited to cells that are already tumorigenic and that EGF/TGF-alpha is not effective in making nontumorigenic cells become tumorigenic (or in making nontumorigenic cells grow in soft agar).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Proto-Oncogene Proteins; Rats; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Transforming growth factor-beta 1 (TGF-beta 1), a potent growth modulator produced by a variety of tumor cells, as well as by platelets, has pleiotropic effects on cell-extracellular matrix interactions and may influence tumor cell invasion and metastasis.. Our purpose was to characterize the effects of TGF-beta 1 on the adhesion, motility, and invasiveness of a metastatic human pulmonary carcinoma (A549 cell line) in vitro.. A549 cells were seeded onto type I collagen gels, and invasion over a 9-day period was measured in the presence or absence of TGF-beta 1 (0.1-10 ng/mL). In addition, cell adhesion to substrata coated with type I collagen (1-100 nM) as well as haptotactic migration through filters coated with type I collagen (100 micrograms/mL) were measured following a 24-hour treatment with TGF-beta 1 (1-10 ng/mL).. TGF-beta 1 stimulated the invasion of A549 cells into type I collagen gels in a dose-dependent manner. Both the number of cells entering the gel and the depth of invasion into the gel were increased. In addition, the effects of TGF-beta 1 were blocked in a dose-dependent manner by a purified polyclonal IgG against TGF-beta 1 but not by normal rabbit IgG. A549 cell invasion was accompanied by dramatic changes in A549 cell morphology that included the appearance of numerous long pseudopodia, consistent with a change in the motile behavior of these cells. TGF-beta 1 stimulated by approximately fourfold the haptotactic migration of A549 cells on polycarbonate filters coated with type I collagen. The TGF-beta 1-mediated increase in invasion and motility was accompanied by a fourfold increase in A549 cell adhesion to type I collagen.. The results suggest that TGF-beta 1 can influence cellular recognition of extracellular matrix components and can modulate cellular adhesion and migration on these components, leading to increased invasive potential.. Given the wide-spread tissue distribution of TGF-beta 1 and its secretion by a variety of tumor cells as well as by platelets, TGF-beta 1 may be an important autocrineparacrine regulator of the invasive phenotype in vivo.

Topics: Adenocarcinoma; Cell Adhesion; Cell Movement; Collagen; Filtration; Humans; Lung Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Adenocarcinoma; Antibodies, Anti-Idiotypic; Female; Humans; Lung Neoplasms; Middle Aged; Scleroderma, Localized; Transforming Growth Factor beta

We have compared the cell-specific expression and regulation of the receptor for urokinase-type plasminogen activator (u-PAR) by transforming growth factor beta type 1 (TGF-beta 1) in 10 human cell lines derived from both normal and neoplastic tissues. The basal expression of u-PAR mRNA as well as its response to TGF-beta 1 varied strongly between different cell lines; however, five out of the 10 cell lines responded to TGF-beta 1 by an increase in the u-PAR mRNA level. Among these, A549 cells were selected for a detailed elucidation of the molecular mechanism involved in TGF-beta 1 regulation of u-PAR mRNA expression. TGF-beta 1 caused an early increase in u-PAR mRNA level, with a maximal 15-fold enhancement after 24 h of treatment. This was paralleled by an increase in u-PAR protein as detected by crosslinking studies with radiolabeled ligand, and also resulted in an increase in cell surface plasmin generation. The protein synthesis inhibitor cycloheximide also increased the level of u-PAR mRNA in a time-dependent fashion and when both cycloheximide and TGF-beta 1 were used, an additive effect was seen. Nuclear run-on experiments demonstrated only a moderate (3-fold) increase in the u-PAR gene transcription rate after exposure of the cells to TGF-beta 1 for 3 h compared with a 12-fold increase in the mRNA level. TGF-beta 1 also caused an increase of both u-PA and PAI-1 antigens, while there was no detectable effect on t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blotting, Northern; Cycloheximide; Enzyme-Linked Immunosorbent Assay; Humans; Lung Neoplasms; Plasminogen Activators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

In vitro and in vivo metastatic variants derived from Lewis lung carcinoma (3LL) were examined for the level of the expression of several growth-regulated genes, oncogenes, and transforming growth factor (TGF) genes. To determine whether the proliferative advantage of metastatic cells is due to an increased growth fraction of the cell population or to a deregulated expression of some growth-regulated genes, the mRNA levels of the S-phase-specific H3 histone gene were compared with that of some cell cycle-related genes (vimentin, calcyclin, c-myc, and p53) and oncogenes (Ki-ras, Ha-ras, c-sis, c-src, c-fes, and c-erb). In addition, to evaluate whether an autocrine pattern of cell proliferation is responsible for the proliferative advantage of metastatic cells, the level of the expression of TGF genes (alpha and beta 1) was studied. Northern blot analysis demonstrated that in 3LL metastatic variants the expression of TGF-alpha as well as the expression of all growth-regulated genes and oncogenes studied are similar. Only the TGF-beta 1 gene is expressed at higher levels in highly metastatic 3LL variants maintained either in vitro or in vivo. Data suggest that the proliferative advantage of 3LL metastatic cells is not due to a deregulated expression of some growth-regulated genes and oncogenes, but more likely is acquired through the expression of genes which might interfere with the ability of the tumor cells to escape hostile microenvironmental conditions.

Topics: Animals; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Proto-Oncogenes; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

We examined for transforming growth factor alpha (TGF alpha) in adenocarcinomatous lesions of the lung tissues excised from 138 patients, with use of the avidin-biotin-peroxidase complex (ABC) method. TGF alpha was present in the cytoplasm of the adenocarcinoma. Our objective was to determine if TGF alpha could serve as a prognostic parameter. We divided 138 patients into two groups according to the concentration of TGF alpha. Ninety-two patients had a high concentration of TGF alpha, in over 75% of the tumour cells, while 46 had a low concentration, that is in less than 75% of the cells. The 5-year survival rates of patients with high TGF alpha and low TGF alpha were 39% and 64%, respectively (P less than 0.05). Our data suggest that evidence of a high immunoreactivity of TGF alpha can serve as a prognostic parameter in adenocarcinoma of the lung.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cytoplasm; Female; Humans; Immunoenzyme Techniques; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Prognosis; Transforming Growth Factor beta

We have examined the possible role of transforming growth factor-beta (TGF-beta) in metastatic malignancy by analyzing the production and activation of TGF-beta 1 and -beta 2 and the regulation of TGF-beta-responsive genes in oncogene-transformed metastatic fibrosarcomas. All transformed lines derived from either 10T1/2 or NIH 3T3 by either H-ras or protein-kinase encoding oncogenes produced more TGF-beta than parental cells. However, only highly metastatic fibrosarcomas secreted activated TGF-beta at rates that were greater than parental fibroblasts. Immunohistochemical staining for TGF-beta 1 showed widespread intra- and extracellular distribution in metastatic lung nodules and adjacent tissue. Cells isolated from tumors successfully metastasizing to the lung had TGF-beta 1 mRNA levels which were increased 19-fold over in vitro controls. Despite the greatly enhanced rate of secretion of activated TGF-beta, metastatic cells exhibited markedly altered responses of TGF-beta 1 and TGF-beta 2, being unable to either increase collagen secretion or enhance collagen alpha 2(1) or TGF-beta 1 mRNA levels. This lack of response was not due to either altered TGF-beta receptor affinity or numbers. Metastatic progression was, therefore, associated with an increase in the secretion of activated TGF-beta 1 and a loss of the ability to deregulate TGF-beta-responsive genes.

Topics: Animals; Cell Line, Transformed; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Lung Neoplasms; Mice; Mice, Inbred C3H; Oncogenes; Protein Kinases; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured