transforming-growth-factor-beta and Mesothelioma

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

Malignant mesothelioma (MM) is an asbestos-associated cancer that is increasing in incidence worldwide and is refractory to conventional therapy. MM cells are potent sources of a number of cytokines, some of which have recently been shown to be directly involved in the aggressive growth and spread of MM tumors. Emerging data also suggest involvement of MM-derived cytokines in systemic paraneoplastic syndromes including immunosuppression, thrombocytosis, cachexia, amyloidosis, and hypoglycemia. Additional characterization of the expression of cytokines and cytokine receptors in situ in MM tumors may provide a more complete picture of the autocrine and paracrine processes which occur in MM. Improved therapy of MM, particularly cytokine-based approaches, is likely to benefit from further elucidation of the patterns and regulation of cytokine expression associated with MM tumors.

Topics: Cytokines; Growth Substances; Humans; Interleukin-6; Mesothelioma; Platelet-Derived Growth Factor; Pleural Neoplasms; Receptors, Cytokine; 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

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

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

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

Malignant mesothelioma (MM) is an incurable malignancy that is caused by exposure to asbestos and is accompanied by severe fibrosis. Because MM is usually diagnosed at an advanced stage and clinical identification of early lesions is difficult, its molecular pathogenesis has not been completely elucidated. Nearly 75% of MM cases have inactivating mutations in the NF2 (neurofibromatosis type 2; Merlin) gene or in downstream signaling molecules of the Hippo signaling cascade, which negatively regulates the transcription factor Yes-associated protein (YAP). In this study, we demonstrate a functional interaction between the Hippo and TGF-β pathways in regulating connective tissue growth factor (CTGF). Expression of CTGF in MM cells was induced by the formation of a YAP-TEAD4-Smad3-p300 complex on the CTGF promoter. Knocking down CTGF expression in MM cells prolonged the survival of xenografted mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. We further suggest that CTGF may influence the malignancy of mesothelioma because of the different histological expression patterns observed in human MM tissues. These data suggest that CTGF is an important modulator of MM growth and pathology and represents a novel therapeutic target for this disease.

Topics: Animals; Base Sequence; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; DNA, Neoplasm; Extracellular Matrix; Female; Gene Expression; Genes, Neurofibromatosis 2; Humans; Mesothelioma; Mice; Mice, Nude; Molecular Sequence Data; Nuclear Proteins; Promoter Regions, Genetic; Signal Transduction; Smad Proteins; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta

Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4-Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.

Topics: Animals; Connective Tissue Growth Factor; Epithelium; Female; Humans; Mesothelioma; Mice; Mice, Nude; Molecular Targeted Therapy; Mutation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta

Despite having potent oncolytic activity, in vitro, direct intratumoral injection of oncolytic vesicular stomatitis virus (VSV) into established AE17ova mesothelioma tumors in C57Bl/6 mice had no therapeutic effect. During studies to combine systemic cyclophosphamide (CPA) with VSV to suppress the innate immune reaction against VSV, we observed that CPA alone had highly significant antitumor effects in this model. However, against our expectations, the combination of CPA and VSV consistently reduced therapeutic efficacy compared to CPA alone, despite the fact that the combination increased intratumoral VSV titers. We show here that CPA-mediated therapy against AE17ova tumors was immune-mediated and dependent upon both CD4 T cells and natural killer (NK) cells. However, intratumoral VSV induced a transforming growth factor-β (TGF-β)-dependent suppressive activity, mediated by CD11b(+)GR-1(+) cells that significantly inhibited both antigen-specific T-cell activation, and CPA-activated, NK-dependent killing of AE17ova tumor cells. Overall, our results show that treatment with oncolytic viruses can induce a variety of immune-mediated consequences in vivo with both positive, or negative, effects on antitumor therapy. These underexplored immune consequences of treatment with oncolytic viruses may have significant, and possibly unexpected, impacts on how virotherapy interacts in combination with other agents which modulate antitumor immune effectors.

Topics: Animals; CD4-Positive T-Lymphocytes; Combined Modality Therapy; Cyclophosphamide; Genetic Therapy; Killer Cells, Natural; Melanoma, Experimental; Mesothelioma; Mice; Mice, Inbred C57BL; Oncolytic Virotherapy; Oncolytic Viruses; Transforming Growth Factor beta; Vesicular stomatitis Indiana virus; Virus Replication

Malignant mesothelioma is an aggressive cancer of the pleura with poor prognosis. There is a need to identify new biomarkers and therapeutic targets for this invasive and fatal disease. Transforming growth factor β (TGF-β) can promote mesothelioma tumorigenesis through multiple mechanisms. Latent TGF-β binding proteins (LTBPs) regulate TGF-β activation by targeting the growth factor into the extracellular matrix from where it can be released and activated. We investigated here the expression patterns of different LTBP isoforms in malignant mesothelioma tissues and in 2 established malignant mesothelioma cell lines. All LTBPs were expressed, but LTBP-3 was the main isoform in healthy pleura and in cultured nonmalignant mesothelial cells. We observed down-regulation of LTBP-3 expression in malignant mesothelioma, which was associated with high P-Smad2 levels indicative of TGF-β activity specifically in the tumor tissue. Small interfering RNA-mediated suppression of LTBP-3 expression in mesothelioma cells increased the secretion of TGF-β activity. Immunoreactivity of LTBP-1, on the other hand, was markedly strong in the tumor stroma, which showed significantly lower levels of P-Smad2. A strong negative correlation between LTBP-1 and P-Smad2 immunoreactivity was found, implying that LTBP-1 is not likely to contribute directly to the increased levels of TGF-β activity in malignant mesothelioma. Current results suggest that LTBPs 1 and 3 may have specific roles in malignant mesothelioma pathogenesis through the regulation of TGF-β activation in the tumor tissue and the structure of the tumor stroma.

Topics: Cell Line, Tumor; Down-Regulation; Gene Expression; Gene Silencing; Humans; Kaplan-Meier Estimate; Latent TGF-beta Binding Proteins; Mesothelioma; Pleural Neoplasms; RNA, Messenger; RNA, Small Interfering; Smad2 Protein; Transforming Growth Factor beta

As an uncommon cancer, mesothelioma is very hard to treat with a low average survival rate owing to its usual late detection and being highly invasive. The link between asbestos exposure and the development of mesothelioma in humans is unequivocal. TGFBI, a secreted protein that is induced by transforming growth factor-β in various human cell types, has been shown to be associated with tumorigenesis in various types of tumors. It has been demonstrated that TGFBI expression is markedly suppressed in asbestos-induced tumorigenic cells, while an ectopic expression of TGFBI significantly suppresses tumorigenicity and progression in human bronchial epithelial cells. In order to delineate a potential role of TGFBI in mediating the molecular events that occur in mesothelioma tumorigenesis, we generated stable TGFBI knockdown mutants from the mesothelium cell line Met-5A by using an shRNA approach, and secondly created ectopic TGFBI overexpression mutants from the mesothelioma cell line H28 in which TGFBI is absent. We observed that in the absence of TGFBI, the knockdown mesothelial and mesothelioma cell lines exhibited an elevated proliferation rate, enhanced plating efficiency, increased anchorage-independent growth, as well as an increased cellular protein synthesis rate as compared with their respective controls. Furthermore, cell cycle regulatory proteins c-myc/cyclin D1/phosphor-Rb were upregulated; a more active PI3K/Akt/mTOR signaling pathway was also detected in TGFBI-depleted cell lines. These findings suggest that TGFBI may repress mesothelioma tumorigenesis and progression via the PI3K/Akt signaling pathway.

Topics: Asbestos; Cell Cycle; Cell Growth Processes; Cell Line; Cell Line, Tumor; Cyclin D1; Disease Progression; DNA-Binding Proteins; Epidermal Growth Factor; Extracellular Matrix Proteins; Gene Knockdown Techniques; Humans; Mesothelioma; Mutation; Neoplasms, Mesothelial; Oncogene Proteins v-mos; Phosphatidylinositol 3-Kinases; Protein Biosynthesis; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factors; Transforming Growth Factor beta

The plasminogen activator inhibitor type-1 (PAI-1) effectively blocks the activities of free and receptor-bound urokinase-type plasminogen activator. Incubation of cultured human pleural mesothelial (Met5A) cells with TGF-beta increased PAI-1 protein. TGF-beta, phorbol myristate acetate, and the translation inhibitor cycloheximide induced PAI-1 mRNA and slowed its degradation, suggesting that PAI-1 mRNA could be regulated by interaction of a PAI-1 binding protein (PAI-1 mRNABp) with PAI-1 mRNA. We found that an approximately 60 kD cytoplasmic PAI-1 mRNABp is detectable in cytoplasmic extracts of MeT5A human pleural mesothelial and malignant mesothelioma cells. The PAI-1 mRNABp specifically binds to a 33-nt sequence in the 3' untranslated region of PAI-1 mRNA. Insertion of this 33-nt sequence destabilizes otherwise stable beta-globin mRNA, indicating that the binding sequence accelerates decay of endogenous PAI-1 mRNA. Competitive inhibition by overexpression of the 33-nt binding sequence in MeT5A cells reduced PAI-1 mRNA decay and increased PAI-1 protein and mRNA expression, indicating that the PAI-1 mRNABp destabilizes PAI-1 mRNA by its interaction with the endogenous 33-nt binding sequence. Incubation of Met5A cells with TGF-beta attenuated the interaction of the PAI-1 mRNABp with the 33-nt sequence. By conventional and affinity purification, we isolated the PAI-1 mRNABp and confirmed its identity as 6-phospho-d-gluconate-NADP oxidoreductase, which specifically interacts with the full-length and the 33-nt sequence of the PAI-1 mRNA 3' untranslated region. This newly recognized pathway could influence expression of PAI-1 by mesothelial or mesothelioma cells at the level of mRNA stability in the context of pleural inflammation or malignancy.

Topics: Blotting, Northern; Blotting, Western; Carcinogens; Cells, Cultured; Cycloheximide; Epithelium; Gene Expression Regulation; Humans; Mesothelioma; Plasminogen Activator Inhibitor 1; Pleura; Pleural Neoplasms; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta

We have reported previously that a combined intratumoral treatment with anti-CD25mAb/transforming growth factor-β (TGF-β) soluble receptor induced regression of established and subcutaneous AE17 murine mesotheliomas. Here, we have investigated the mechanisms underlying this observation by analyzing the concentrations of interferon-γ (IFN-γ) and TGF-β within tumors at various time points following single regulatory T-cell (T(reg)) depleting anti-CD25mAb, TGF-β soluble receptor, or combined anti-CD25mAb/TGF-β soluble receptor treatment. The combined treatment maintains the intratumoral TGF-β concentration at a significantly lower level than either the untreated controls or the single anti-CD25mAb treatment alone. Also, the lower level of TGF-β correlated with a significantly higher concentration of IFN-γ compared with the single anti-CD25mAb treatment. It was hypothesized that TGF-β was the master regulator of immune suppression in the AE17 model of mesothelioma. However, it was found that although important, this cytokine alone is not responsible for maintaining immune suppression and that multiple mechanisms of suppression exist. Specifically, we have shown that the presence of T(regs) in the tumor draining lymph nodes alters the phenotype of dendritic cells in the same location. These data suggest that because the antitumor immune response is inhibited by multiple mechanisms of suppression, development of immunotherapeutic treatment regimes will be more successful if these mechanisms can be simultaneously inhibited.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Dendritic Cells; Female; Immunosuppression Therapy; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Lymphocyte Depletion; Mesothelioma; Mice; Mice, Inbred C57BL; Receptors, Transforming Growth Factor beta; Remission Induction; Skin Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape

Suppression of an anti-tumor immune response by regulatory T cells (T(regs)) in tumor-bearing hosts is now well established. Previously, we have reported that the intratumoral administration of T(reg)-depleting anti-CD25 monoclonal antibody every 10 days is highly effective at inhibiting the further development of established murine mesotheliomas. Here we investigate the dosage, kinetics, and immunology of this treatment. Further, we show that by precisely timing neutralization of transforming growth factor-β (TGF-β) within treated tumors using a TGF-β-soluble receptor, the efficacy of the treatment can be significantly improved, resulting in tumor regression. We suggest that this combined intratumoral treatment approach can be applied clinically for the treatment of mesothelioma.

Topics: Animals; Antibodies, Monoclonal; Female; Mesothelioma; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

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

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

Pleural malignant mesothelioma is a locally aggressive tumor of mesothelial cell origin. In other tumor types high expression of matrix metalloproteinase (MMP)-2, together with membrane-type1-MMP (MT1-MMP), and low levels of the tissue inhibitor of MMP (TIMP)-2 have been correlated with aggressive tumor progression and low survival rates. Therefore, we compared the expression and activation of these three factors and their regulation by two mesothelioma associated growth factors, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-beta1 in six human mesothelioma and one mesothelial cell line. Polymerase chain reaction (PCR), immunoblotting, zymography, and small inhibitory RNAs (siRNA) were used to study gene expression, protein activation, and signal transduction. To proof the relevance of our in vitro data immunohistochemistry was performed in tissue sections. PDGF-BB induced, while TGF-beta1 inhibited cell proliferation. PDGF-BB was a chemoattractant for mesothelial cells, and its effect was increased in the presence of TGF-beta1. TGF-beta1 stimulated the de novo synthesis of pro-MMP-2 in both cell types. Pro-MMP-2 synthesis involved p38 MAP kinase. In cell culture and tissue sections only mesothelial cells expressed MT1-MMP. Migration of mesothelioma cells was dependent on the presence of MT1-MMP. Migration, but not proliferation of mesothelioma cells was inhibited by oleoyl-N-hydroxylamide, TIMP-2, and siRNA for MT1-MMP. Our data suggest that in mesothelioma cells the phosphorylation of p38 MAP kinase is deregulated and is involved in pro-MMP-2 expression. Mesothelioma progression depends on an interaction with mesothelial cells that provide MT1-MMP necessary to activate pro-MMP-2 to facilitate migration through an extracellular matrix (ECM) layer.

Topics: Becaplermin; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial Cells; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Mesothelioma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Platelet-Derived Growth Factor; Protease Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; RNA, Small Interfering; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF)-beta blockade has been proposed as an anticancer therapy; however, understanding which tumor patients might benefit most from such therapy is crucial. An ideal target of such inhibitory therapy might be malignant mesothelioma (MM), a highly lethal, treatment-resistant malignancy of mesothelial cells of the pleura and peritoneum that produces large amounts of TGF-beta. The purpose of this study was to explore the possible therapeutic utility of TGF-beta blockade on MM.. To evaluate this hypothesis, we tested the effects of a soluble TGF-beta type II receptor (sTGF-beta R) that specifically inhibits TGF-beta1 and TGF-beta 3 in three different murine MM tumor models, AB12 and AC29 (which produce large amounts of TGF-beta) and AB1 (which does not produce TGF-beta).. Tumor growth of both established AB12 and AC29 tumors was inhibited by sTGF-beta R. In contrast, AB1 tumors showed little response to sTGF-beta R. The mechanism of these antitumor effects was evaluated and determined to be primarily dependent on immune-mediated responses because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice or mice depleted of CD8(+) T cells and (b) CD8(+) T cells isolated from spleens of mice treated with sTGF-beta R showed strong antitumor cytolytic effects, whereas CD8(+) T cells isolated from spleens of tumor-bearing mice treated with of control IgG2a showed no antitumor cytolytic effects.. Our data suggest that TGF-beta blockade of established TGF-beta-secreting MM should be explored as a promising strategy to treat patients with MM and other tumors that produce TGF-beta.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; fas Receptor; Female; Genes, MHC Class I; Genes, MHC Class II; Lymphocyte Depletion; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mice, SCID; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Spleen; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Tumor Cells, Cultured

Besides acting complexly on both normal and tumor cells, transforming growth factor-beta (TGF-beta) can determine the nature of the response to the antigen, strongly inhibiting the differentiation of naive CD4+ T-cells toward a T helper-1 (Th-1) phenotype; in a number of experimental models, TGF-beta also appeared to be a potent immunosuppressant factor. TGF-beta was shown to be released by some human malignant mesothelioma (MMe) cells, which affects the immune response to this tumor. Thus, for a better understanding of the role of TGF-beta in the immune response to MMe cells, we evaluated the production of a Th-1 cytokine (IFN-gamma) and of a Th-2 cytokine (IL-4), following Purified Protein Derivative (PPD) recall antigen presentation by human MMe cells to a class-II major histocompatibility complex (MHC-II)-matched PPD clone (PPD clone). Our data confirm that human MMe cells possess the unusual capability of presenting a common recall antigen to CD4+ lymphocytes but also show that these tumor cells can abrogate Th-1 immune response, as evidenced by a shift in favor of the production of IL-4 over that of IFN-gamma, through a TGF-beta-mediated pathway; only the simultaneous block of TGF-beta1 and beta2 effects can significantly restore a typical Th-1 pattern of cytokine production by PPD clone in response to PPD presentation by MMe. Even though the role of TGF-beta in the promotion of MMe growth should be further and better defined, this effect should be considered when designing new therapeutical approaches aimed at improving the immune response to MMe.

Topics: CD4-Positive T-Lymphocytes; Cell Division; Histocompatibility Antigens Class II; Humans; Interferon-gamma; Interleukin-4; Karyotyping; Mesothelioma; Transforming Growth Factor beta; Tuberculin

Tenascin-C is an extracellular matrix glycoprotein known to have anti-adhesive characteristics and to be expressed in various human malignant neoplasms. We hypothesized that the expression of tenascin-C would be increased in human malignant pleural mesothelioma, and its accumulation associated with the prognosis of the patients with this disease.. Thirty-seven cases of mesothelioma were studied by immunohistochemically using a monoclonal antibody against tenascin-C, and with a semiquantitative scoring system for tenascin-C in different areas of the tumours. In 10 selected cases tenascin-C mRNA in-situ hybridization was also analysed. Since transforming growth factor-beta (TGF-beta) is known to induce both the synthesis of tenascin-C and the growth of mesotheliomas, an immunohistochemical analysis of TGF-beta 1, -beta 2 and -beta 3 was also performed. Normal pleura (n = 7) and metastatic pleural adenocarcinomas (n = 7) were used as controls. Tenascin-C protein was expressed in every histological subtype of malignant mesothelioma, being most prominent in the fibrotic stroma of a tumour, around tumour cells and at the invasive border, whereas tenascin-C mRNA was scarce in tumour cells. The patients with less immunohistochemical expression for tenascin-C tended to live longer (P = 0.028 by Fishers' exact probability test). All mesotheliomas showed positivity for at least one isoform of TGF-beta.. In conclusion, high expression of tenascin-C protein in malignant pleural mesotheliomas may play a role in its invasive growth, and might serve as a prognostic marker of the disease.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization; Male; Mesothelioma; Middle Aged; Neoplasm Proteins; Pleural Neoplasms; Prognosis; RNA, Messenger; RNA, Neoplasm; Tenascin; Transforming Growth Factor beta

Malignant mesothelioma (MM) is a lethal tumor linked with a prior exposure to asbestos in which limited progress has been made so far using conventional therapies. MM is an example of a "nonimmunogenic" tumor characterized by a fibrous stroma and an absence of infiltrating T lymphocytes. High levels of transforming growth factor-beta (TGF-beta) produced by mesothelioma cells have been related to the immune tolerance towards the tumor. In order to evaluate the effect of local delivery of cytokines such as interferon gamma (IFN-gamma) by gene transfer, we characterized and used a murine model, AK7, which appeared very similar to human mesothelioma. AK7 cells expressed low levels of major histocompatibility class I and class II antigens and secreted high levels of latent TGF-beta. The TGF-beta pathway in AK7 cells is operative but inefficient because endogenous TGF-beta is predominantly inactive. Treatment of pre-established AK7 tumors by direct intratumoral injection of an adenovirus vector expressing murine IFN-gamma, Ad.mIFN-gamma, led to significant tumor regression. Peripheral tumor infiltration by CD4+ and CD8+ T lymphocytes in the treated tumors appeared to be because of the induction of an immune response. Tumor relapse was observed, which could be due to local TGF-beta secretion by remaining tumor cells.

Topics: Adenoviridae; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Line, Tumor; Cell Separation; Cytokines; DNA, Complementary; Down-Regulation; Female; Flow Cytometry; Gene Transfer Techniques; Genetic Therapy; Immunohistochemistry; Interferon-gamma; Mesothelioma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Phosphorylation; Polymerase Chain Reaction; RNA; T-Lymphocytes; Time Factors; Transforming Growth Factor beta; Up-Regulation

We investigated whether transforming growth factor beta (TGF-beta) is involved in the growth of malignant mesothelioma (MM) cells in culture. TGF-beta production was examined in two mesothelioma cell lines (MeET-4 and -6) that were established from rat spontaneous MM in our laboratory. TGF-beta bioactivity in conditioned medium of these cell lines was analyzed using a CCL64 mink lung epithelial cell growth inhibition assay and found to be 30-70 times higher than that of normal rat mesothelial cells (MCs). The MM cell lines also showed considerably higher levels of TGF-beta mRNA expression when compared with MCs. The bioactivity and mRNA expression level were greater in MeET-4 than MeET-6. When MeET-4 was treated with antisense TGF-beta1 oligonucleotide (ODN), a significant decrease in both anchorage-dependent and -independent growth was observed. Treatment with exogenous TGF-beta resulted in no effects on the growth pattern of the MM cell lines, while proliferation of the MCs was slightly induced. It is considered that TGF-beta appears to be produced by rat spontaneous MM cells through an autocrine mechanism and could modulate the malignant growth of the tumor cells.

Topics: Animals; Cell Count; Cell Division; Culture Media, Conditioned; DNA Primers; Dose-Response Relationship, Drug; Epithelium; Mesothelioma; Molecular Sequence Data; Oligonucleotides, Antisense; Peritoneum; Polymerase Chain Reaction; Rats; Rats, Inbred F344; Retroperitoneal Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

Vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors (FGF-1 and -2), and transforming growth factor beta (TGFbeta) are potent angiogenic cytokines. Malignant mesothelioma of the pleura presents with a high intra-tumoural microvascular density (IMD) which also has prognostic relevance. This study was designed to verify the immunohistochemical expression of the angiogenic cytokines in mesothelioma as well as in non-neoplastic human mesothelial cells and to study the individual as well as the combined expression of these cytokines in mesothelioma in relation to both IMD and prognosis. In addition, four mesothelioma cell lines were studied by ELISA for the secretion of VEGF and FGF-2 in their supernatants and were shown to contain high levels of both of these cytokines. Immunohistochemically, VEGF, FGF-1 and -2, and TGFbeta immunoreactivity was present in 81, 67, 92 and 96 per cent of mesotheliomas, and in 20, 50, 40, and 10 per cent of samples of the non-neoplastic mesothelium, respectively. Co-ordinate expression of the cytokines was observed whereby mesotheliomas expressed more than one cytokine. The combined immunohistochemical expression levels for all four cytokines correlated significantly with both IMD (p=0.01) and prognosis (p=0. 0013). When studied individually, high FGF-2 expression correlated best with more tumour aggressiveness and worse prognosis for mesothelioma (p=0.0011). There was no significant correlation between prognosis and immunoexpression of VEGF (p=0.07), FGF-1 (p=0.3), or TGFbeta (p=0.1), or between IMD and any of the cytokines studied individually. These data support the assertion that selective angiogenic cytokines might contribute to the progressive changes of mesothelioma by tumour angiogenesis.

Topics: Biomarkers, Tumor; Cytokines; Endothelial Growth Factors; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunohistochemistry; Lymphokines; Male; Mesothelioma; Middle Aged; Neovascularization, Pathologic; Pleural Neoplasms; Survival Analysis; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

An autopsy case of biphasic malignant mesothelioma with osseous and cartilaginous differentiation diffusely involving the peritoneal cavity was confirmed by light microscopic histochemistry, immunohistochemistry, and electron microscopy. A reverse transcription-polymerase chain reaction using specific primers for bone morphogenetic protein-2 (BMP-2) revealed weak positive transcript in normal mesothelial cells and up-regulated expression around bone-forming malignant mesothelioma tissue. However, BMP-2 protein expression was detected only in the marginal zone of bone trabeculae and spindle-shaped mesothelioma cells distributed around bone trabeculae in tumor tissue. The distribution of type IV collagen in tumor tissue was in accordance with the BMP-2 expression. Normal mesothelial cells and tumor cells expressed BMP-2 mRNA, but the BMP-2 protein expression was restricted to the bone-forming area in the malignant mesothelioma.

Topics: Abdominal Neoplasms; Aged; Bone and Bones; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage; Epithelium; Fatal Outcome; Humans; Immunohistochemistry; Male; Mesothelioma; RNA, Messenger; Tomography, X-Ray Computed; Transforming Growth Factor beta

Treatment of human pleural mesothelioma (MS-1) cells with phorbol myristate acetate (PMA) and cycloheximide results in 17- and 10-fold, respectively, increases in steady-state expression of urokinase-type plasminogen activator receptor (uPAR) mRNA. Studies of transcriptional inhibition by actinomycin D showed four- and sixfold extensions of uPAR mRNA half-life in MS-1 cells treated with PMA and cycloheximide, respectively, suggesting that uPAR gene expression involves a posttranscriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 50-kDa uPAR mRNA binding protein (uPAR mRNABp) that selectively bound to a 51-nucleotide (nt) fragment of mRNA corresponding to the uPAR coding region. We investigated the possibility that this 51-nt protein binding fragment of uPAR mRNA contains regulatory information for message stability. Chimeric beta-globin/uPAR/beta-globin mRNA containing the 51-nt protein binding fragment was able to destabilize otherwise stable beta-globin mRNA. Conversely, a control chimeric beta-globin/uPAR/beta-globin mRNA containing a 51-nt fragment of the uPAR coding region that does not bind uPAR mRNABp was stable under identical conditions. Binding of uPAR mRNABp to uPAR mRNA was abolished after treatment with cycloheximide and rapidly down-regulated by PMA. These data suggest that the 51-nt protein binding fragment of uPAR mRNA may be involved in mRNA turnover as well as in cycloheximide-induced uPAR message stabilization. Our results indicate a novel mechanism of uPAR gene regulation in which cis elements within a 51-nt coding region interact with a uPAR mRNABp to regulate uPAR message stability.

Topics: Base Sequence; Binding Sites; Cycloheximide; Dactinomycin; Epithelial Cells; Epithelium; Gene Expression; Globins; Humans; Lipopolysaccharides; Mesothelioma; Molecular Sequence Data; Nucleic Acid Synthesis Inhibitors; Pleural Neoplasms; Protein Synthesis Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA; RNA-Binding Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Transforming growth factor beta (TGF-beta) is a potent growth-regulatory and immunomodulatory cytokine that exerts a diverse range of effects on many types of cells. High levels of TGF-beta are produced by several human and mouse malignant mesothelioma (MM) cell lines, and it is known to act as a growth factor for these cells. Antisense oligonucleotides (ODNs), targeted against specific TGF-beta mRNA, were used to block TGF-beta production from MM cells in vitro and in vivo. TGF-beta antisense ODNs were encapsulated in liposomes and transfected into MM cells or delivered intratumorally. TGF-beta2 mRNA levels, assessed by semiquantitative PCR, and TGF-beta2 protein secretion were reduced after TGF-beta2 antisense ODN transfection. MM cell proliferation, assessed by tritiated thymidine uptake, was specifically inhibited by both TGF-beta1- and TGF-beta2-specific antisense ODNs. In vivo administration of TGF-beta2 antisense ODNs, delivered locally, reduced tumor growth. These data show that the blockade of TGF-beta2 within this tumor reduces tumor growth and raises the possibility that TGF-beta2 antisense ODNs may be useful as a therapy for this disease.

Topics: Animals; Bromodeoxyuridine; Cell Division; Female; Injections, Intralesional; Injections, Subcutaneous; Mesothelioma; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Oligonucleotides, Antisense; RNA, Messenger; Survival Rate; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects and the mechanisms of action of transforming (TGF-beta 2) and basic fibroblast (bFGF) growth factors on the synthesis of hyaluronan and proteoglycans (PGs) in human malignant mesothelioma cells have been studied. Two cell lines, one with an epithelial differentiation (STAV-AB) and the other with a fibroblast-like phenotype (STAV-FCS) have been examined. Using monoclonal antibodies to growth factor receptors, the presence of a high density of the specific receptors for TGF-beta 2 and bFGF was immunochemically demonstrated in both mesothelioma cell lines. These cell lines synthesize hyaluronan, galactosaminoglycan containing PGs (GalAGPGs) and heparan sulphate containing PGs (HSPGs) at different levels; the epithelial differentiated cells produce a 6-8 times higher amount than those with the fibroblast-like morphology. In both cell lines the rates of proteoglycan synthesis were affected in a dose-dependent mode by TGF-beta 2 and bFGF. Maximal synthetic levels of both secreted and cell-bound proteoglycans were reached at 10 ng/mL whereafter they remained constant. TGF-beta 2 stimulated the synthesis of hyaluronan, GalAGPGs and of HSPGs in STAV-FCS, whereas this effect was pronounced only for GalAGPGs in STAV-AB. bFGF showed stimulatory effects for the synthesis of hyaluronan and cell associated GalAGPGs in STAV-FCS, whereas no significant enhancement was recorded for HSPGs. In the STAV-AB cell line the synthesis of hyaluronan and GalAGPGs remained unaltered by the addition of both growth factors. Although the synthesis of total HSPGs remained constant, this was due to a decrease in the secreted product and a similar increase of the cell-associated proteoglycan. The stimulatory mechanisms of both growth factors was examined by using the specific protein tyrosine kinase inhibitor genistein. Incubation of both cell lines with this isoflavonoid inhibited the enhanced synthesis of hyaluronan and all PGs induced by TGF-beta 2 and bFGF. It is suggested that most, if not all, of the stimulatory effects on the hyaluronan and PGs synthesis are mediated via protein tyrosine kinase activity elicited by receptor-ligand complexes. Decreased synthetic rates obtained when giving genistein to unstimulated mesothelioma cells may indicate the relation of hyaluronan and PGs synthesis with an autocrine stimulatory mechanisms.

Topics: Fibroblast Growth Factor 2; Humans; Hyaluronic Acid; Intracellular Fluid; Mesothelioma; Pleural Neoplasms; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Fibroblast Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

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

In earlier studies we showed that the expression of patterns of platelet-derived growth factor (PDGF) alpha- and beta-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF alpha-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF beta-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF beta-receptor mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF beta-receptor transcription but of alpha-receptor transcription as well, as evidenced from run off analysis and RT-PCR using alpha-receptor specific primers. However, the fact that PDGF alpha-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF alpha-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF alpha-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF beta-receptor mRNA expression was seen. The half-life of the PDGF beta-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF beta-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in beta-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF beta-receptor transcripts in these cells. Transforming growth factor beta-1 (TGF-beta 1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-beta 1 revealed decreased PDGF alpha-receptor mRNA expression in normal mesothelial cells. The effect on PDGF beta-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-beta 1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-beta 1 may nevertheless play a role in downregulation of an (already) low PDGF alpha-receptor mRNA level in malignant mesothelioma cell lines.

Topics: Base Sequence; Cell Line; DNA Primers; Epithelial Cells; Epithelium; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Mesothelioma; Molecular Sequence Data; Polymerase Chain Reaction; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de-activated upon infiltration of the tumor micro-environment.

Topics: Animals; Base Sequence; Cytokines; DNA Primers; Female; Gene Expression; Immunohistochemistry; Interferon-gamma; Interleukin-3; Interleukin-4; Lymphocytes, Tumor-Infiltrating; Macromolecular Substances; Mesothelioma; Mice; Mice, Inbred CBA; Molecular Sequence Data; Polymerase Chain Reaction; Receptor-CD3 Complex, Antigen, T-Cell; RNA, Antisense; RNA, Messenger; T-Lymphocytes; Time Factors; Transcription, Genetic; Transforming Growth Factor beta

Malignant mesothelioma is an aggressive tumor, usually induced by asbestos exposure, that has a poor prognosis and is unresponsive to conventional therapy. The present study was aimed at assessing the potential for interferon-alpha (IFN-alpha)-based therapies in a murine model for malignant mesothelioma. The effect of recombinant human IFN-alpha B/D on tumor growth, alone and in combination with either of two immunomodulatory and antiproliferative agents beta-carotene or alpha-difluoromethylornithine (DFMO), was assessed. The data suggest that IFN-alpha treatment is most efficacious when commenced early in tumor development. Combination of IFN-alpha with either DFMO or dietary beta-carotene supplementation improved the effect of an otherwise suboptimal IFN-alpha therapy regimen. Both IFN-alpha and beta-carotene had in vivo stimulatory effects on immune cells, perhaps indirectly by inhibiting TGF-beta generation. The immunomodulatory effects may contribute, at least in part, to the positive antitumor and clinical activities of the treatments in this model.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Combined Chemotherapy Protocols; beta Carotene; Carotenoids; Eflornithine; Female; Interferon Type I; Lymphocytes, Tumor-Infiltrating; Macrophages; Mesothelioma; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Urokinase (uPA) interacts with its receptor (uPAR) to promote proteolysis and tumor migration, functions of potential importance in the pathogenesis of malignant mesothelioma. Immunohistochemistry of human malignant mesothelioma tissue and mesothelioma cells (MS-1) showed that mesothelioma cells express uPAR. We isolated uPAR from MS-1 cells by metabolic labeling and showed that it could be induced by phorbol myristate acetate (PMA), lipopolysaccharide (LPS), a transforming growth factor-beta (TGF-beta) or tumor necrosis factor-alpha (TNF-alpha). Experiments with MS-1 cells showed that uPA binding was saturable, specific, and reversible with a mean dissociation constant (Kd) of 5.4 +/- 1.1 nM. Binding was inhibited by a blocking antibody to uPAR and by the uPA amino-terminal fragment (ATF), but not by low molecular weight uPA. uPAR expression was regulated transcriptionally and translationally; antisense oligonucleotides blocked expression of uPAR protein. Plasminogen activator inhibitor-1 (PAI-1) inhibited PA activity of preformed uPA/uPAR complexes and increased cycling of the receptor from the cell surface. Stimulation of subconfluent MS-1 cells by high molecular weight or recombinant uPA, but not ATF or low molecular weight fragment, caused concentration-dependent incorporation of [3H]thymidine. These data indicate a novel mechanism by which malignant mesothelioma cells localize pericellular proteolysis and concurrently regulate tumor cell proliferation.

Topics: Cell Cycle; Cell Division; Cell Line; Gene Expression; Humans; Immunohistochemistry; Iodine Radioisotopes; Kinetics; Mesothelioma; Methionine; Protein Biosynthesis; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymidine; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Two human mesothelioma cell sublines with fibroblast-like and epithelial morphology produce hyaluronan, galactosaminoglycans, and heparan sulfate in amounts varying with their cell phenotype. The epithelially differentiated cells synthesize these glycosaminoglycans in 6- to 8-fold higher amounts than the fibroblast-like cells. Hyaluronan is mainly a secretory product (> 90%), a considerable proportion of galactosaminoglycans is present in the extracellular medium (> 80%), while more of the heparan sulfate (50-70%) is cell-associated. In both cell lines the rates of synthesis of glycosaminoglycans are affected by the addition of the exogenous growth factors. In fibroblast phenotype cells, TGF-beta 2, EGF, and bFGF increase the production of glycosaminoglycans from 1.6- to 2.0-fold, with the exception of HS, which is suppressed by the addition of bFGF. The combination of these growth factors with PDGF-BB showed that only EGF causes a synergistic action in the synthesis of all glycosaminoglycans and that no additive effect is obtained when PDGF-BB is combined with TGF-beta 2 and bFGF. In epithelially differentiated cells, the addition of exogenous TGF-beta 2 and bFGF has no significant effect on hyaluronan synthesis, which is increased by EGF to 45%. The synthesis of galactosaminoglycans is stimulated by EGF and TGF-beta 2 approximately 35%, whereas bFGF has no significant effect. Heparan sulfate production is considerably increased by the addition of EGF by 50%, whereas bFGF has no significant effect and TGF-beta 2 suppresses this synthesis. The combination of the various growth factors with PDGF-BB showed that only heparan sulfate synthesis is affected. Thus, combining PDGF-BB with TGF-beta 2 and EGF this synthesis is increased by 35 and 25%, whereas the combination of bFGF with PDGF-BB has no further effect. The remarkable differences found between the two mesothelioma sublines may well be related to the importance of glycosaminoglycan-growth factor interactions as a key factor in phenotypic cell differentiation.

Topics: Epidermal Growth Factor; Epithelial Cells; Epithelium; Fibroblast Growth Factors; Fibroblasts; Glycosaminoglycans; Growth Substances; Humans; Infant, Newborn; Mesothelioma; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is produced by a number of tumor cell types including human malignant mesothelioma (MM), but its role as a direct or indirect factor in tumorigenesis is incompletely understood. We have investigated the expression of TGF-beta isoforms by human and murine MM cells and have analysed the effects of inducible antisense RNA-mediated inhibition of TGF-beta expression on murine MM in vitro and in vivo. The results showed that (a) TGF-beta 1 and -beta 2 were produced by both human and mouse MM cells, (b) antisense RNA against either TGF-beta 1 or -beta 2 cross-inhibited both TGF-beta 1 and -beta 2 expression, (c) inhibition of TGF-beta expression reduced the anchorage-independent growth of MM cells in vitro and the tumorigenicity of MM cells in vivo, and (d) inhibition of TGF-beta expression led to increased T lymphocyte infiltration into tumors. The data suggest that TGF-beta has multiple tumor-enhancing effects in MM.

Topics: Animals; Base Sequence; Cell Adhesion; Cell Division; DNA Primers; Female; Genetic Vectors; Humans; Lymphocytes, Tumor-Infiltrating; Mesothelioma; Mice; Mice, Inbred CBA; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

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

Malignant mesothelioma (MM) is an aggressive, uniformly fatal serosal tumour, usually associated with asbestos exposure, for which there currently is no effective treatment. In order to gain insight into the mechanism(s) whereby MM might escape immune surveillance, a murine model for MM was used (a) to characterise the tumour-infiltrating lymphocytes (TIL) and macrophages (TIM) phenotypically, (b) to examine systemic immune recognition of MM, and (c) to examine the possible influence of tumour-derived cytokines on systemic and local pathobiological manifestations of MM. A profound down-regulation of lymphocyte surface markers, known to be involved in T cell activation, was found in TIL. Likewise, although TIM were present in large numbers, their expression of MHC class II antigen and integrins was weak or absent, suggestive of altered functional activity. Significant amounts of cytokines, in particular transforming growth factor beta, interleukin-6 (IL-6), IL-1 and tumour necrosis factor were produced during the course of MM tumour development-directly by the MM cells and/or indirectly in response to tumour growth. These factors may contribute both to derangement of antitumour effector mechanisms and to the clinical and pathological manifestations of the disease.

Topics: Animals; Antigens, CD; Cytokines; Female; Immunophenotyping; Lymphocytes, Tumor-Infiltrating; Macrophages; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Transforming Growth Factor beta

The mesothelium contains both procoagulant and fibrinolytic activities. An imbalance between these activities could account for the abnormal fibrin turnover and pleural fibrin deposition that is characteristic of pleural inflammation. Procoagulant activity of human pleural mesothelial cells (HPMC) is in part due to tissue factor, and the prothrombinase complex can also assemble at the HPMC surface. HPMC express tissue plasminogen activator (tPA) but no detectable fibrinolytic activity in a fibrin plate assay. Inhibition of HPMC fibrinolytic activity is due, in part, to elaboration of plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) as well as antiplasmins. Synthesis of PAI-1 and PAI-2 is inhibited by actinomycin D and cyclohexamide. HPMC PAI-1 is increased by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), as is tPA release, while PAI-1 mRNA is unchanged and tPA mRNA is increased. PAI-2 release is induced by TNF-alpha and TGF-beta. Because they are a rich source of PAI-1 and PAI-2, HPMC may contribute to the high levels of these inhibitors in pleural exudates. Stimulation of HPMC by TNF-alpha or TGF-beta in vitro did not alter HPMC procoagulant activity nor the balance of elevated PAI and antiplasmins relative to PA, changes that collectively favor formation and persistence of pericellular fibrin.

Topics: Base Sequence; Blood Coagulation Factors; Cells, Cultured; Cycloheximide; Dactinomycin; Epithelium; Fibrin; Fibrinolysis; Fibroblasts; Humans; Inflammation; Lung; Mesothelioma; Molecular Sequence Data; Oligonucleotide Probes; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Pleural Effusion; Prothrombin; RNA, Messenger; Tissue Plasminogen Activator; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator