cyclin-d1 and Mesothelioma

To examine the potential of cyclin D1/podoplanin dual immunohistochemical stain to differentiate malignant mesothelioma from reactive mesothelial proliferations.. Cyclin D1/podoplanin dual immunohistochemistry was performed on 34 surgical cases of reactive mesothelial proliferations, malignant mesothelioma, and nonmesothelioma malignancies.. All 15 reactive mesothelial proliferations demonstrated less than 50% cyclin D1 staining with variable to diffuse podoplanin staining. In 6 (60%) of 10 cases of epithelioid malignant mesothelioma, the dual stain supported the diagnosis. Less than 50% cyclin D1 staining was noted in the remaining four cases, including small biopsy specimens or cases with focal papillary architecture. The five cases of sarcomatoid/desmoplastic/biphasic mesothelioma showed more than 50% cyclin D1 staining with focal to absent podoplanin staining. Well-differentiated papillary mesothelioma appears to demonstrate less than 25% cyclin D1 staining.. The cyclin D1/podoplanin dual stain is reliable and may be used to aid in differentiation of benign mesothelial proliferations from malignant tumors. In addition, histologic features and other ancillary testing may support the classification of cases with an inconclusive cyclin D1/podoplanin staining.

Topics: Biomarkers, Tumor; Coloring Agents; Cyclin D1; Diagnosis, Differential; Humans; Mesothelioma; Mesothelioma, Malignant

Pleural mesothelioma (PM) is an aggressive cancer with poor prognosis and no effective therapies, mainly caused by exposure to asbestos. Antagonists of growth hormone-releasing hormone (GHRH) display strong antitumor effects in many experimental cancers, including lung cancer and mesothelioma. Here, we aimed to determine whether GHRH antagonist MIA-690 potentiates the antitumor effect of cisplatin and pemetrexed in PM. In vitro, MIA-690, in combination with cisplatin and pemetrexed, synergistically reduced cell viability, restrained cell proliferation and enhanced apoptosis, compared with drugs alone. In vivo, the same combination resulted in a strong growth inhibition of MSTO-211H xenografts, decreased tumor cell proliferation and increased apoptosis. Mechanistically, MIA-690, particularly with chemotherapeutic drugs, inhibited proliferative and oncogenic pathways, such as MAPK ERK1/2 and cMyc, and downregulated cyclin D1 and B1 mRNAs. Inflammatory pathways such as NF-kB and STAT3 were also reduced, as well as oxidative, angiogenic and tumorigenic markers (iNOS, COX-2, MMP2, MMP9 and HMGB1) and growth factors (VEGF and IGF-1). Overall, these findings strongly suggest that GHRH antagonists of MIA class, such as MIA-690, could increase the efficacy of standard therapy in PM.

Topics: Cell Line, Tumor; Cisplatin; Cyclin D1; Cyclooxygenase 2; Growth Hormone-Releasing Hormone; HMGB1 Protein; Humans; Insulin-Like Growth Factor I; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mesothelioma; Mesothelioma, Malignant; NF-kappa B; Pemetrexed; Pleural Neoplasms; Vascular Endothelial Growth Factor A

Topics: Cell Line, Tumor; Cell Proliferation; Computational Biology; Cyclin D1; Cytosol; E2F Transcription Factors; Female; Gene Expression Regulation, Neoplastic; Humans; Hydrogen-Ion Concentration; Male; Mesothelioma; Metabolomics; Mitosis; Subcellular Fractions; Transcription Factors

Malignant mesothelioma (MM) is an asbestos-induced cancer arising on the mesothelial surface of organ cavities. MM is essentially incurable without a means of early diagnosis and no successful standard of care. These facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome-contained microRNAs (miRNAs). We discovered that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly miR-16-5p. This observation lead us to hypothesize that MM cells preferentially secreted tumor-suppressor miRNAs via exosomes. Through separate avenues of potential therapeutic advance, we embarked on an innovative strategy to kill MM tumor cells. We employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16-5p exosome loss and replenishing cellular miR-16-5p leading to reduced tumorigenic capacity and miR-16-5p target oncoproteins CCND1 and BCL2. Additionally, we force-fed MM tumor exosomes back to MM tumor cells, which led to cell death, and a reduction in the same oncoproteins. We recapitulated these results with direct transfection of miR-16-5p, confirmed that this is a cancer-cell specific effect, and elucidated a part of the miR-16-5p mechanism of exosome loading.

Topics: Aniline Compounds; Antineoplastic Agents; Benzylidene Compounds; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Exosomes; Gene Expression Regulation, Neoplastic; Humans; Indoles; Lung Neoplasms; Maleimides; Mesothelioma; Mesothelioma, Malignant; MicroRNAs; Molecular Targeted Therapy; Ornithine; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Small Molecule Libraries; Transfection

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

Manumycin A (Manu A) is a natural product isolated from Streptomyces parvulus and has been reported to have anti-carcinogenic and anti-biotic properties. However, neither its molecular mechanism nor its molecular targets are well understood. Thus, the aim of the present study was to explore the possibility that Manu A has cancer preventive and chemotherapeutic effects on malignant pleural mesothelioma (MPM) through regulation of Sp1 and induction of mitochondrial cell death pathway. Manu A inhibited the cell viability of MSTO-211H and H28 cells in a concentration‑dependent manner as determined by MTS assay. IC50 values were calculated as 8.3 and 4.3 µM in the MSTO-311H and H28 cells following 48 h incubation, respectively. Manu A induced a significant increase in apoptotic indices as shown by DAPI staining, Annexin V assay, multi-caspase activity and mitochondrial membrane potential assay. The downregulation of Sp1 mRNA and protein expression by Manu A led to apoptosis by suppressing Sp1-regulated proteins (cyclin D1, Mcl-1 and survivin). Manu A decreased the protein levels of BID, Bcl-xL and PARP while it increased Bax levels. Manu A caused depolarization of the mitochondrial membrane with induction of CHOP, DR4 and DR5. Our results demonstrated that Manu A exerted anticancer effects by inducing apoptosis via inhibition of the Sp1-related signaling pathway in human MPM.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cell Survival; Cyclin D1; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Membrane Potential, Mitochondrial; Mesothelioma; Mesothelioma, Malignant; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Pleural Neoplasms; Poly(ADP-ribose) Polymerases; Polyenes; Polyunsaturated Alkamides; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sp1 Transcription Factor; Survivin; Transcription Factor CHOP

Malignant mesothelioma (MM), which is associated with asbestos exposure, is one of the most deadly tumors in humans. Early MM is concealed in the serosal cavities and lacks specific clinical symptoms. For better treatment, early detection and prognostic markers are necessary. Recently, CD146 and insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) were reported as possible positive markers of MM to distinguish from reactive mesothelia in humans. However, their application on MM of different species and its impact on survival remain to be elucidated. To disclose the utility of these molecules as early detection and prognostic markers of MM, we injected chrysotile or crocidolite intraperitoneally to rats, thus obtaining 26 peritoneal MM and establishing 11 cell lines. We immunostained CD146 and IMP3 using paraffin-embedded tissues and cell blocks and found CD146 and IMP3 expression in 58% (15/26) and 65% (17/26) of MM, respectively, but not in reactive mesothelia. There was no significant difference in both immunostainings for overexpression among the three histological subtypes of MM and the expression of CD146 and IMP3 was proportionally associated. Furthermore, the overexpression of CD146 and/or IMP3 was proportionally correlated with shortened survival. These results suggest that CD146 and IMP3 are useful diagnostic and prognostic markers of MM.

Topics: Animals; Asbestos; Biomarkers, Tumor; CD146 Antigen; Cyclin D1; Female; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Insulin-Like Growth Factor Binding Protein 3; Ki-67 Antigen; Male; Mesothelioma; Peritoneal Neoplasms; Prognosis; Rats; Rats, Inbred BN; RNA, Messenger

New therapeutic approaches are still needed for effective malignant pleural mesothelioma treatment. The use of classical chemotherapy agents in combination with newly developed molecules may shed light on new therapeutic approaches. We aimed to determine the efficacy of panobinostat, alone and in combination with cisplatin, on cell survival and mRNA expression of FOXO3A, CCND1, and CASP9 genes in both mesothelioma and healthy mesothelial cell lines. Cells were treated with 1-100 µM cisplatin and 25-1000 nM panobinostat. Methylthiazol tetrazolium assays were performed to determine cell viability. mRNA expression levels of genes were analyzed with quantitative real-time polymerase chain reaction. Cisplatin and panobinostat exposure of the cells for 24 h resulted in decreased cell survival. The combined treatment was found to be more effective. No significant changes were observed with respect to CCND1 expression after exposure to agents alone or in combination. However, agents in combination resulted in upregulation of FOXO3A and CASP9 in MSTO-211H cells. Gene expression levels were not affected by any agents in healthy cells. Use of cisplatin in combination with new chemotherapeutic agents may reduce the toxic effects of cisplatin in normal cells and result in more effective removal of tumor cells.

Topics: Antineoplastic Agents; Caspase 9; Cell Line, Tumor; Cell Survival; Cisplatin; Cyclin D1; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Hydroxamic Acids; Indoles; Mesothelioma; Panobinostat; RNA, Messenger; Transcription, Genetic

Dietary phytochemicals as adjuvants have been suggested to play important roles in enhancing chemotherapeutic potential owing to multitargeted chemopreventive properties and lack of substantial toxicity. Here, we investigated the efficacy of the combined treatment of various phytochemicals with the anticancer drug clofarabine in malignant mesothelioma MSTO-211H cells and normal mesothelial MeT-5A cells. The combined treatment of resveratrol and clofarabine produced a synergistic antiproliferative effect in MSTO-211H cells, but not in MeT-5A cells. In MSTO-211H cells, the nuclear accumulation of Sp1 and the levels of p-Akt, Sp1, c-Met, cyclin D1, and p21 were effectively decreased by the combined treatment of them. In combination with clofarabine, the ability of resveratrol to reduce the contents of Sp1 and its target gene products was also evident in a time- and dose-dependent experiment. The inhibition of phosphoinositide 3-kinase using Ly294002 augmented a decrease in the p21 level induced by their combination, but it showed no significant effects on expression of Sp1 and cyclin D1. Taken together, the data provide evidence that the synergistic antiproliferative effect of resveratrol and clofarabine is linked to the inhibition of Akt and Sp1 activities, and suggest that this combination may have therapeutic value in treatment of malignant mesothelioma.

Topics: Adenine Nucleotides; Antineoplastic Combined Chemotherapy Protocols; Arabinonucleosides; Cell Line, Tumor; Cell Proliferation; Chromones; Clofarabine; Cyclin D1; Enzyme Inhibitors; Epithelial Cells; Humans; Mesothelioma; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Resveratrol; Sp1 Transcription Factor; Stilbenes

Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes for which there is currently no effective curative modality. Recent data suggest that hyperactivation of the tyrosine kinase SRC has a key role in MM development and therefore this kinase represents an important molecular target for MM therapy. We tested new pyrazolo[3,4-d]pyrimidine SRC inhibitors on a panel of MM cell lines expressing the active form of SRC. These SRC inhibitors exerted a significant proapoptotic effect on MM cells without affecting the normal mesothelial cell line MET-5A, supporting a possible use of these SRC inhibitors for a safe treatment of MM. We also showed that SRC inhibitor-induced apoptosis occurred concomitantly with an increase in the nuclear stability of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that loss of nuclear p27 expression is a well-established adverse prognostic factor in MM, and p27 nuclear localization is crucial for its tumor-suppressive function. Consistently, SRC inhibition seems to promote the increase in p27 nuclear level also by inactivating the AKT kinase and downregulating cyclin D1, which would otherwise delay p27 nuclear import and provoke its cytoplasmic accumulation. To determine whether p27 stabilization has a direct role in apoptosis induced by SRC inhibition, we stably silenced the CDKN1B gene, encoding p27, in MSTO-211H and REN mesothelioma cells by transduction with lentiviral vectors expressing short hairpin RNAs against the CDKN1B transcript. Strikingly, p27 silencing was able to suppress the apoptosis induced by these SRC inhibitors in both MM cell lines, suggesting that p27 has a crucial proapoptotic role in MM cells treated with SRC inhibitors. Our findings reveal a new mechanism, dependent on p27 nuclear stabilization, by which SRC inhibition can induce apoptosis in MM cells and provide a new rationale for the use of SRC inhibitors in MM therapy.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Enzyme Stability; Flow Cytometry; Humans; Mesothelioma; Molecular Structure; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; RNA Interference; src-Family Kinases

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2) -tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Mesothelioma; Nuclear Proteins; TEA Domain Transcription Factors; Transcription Factors; Transcriptome; Up-Regulation

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Humans; Inhibitor of Apoptosis Proteins; Membrane Proteins; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Pleural Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Resveratrol; RNA, Messenger; Sp1 Transcription Factor; Stilbenes; Survivin; Transplantation, Heterologous

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

Cyclin D1 overexpression exists in multiple types of cancer and is a potential chemopreventive or therapeutic target.. Non-small cell lung cancer and mesothelioma cells were incubated with antisense oligonucleotides (ASO) to cyclin D1 (CD1) and evaluated for effects on cellular proliferation, apoptosis, expression of cell cycle-specific proteins, and protein phosphorylation states.. ASO to CD1 inhibited proliferation of non-small lung cancer cells and mesothelioma cells. ASO induced apoptosis as determined by TUNEL assay. Western blot analysis of cell lysate showed that ASO inhibited the de novo synthesis of CD1, CD3, and CDK2 in multiple cell lines. Immunoprecipitation and immunoblotting with phosphoantibodies demonstrated that CD1, CD3, and CDK2 exist in a phosphorylated state.. The work demonstrates that non-small cell lung cancer and mesothelioma cells respond to ASO-mediated cellular growth inhibition. These findings make ASO to CD1 attractive as a potential therapeutic for mesothelioma and non-small cell lung cancer.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Humans; Immunoprecipitation; Lung Neoplasms; Mesothelioma; Oligonucleotides, Antisense; Phosphorylation; Tumor Cells, Cultured

Integrin signaling promotes, through p21-activated kinase, phosphorylation and inactivation of the tumor suppressor merlin, thus removing a block to mitogenesis in normal cells. However, the biochemical function of merlin and the effector pathways critical for the pathogenesis of malignant mesothelioma and other NF2-related malignancies are not known. We report that integrin-specific signaling promotes activation of mTORC1 and cap-dependent mRNA translation. Depletion of merlin rescues mTORC1 signaling in cells deprived of anchorage to a permissive extracellular matrix, suggesting that integrin signaling controls mTORC1 through inactivation of merlin. This signaling pathway controls translation of the cyclin D1 mRNA and, thereby, cell cycle progression. In addition, it promotes cell survival. Analysis of a panel of malignant mesothelioma cell lines reveals a strong correlation between loss of merlin and activation of mTORC1. Merlin-negative lines are sensitive to the growth-inhibitory effect of rapamycin, and the expression of recombinant merlin renders them partially resistant to rapamycin. Conversely, depletion of merlin restores rapamycin sensitivity in merlin-positive lines. These results indicate that integrin-mediated adhesion promotes mTORC1 signaling through the inactivation of merlin. Furthermore, they reveal that merlin-negative mesotheliomas display unregulated mTORC1 signaling and are sensitive to rapamycin, thus providing a preclinical rationale for prospective, biomarker-driven clinical studies of mTORC1 inhibitors in these tumors.

Topics: Antibiotics, Antineoplastic; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; G1 Phase; Humans; Immunoblotting; Integrins; Mechanistic Target of Rapamycin Complex 1; Mesothelioma; Multiprotein Complexes; Neurofibromin 2; Protein Biosynthesis; Proteins; RNA Caps; RNA Interference; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transfection

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.

Topics: Adenoviridae; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Silencing; Genetic Vectors; Humans; Mesothelioma; Mutation; Neurofibromin 2; Oligonucleotide Array Sequence Analysis; p21-Activated Kinases; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Transfection

The epidemiologic association between asbestos exposure and human malignant mesothelioma is well established. However, the molecular mechanisms linking asbestos exposure of humans and the subsequent mesothelioma formation is not well understood. The most frequent genetic changes found so far in human malignant mesothelioma (HMM) are deletions and point mutations in the tumor suppressor genes p16INK4a and NF2. Whereas homozygous deletions appear to be the predominant mechanism leading to p16/CDKN2A inactivation, inactivating point mutations coupled with allelic loss mainly occur at the NF2 locus. In the present study, asbestos-treated human mesothelial cells (HMC), SV40-transformed human mesothelial cells (MeT-5A) and a human mesothelioma cell line (COLO) were investigated for genetic changes of cell cycle genes (cyclin D1, p16INK4a, RB1, CDK2) using multicolor fluorescence in situ hybridization (mFISH) in interphase cells. The results show that cyclin D1 is unaffected in all investigated cells. The p16INK4a gene locus was shown to be mutated in COLO cells but not in HMC. After labeling of CDK2 and RB1, hemizygous loss of one allele of each gene was observed in asbestos-treated HMC whereas gene amplification of these genes was detectable in MeT-5A and COLO cells. Our data indicate that disarrangement of the RB1 dependent pathway seems to be involved in mesothelioma formation.

Topics: Asbestos; Cell Culture Techniques; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Epithelium; Genes, Tumor Suppressor; Humans; In Situ Hybridization, Fluorescence; Mesothelioma; Nucleic Acid Hybridization; Point Mutation; Sequence Deletion; Simian virus 40; Tumor Cells, Cultured