MPM Cell Lines Research Articles

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

IntroductionMalignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. MethodsThe mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. ResultsRNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. ConclusionsThese results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.

Abstract B139: Arginine deprivation with pegylated arginine deiminase regulates key metabolic genes in RNA and DNA synthesis in ASS1-deficient malignant mesothelioma cells: Clinical implications.

Abstract Tumors deficient in argininosuccinate synthetase-1 (ASS1), a key enzyme involved in arginine synthesis, are auxotrophic for arginine and sensitive to amino acid deprivation. Currently, several trials are testing the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20) in patients with cancer, including a randomised phase II multicenter UK study in patients with ASS1-deficient mesothelioma (NCT01279967). Here, we sought to identify key pathways involved in the mechanism of action of ADI-PEG20 using a panel of ASS1-deficient malignant mesothelioma (MPM) cell lines as our model. Epithelioid (2591), biphasic (MSTO) and sarcomatoid (JU77) mesothelioma cells were exposed to ADI-PEG20 and analysed using Affymetrix gene expression profiling with validation of candidate genes by qPCR and western blotting, and metabolic studies by LC-MS and 1H-NMR. ADI-PEG20 modulated several metabolic genes involved in nucleotide synthesis in the MPM cell lines. We identified suppression of ribonucleotide reductase (M1 and M2 isoforms), thymidylate synthase and dihydrofolate reductase by 24hrs, the latter enzymes being known targets of pemetrexed, a multitargeted antifolate used in the first-line therapy of mesothelioma. Moreover, metabolic profiling revealed upregulation of several modified nucleosides, including ribothymidine and downregulation of the nucleotide, guanosine monophosphate. Lastly, in vitro studies confirmed potentiation between ADI-PEG20 and pemetrexed and sequential therapy led to reduced tumor growth in vivo. In conclusion, arginine depletion modulates several key enzymes involved in nucleotide synthesis, providing a rationale for combining ADI-PEG20 with pemetrexed-containing regimens in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B139.

Abstract C37: BMAL1 depletion represses growth of mesothelioma through induction of mitotic catastrophe

Abstract Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the pleura. Asbestos exposure is considered the most important carcinogenic factor that has been mentioned in relation to MPM. Although the incidence of MPM continues to increase, it remains resistant to the currently available therapeutic modalities. Thus, identification of more efficient therapeutics based on its molecular abnormalities is highly recommended. Recently, a genome-wide expression analysis showed deregulation of the circadian rhythm pathway in MPM, including overexpression of BMAL1, a central player in the circadian clock machinery. In this study we explored the consequences of targeting BMAL1 in MPM. This was accomplished by genetic knockdown of BMAL1 with small interference RNA (siRNA) and short hairpin lentiviral expression system. The majority of MPM cell lines expressed higher levels of BMAL1 than the nontumorigenic mesothelial cell line (Met-5A). Immunohistochemical analysis revealed that BMAL1 is constitutively expressed in the cytoplasm with granular pattern in a subset of clinical MPM specimens. Long-term depletion of BMAL1 induced senescence and downregulation of IL-8 and BcL-2 in ACC-MESO-1-cells. Notably, RNAi-mediated knockdown of BMAL1 resulted in suppression of cell growth and induction of apoptosis in MPM cell lines with limited consequences in Met5A. Moreover, ACC-MESO-1 cells, which expressed the highest level of BMAL1, underwent drastic morphological changes including micronucleation, multiple nuclei, and increased cellular volume. Consistently, cell cycle profiling revealed substantial increase in apoptotic and polyploidy cells with concomitant decrease in Wee1, cyclin B, p21, and increase in cleaved caspase 3 and cyclin E proteins by Western blot analysis. Taken together, these results suggest that mitotic catastrophe could be the main cause of cells death in ACC-MESO-1 cells after BMAL1 knockdown. BMAL1 plays an important role in MPM and serves as an attractive therapeutic target for MPM with high therapeutic index. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C37.

Targeting Wnt-2 in Mesothelioma and Lung Cancer

Malignant mesothelioma of the pleura (MPM) and lung cancer are highly aggressive and challenging carcinomas, often being diagnosed at late stages, with poor prognosis and limited treatment options. A greater understanding of the pathogenesis of these diseases is essential to developing alternative therapeutic strategies. Wnt pathway activation in MPM was recently demonstrated in our laboratory with the observation of disheveled protein overexpression. To extend our knowledge of Wnt signaling activation in MPM, we performed Wnt-specific microarrays in normal pleura and MPM. We found that the most common event in MPM was upregulation of Wnt-2. This was confirmed by analysis of MPM cell lines and tissues. Similarly, a screen of 50 freshly resected lung cancer tumor samples with their corresponding matched normal lung controls demonstrated Wnt-2 upregulation in > 70% of tumor samples. Inhibition of Wnt-2 expression by Wnt-2—specific small interfering RNA (siRNA) induced programmed cell death in MPM cells and human non—small-cell lung cancer (NSCLC) cell lines that overexpress Wnt-2 protein. Wnt-2 signaling blockade by the Wnt-2—specific siRNA was confirmed by downregulation of cytosolic β-catenin and reduction in Tcf-dependent transcriptional activity (TOPFLASH assay) in NSCLC cell lines. Additionally, we assessed the antiproliferative effects of the Wnt-2 inhibitor and pemetrexed, one of the current standard treatments of MPM. We found that, although inhibiton by itself had less antiproliferative potency than pemetrexed, the 2 in combination had substantially more activity than pemetrexed alone. Hence, the studies presented here demonstrate the significance of Wnt pathway activation in the propagation of these cancers and that inhibition of Wnt-2 is of therapeutic interest in the development of more effective treatments for patients with MPM and lung cancer.

Phosphorylation and nuclear localisation of Akt is induced by hypoxia and associated with a poor prognosis in malignant pleural mesothelioma

22034 Background: The phosphatidylinositol-3 kinase (PI3K)/Akt (PKB) pathway is activated in many solid tumours, and plays a central role in cell survival. Recent evidence indicates that hypoxia induces upregulation and activation of the PI3K/Akt pathway. Once activated, Akt phosphorylates a variety of downstream substrates, including the pro-apoptotic transcription factor FOXO3a, facilitating cell survival by blocking apoptosis. This study examined the interaction between phosphorylated Akt (pAkt) and hypoxia in mesothelioma cell lines and patient tumour samples. Methods: Western blot analysis was used to examine the effect of hypoxia on Akt and pAkt expression in 4 MPM cell lines (JU77; ONE58; CRL5915; CRL5820) in the presence or absence of the PI3K inhibitor, LY294002. High-content screening (HCS) analyses, using the Incell 1000 analyser, was used to quantify changes in subcellular localisation of pAkt and FOXO3a in the cells. FACs and HCS were used to quantify levels of apoptosis. Tumour samples from 187 patients with MPM were stained using pAkt and carbonic anhydrase (CA)-IX (a surrogate marker of hypoxia) specific antibodies. Results: Hypoxia induced the activation of Akt in the MPM cell lines with an increase in nuclear pAkt expression. This correlated with a reduction in nuclear FOXO3a expression. An increase in the level of apoptosis was seen under hypoxic conditions when Akt phosphorylation was blocked by LY294002. This correlated with an increase in nuclear FOXO3a. A positive association between pAkt and CA-IX immunostaining was observed in the tumour samples, supporting the in vitro findings that intra-tumoural hypoxia is a stimulus for Akt phosphorylation. On multivariate analysis increased expression of nuclear pAkt, sarcomatoid histology, weight loss >5% and increasing CALGB score emerged as independent prognostic variables predicting a poor survival. Conclusions: The in vitro and tumour sample work demonstrates that hypoxia induces activation of Akt in MPM. The association between increased nuclear pAkt and reduced nuclear FOXO3a, reversed by LY294002, indicates that pAkt plays a central role in the inhibiton of apoptosis under hypoxic conditions and therefore may represent a novel therapeutic target in MPM. No significant financial relationships to disclose.

Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion

Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non-receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy.

Characterization of human malignant mesothelioma cell lines orthotopically implanted in the pleural cavity of immunodeficient mice for their ability to grow and form metastasis

BackgroundMalignant pleural mesothelioma (MPM) is a tumor known to be resistant to conventional therapies. Thus, an in vivo model can represent an important tool for assessing the efficacy of novel approaches in the treatment of MPM.Presently, human MPM cells have been grown orthotopically in mice upon transplantation of tumor masses or tumor cell suspensions following surgery. In these models however, surgery can interfere with the tumor growth and the early stages of tumor development cannot be easily explored. Finally, results may not be so accurate due to implantation of potentially different tumor samples in different experimental groups.Our work aimed at establishing a nude mouse model xenotransplanted with human MPM cell lines in which tumor progression exhibits some features of the human disease.MethodsThree distinct human MPM cell lines previously established from MPM patients displaying two different phenotypes, biphasic (MM-B1 and IST-Mes3) and epithelioid (IST-Mes2), were directly injected into the pleural cavity of nude mice. At different times, mice were sacrificed for autopsy, tumor nodules were counted and then removed for histology. Presence of metastases in visceral organs was also monitored.ResultsIST-Mes2 cells were unable to grow in nude mice. MM-B1 and IST-Mes3 cells were capable of growing in nude mice and formed tumor nodules in the pleura. Post-mortem examination showed that MPM cells progressively colonized the parietal and visceral pleura, the diaphragm, the mediastinum and, lastly the lung parenchyma. No pneumo-thorax was evidenced in the mice. Pleural effusions as well as lymph node metastases were observed only at later times.ConclusionThis model mimics the progression of human malignant mesothelioma and it is easy to perform and reproducible; therefore it can be useful to study human MPM biology and evaluate the efficacy of novel therapies.

061 Wnt2 as a new therapeutic target in malignant pleural mesothelioma

Malignant mesothelioma of the pleura (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treat-ment options. A better understanding of its pathogenesis is essential to developing alternative therapeutic stratégies. We previously demonstrated that the Wnt signaling pathway is activated in MPM through the overexpression of dishevelled proteins. To extend our knowledge of Wnt signaling activation in MPM, we performed Wnt spécifie microarrays in normal pleura and MPM. We inhibited Wnt2 by siRNA and a monoclonal anti-Wnt2 antibody and analyzed their effects on apoptosis and downs-tream signaling effectors. We then assessed the anti-proliferative effects of the Wnt2 antibody and Alimta ® , one of the current standard treatments of MPM. We found that the most common event in MPM was the upregulation of Wnt2. We confirmed Wnt2 overexpression at the mRNA and protein level in MPM cell lines and tissues. We then demonstrated that inhibition of Wnt2 by siRNA or a monoclonal antibody induces programmed-cell death in MPM cells. We next analyzed the effects of the anti-Wnt2 antibody and of Alimta on MPM cell prolifération. We found that although Wnt2 antibody by itself had less anti-proliferative potency than Alimta ® , the two in combination had substantially more activity than Alimta ® alone. We thus propose that inhibition of Wnt2 is of the rapeutic interest in the development of more effective treatments for MPM.

Wnt2 as a new therapeutic target in malignant pleural mesothelioma

Malignant mesothelioma of the pleura (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treatment options. A better understanding of its pathogenesis is essential to developing alternative therapeutic strategies. We previously demonstrated that the Wnt signaling pathway is activated in MPM through the overexpression of disheveled proteins. To extend our knowledge of Wnt signaling activation in MPM, we performed Wnt-specific microarrays in normal pleura and MPM. We found that the most common event in MPM was the upregulation of Wnt2. We inhibited Wnt2 by siRNA and a monoclonal anti-Wnt2 antibody and analyzed their effects on apoptosis and downstream signaling effectors. We then assessed the antiproliferative effects of the Wnt2 antibody and Alimta, one of the current standard treatments of MPM. We confirmed Wnt2 overexpression at the mRNA and protein level in MPM cell lines and tissues. We then demonstrated that inhibition of Wnt2 by siRNA or a monoclonal antibody induces programmed cell death in MPM cells. We next analyzed the effects of the anti-Wnt2 antibody and of Alimta on MPM cell proliferation. We found that although Wnt2 antibody by itself had less antiproliferative potency than Alimta, the two in combination had substantially more activity than Alimta alone. We thus propose that inhibition of Wnt2 is of therapeutic interest in the development of more effective treatments for MPM.

629. VEGF Promoter-Based Conditionally Replicative Adenoviruses Are Useful for the Treatment of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma, MPM is an asbestos-related malignancy characterized by rapidly progressive and diffusely local growth, late metastases, and poor prognosis. Chemotherapy or radiation therapy, alone or in combination, is not effective, and the majority of patients with MPM die within 2 years regardless of treatment. Therefore, new therapeutic approaches are needed. The tumor formation of malingnant mesothelioma depends on angiogenesis in which the VEGF produced by cancer cells plays a pivotal role. In our previous work, we developed a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter, AdVEGFE1. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anti-cancer effect in vitro and in vivo. Since many MPM tumors are known to produce VEGF, VEGF promoter based CRAd has a potential to suppress a VEGF expressing MPM tumor growth. Six MPM cell lines tested showed the significant VEGF mRNA and protein expression. The cancer cell killing effect by AdVEGFE1 was roughly correlated with the extent of VEGF mRNA expression and the results of luciferase assay using AdVEGFLuci. We also investigated the in vivo anticancer effect of AdVEGFE1 to MPM tumor established in the pleural space of the nude mice. In this experiment, only two MPM cell lines were selected by the tumor forming ability after the screening of all six MPM cell lines. When these intact MPM cells were pre-mixed with infected cells by AdVEGFE1 at the ratio of 10%, tumor formation was suppressed significantly. A direct injection of AdVEGFE1 to pleural space of tumor-bearing nude mice at day 15 improved the survival time, and furthermore, repeat injections at day 15, 22, and 29 showed better results. These results suggest the VEGF promoter based CRAd is the promising strategy for the MPM tumors. We are now investigating the viral replication in MPM in vivo.