MPM Cell Research Articles

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. Materials and methodsMicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. ResultsExpression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. ConclusionsThe miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

S127 Chemotherapy sensitises Malignant pleural mesothelioma cells to undergo MSC-TRAIL induced apoptosis

IntroductionMalignant pleural mesothelioma (MPM) is a highly aggressive, incurable, chemoresistant tumour. Recent studies have shown that Mesenchymal stem cells (MSC) can home to and incorporate into the tumour stroma. Their...

Abstract C262: Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with the standard of care chemotherapy is only 12 months from diagnosis. Thus, new therapeutic modalities are urgently needed to improve the prognosis of MPM patients. ∼50% of MPM patients exhibit biallelic inactivation of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Merlin inactivation is a critical step in MPM tumorigenesis and is related, at least in part, to up-regulation of focal adhesion kinase (FAK) activity. FAK has also been demonstrated to play a role in maintenance of cancer stem cells (CSCs), a subpopulation of tumor cells associated with drug resistance and tumor recurrence. Therefore, therapeutic targeting of FAK using defactinib (VS-6063), a potent, orally active FAK kinase inhibitor, may benefit cancer patients through targeting of both CSCs and bulk tumor cells. Among a panel of MPM cell lines in 3-dimensional Matrigel culture, MPM lines lacking expression of merlin were found to be especially sensitive to defactinib. In contrast, MPM cell lines with wild-type merlin expression were less sensitive with average EC50 values of 5.1 μM. Oral dosing of defactinib induced significant tumor growth inhibition in a merlin-negative MPM model pre-implanted in the lungs of mice. Using Aldefluor activity as a marker for CSCs in MPM, we find that chemotherapeutic agents enriched for Aldefluor-positive CSCs, while the FAK inhibitor defactinib reduced the proportion of CSCs. When used in combination with pemetrexed or cisplatin, defactinib blocked the enrichment of CSCs cells by these chemotherapeutic agents. Disruption of cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies allowed us to propose a molecular model to account for the observed merlin-FAK inhibitor synthetic lethal relationship. We find that weak cell-cell adhesions in merlin-negative MPM cells lead to a greater dependence of tumor cell survival and proliferation on cell-ECM-induced FAK signaling rendering merlin-negative cells especially vulnerable to FAK inhibitor treatment. Taken together, these data support the clinical development of defactinib in an ongoing Phase 2 registration-directed clinical trial evaluating the activity of defactinib in MPM patients (stratified by merlin status) with a confirmed response (PR or SD) following first line platinum/pemetrexed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C262. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Yuwaraj Kadariya, Christian M. Vidal, Quentin G. Wright, Jennifer E. Ring, Craig Menges, Joseph R. Testa, Qunli Xu, Jonathan A. Pachter. Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C262.

Serum HMGB1 as a prognostic marker for malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive malignant tumor of mesothelial origin that shows a limited response to conventional chemotherapy and radiotherapy. Therefore, diagnosing MPM early is very important. Some researchers have previously reported that high-mobility group box 1 (HMGB1) was correlated with pulmonary fibrosis. MPM involves the malignant transformation of mesothelial cells, which originate from mesenchymal cells similar to lung fibroblasts. Here, we investigated serum levels of HMGB1 in patients with MPM and compared them with those of a population that had been exposed to asbestos without developing MPM.MethodsHMGB1 production from MPM cell lines was measured using ELISA. Serum HMGB1 levels were also examined in 61 MPM patients and 45 individuals with benign asbestos-related diseases.ResultsHMGB1 concentrations of 2 out of 4 MPM cell lines were higher than that of normal mesothelial cell line, Met-5A. We demonstrated that patients with MPM had significantly higher serum levels of HMGB1 than the population who had been exposed to asbestos but had not developed MPM. The difference in overall survival between groups with serum HMGB1 levels that were lower and higher than assumed cut-off values was significant.ConclusionsOur data suggest that serum HMGB1 concentration is a useful prognostic factor for MPM.

Abstract 924: FAK inhibition preferentially attenuates growth of Merlin-negative malignant mesotheliomas: role of cancer stem cells.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often caused by asbestos exposure. MPM patients are usually diagnosed at an advanced stage of the disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as pemetrexed are relatively ineffective in increasing median survival time for MPM patients. New therapeutic modalities are urgently needed for MPM patients. Neurofibromatosis 2 (NF2) is a tumor suppressor gene that encodes the protein Merlin. Biallelic inactivation of NF2 by mutation and/or deletion occurs in ∼40% of MPMs leading to inactive or absent Merlin. Merlin has been demonstrated to play roles in cell adhesion, invasion and cell motility in tumor cell lines partially through regulation of focal adhesion kinase (FAK) which in turn mediates signal transduction by integrins and growth factor receptors. Increased activation of FAK has been demonstrated in NF2-mutated mesothelioma cells, indicating that FAK may represent an important therapeutic target for MPM. A potent and selective FAK inhibitor VS-4718 was evaluated in a panel of MPM cell lines with wild-type or mutated NF2. Mutant NF2 MPM cells were found to be especially sensitive to the FAK inhibitor VS-4718 in a 3D Matrigel assay with EC50 values below 100 nM, in contrast to wild type NF2 MPM cell lines which were less sensitive with EC50 values above 1 μM. Decrease in cell growth in response to FAK inhibitor treatment was found to result from reduction of cell proliferation and induction of apoptosis. Ectopic expression of a non-phosphorylatable mutant form of NF2 (NF2-S518A) in NF2 mutant MPM cells abolished the enhanced sensitivity to VS-4718 in both proliferation and apoptosis assays, confirming the hypothesis that Merlin loss confers hypersensitivity to FAK inhibition. In addition, VS-4718 inhibited tumor cell growth in an orthotopic Merlin-negative mesothelioma xenograft model in a dose-dependent manner with corresponding inhibition of tumor FAK autophosphorylation. It has been reported that cancer stem cells (CSCs) are enriched in pemetrexed-resistant mesothelioma. Using Aldefluor activity as a measure of CSCs, we have found that the FAK inhibitor VS-4718 preferentially reduced the percentage of CSCs in Merlin-negative MPM in contrast to the standard-of-care agent pemetrexed which increased the percentage of CSCs. In summary, our results indicate that the FAK inhibitor VS-4718 is especially potent in Merlin-negative MPM tumor cells, and that NF2 status may be a valuable stratification marker for response to FAK inhibition. Furthermore, cancer stem cells in Merlin-negative mesotheliomas appear to be particularly resistant to pemetrexed, but sensitive to VS-4718. These data support the clinical development of a selective FAK inhibitor for treatment of Merlin-negative malignant mesothelioma. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Christian M. Vidal, Jennifer E. Ring, Mitchell Keegan, Qunli Xu, Craig W. Menges, Joseph R. Testa, Jonathan A. Pachter. FAK inhibition preferentially attenuates growth of Merlin-negative malignant mesotheliomas: role of cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2013-924

Abstract 2795: Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma.

Abstract Malignant pleural mesothelioma (MPM) is a poor prognostic malignancy that is distinctly resistant to the conventional therapies. Cell lines are useful materials for studying the biological characteristics of the tumors, thus the establishment of MPM cell lines is valuable for exploring the new therapeutic strategies of MPM. We established 4 MPM cell lines (YUMC8, YUMC44, YUMC63 and YUMC64) from Japanese patients, consisting of 2 epithelioid and 2 sarcomatoid types for histological subtype. We analyzed the status of methylation, mutation, copy number gains (CNGs), and protein expression in representative genes and the sensitivity to several drugs in these 4 cell lines. We detected the complete methylation of P16 in 3 cell lines, in which the protein expression of p16 was lost. Methylation of RASSF1A was observed in 3 of 4 cell lines. Copy number gains (CNGs) in EGFR, HER2 and MET were not detected in all the cell lines. Mutations in various genes including EGFR, KRAS, HER2, BRAF, and PIK3CA, which are frequently detected in non-small cell lung cancer, were not detected in all 4 cell lines. As for microRNA, miR-34b/c was completely methylated in 2 of 4 MPM established cell lines. About cell adhesion molecules, E-cadherin expression was detected in two epithelioid type MPM cell lines, whereas N-cadherin expression was detected in all four established cell lines by Western blot analysis. Vimentin was strongly expressed in two sarcomatoid type MPM cell lines. All established MPM cell lines showed no dramatic responses to the drugs tested including NVP-AUY922, 17-DMAG, Trichostatin A and Vorinostat. Although new molecular findings were not observed in the process of the current characterization of these new MPM cell lines, they seem to be useful for the future extensive analyses of biological behavior of MPM, so as to the development of new therapeutic strategies. Citation Format: Ryuhei Tada, Hiromasa Yamamoto, Masashi Furukawa, Kazuhiko Shien, Shinsuke Hashida, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichi Toyooka, Shinichiro Miyoshi. Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2795. doi:10.1158/1538-7445.AM2013-2795

Anti-Tumour and Chemosensitising Effect of a Combination of Bromelain + N-Acetyl Cysteine with Cisplatin or 5-Fu on Malignant Peritoneal Mesothelioma Cells

normally poor however, cytoreductive surgery and hyperthermic intraperitoneal chemotherapy has increased survival in some patients. Hence, new therapies are needed. MUC1 is a glycosylation dependant protein associated with tumour invasiveness, metastasis and chemo resistance. Bromelain, a cysteine proteinase, hydrolyses glycosidic bonds, whilst N-Acetyl cysteine reduces disulphide bonds in glycoprotein. Hence, we investigated the anti-tumour effect of these agents in MUC 1 expressing MPM cell lines. Materials and Methods: The cell lines were treated to various concentrations of bromelain, NAC and combinations of NAC + bromelain, NAC + bromelain + cisplatin or 5-FU. Their cell viabilities were assessed at 48 hours with sulfhordamine B assay. Finally, with western blotting, the effect of NAC and the combination of NAC + bromelain on cellular survival proteins were investigated. Results: Combination of NAC (10 mM) with bromelain (75 ug/ml) showed 97% and 88% cell proliferation inhibition in YOU and PET cells, respectively. In triple combination, the addition of cisplatin to only 5.0 mM NAC and bromelain increased cytotoxicity in YOU cells but absent at 10.0 mM NAC concentration. However, in PET cells, triple combinations with cisplatin had no effect. The addition of 5-FU in triple combinations showed an increase in cytotoxicity with 5.0 mM NAC and bromelain in YOU cells. No increase in cytotoxicity was seen with addition of 10 mM NAC. In PET cells, the addition 5-FU to 5.0 mM NAC + 50 ug/ml bromelain, enhanced cytotoxicity significantly but was absent at all other combinations. Conclusion: The combination of bromelain and NAC may be developed as anti-tumour agents for treating MPM, with a possible role in combined therapy with current chemotherapeutic agents.

Abstract 3404: Exploring the role of Twist1 in the pathogenesis of malignant pleural mesothelioma (MPM)

Abstract Background: MPM is caused by lethal neoplastic growth of the pleura surrounding lungs. It is resistance to most standard anti-cancer treatment regimens and needs discovery of newer therapeutic approaches. MPM is characterized by massive loco-regional invasion of the malignant pleural cells into the lung parenchyma. Twist1 is a transcription factor, which promotes invasion and metastasis of tumor cells, increases chemotherapeutic resistance and is involved in the pathobiology of many cancers. Also recent studies have highlighted the potential of twist1 as a therapeutic target in cancer. But there is no report investigating its function in mesothelioma. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Bioinformatic analysis of the microarray data using a 2 sample t-test was applied, on a probe-by-probe basis followed by Beta-uniform Mixture for multiple comparisons, to determine the differences between tumor vs normal specimens. The microarray results were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using Taqman assays on the ABI 7300 platform. For all qRT-PCR experiments the twist1 transcript levels were determined relative to endogenous GAPDH as control using ΔΔCT calculation. We performed Western blot analysis on a panel of 16 mesothelioma cell lines including Met-5A (SV-40 immortalized) and HCT-4012 (telomerase immortalized) pleural mesothelial control cell lines. Results: The bioinformatic analysis of microarray data showed that twist1 transcript level was 8.7 fold higher in tumors (p = 1.1E-16) compared to paired normal specimens. Using qRT-PCR, we compared twist1 transcript levels in 12 pairs of tumor vs paired normal tissue specimens and found that twist1 was upregulated to more than 10 fold in MPM tumors (p < E-4). Western blot showed that 10 MPM cancer cell lines had higher expression of twist1 protein compared to Met-5A and HCT-4012 cell lines. The highest expression was seen in 2 of the sarcomatoid cell lines - RS5 and DM3, suggesting a correlation with metastatic phenotype since sarcomatoid tumors are highly metastatic in nature. Conclusion: Our preliminary findings suggest that twist1 is upregulated in MPM tumors and cell lines and may play a role in the development of MPM. Further studies are needed to investigate its role in the process of tumorigenesis and metastasis. Supported by Grants: DoD W81XWH-07-1-0306 (I.I.W and AST), Fleming Foundation, IASLC Young Investigator Award 2011-2013 (MS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3404. doi:1538-7445.AM2012-3404

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.

Abstract 3550: Curcumin suppresses growth of human mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis

Abstract Malignant Pleural Mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis regulatory pathways are frequently altered in MPM and thus remain attractive targets for therapeutic intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. Here we investigated the biological and molecular responses of MPM cells to Curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of Curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose and time dependent manner while pretreatment of MPM cells with Curcumin enhanced Cisplatin efficacy. Curcumin activated stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, Curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of Curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, Curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in development of effective therapeutic treatment options against resistant MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3550. doi:10.1158/1538-7445.AM2011-3550

Abstract 702: RON receptor tyrosine kinase: A potential therapeutic target in malignant pleural mesothelioma

Abstract BACKGROUND: Expression of receptor tyrosine kinases (RTK) and their cognate ligands has been described in mesothelioma, leading to the hypothesis that autocrine growth pathways could be therapeutic targets of tyrosine kinase inhibitors (TKIs). METHODS: Phospho-RTK arrays were used to evaluate phosphorylation of 42 RTKs in 4 mesothelioma cell lines and 11 mesothelioma surgical specimens. Western Blot analysis, IHC, cellular migration and invasion assays were performed. RESULTS: Tumours contained 14 phospho-RTKs. RON emerged as a frequently identifed kinase which has not been previously reported in mesothelioma. An extended panel of sixteen mesothelioma tumour and five benign pleural tissue samples were characterised by western blot analysis. The presence of RON message was confirmed by RT-PCR with specific primers in mesothelioma cell lines Western blot analysis detected RON in 16 tumours and 4 cell lines but not in the SV-40 transformed normal mesothelial MET-5A cells. Mesothelioma expressed different isoforms of RON compared to benign pleural plaques: both benign pleural plaques and mesotheliomas expressed the shortform of RON (sf-RON), whereas, the larger RON variants, Δ160 and Δ165, were seen in mesothelioma samples. IHC was subsequently carried out on a large series of FFPE samples (n>400), with clinical followup to determine if RON expression correlates with prognosis or response to therapy. Scratch assays using antibodies directed against RON prevented MPM cell line migration CONCLUSIONS: Mesothelioma contains multiple activated RTKs including RON. Previously, RON was shown to mediate epithelial mesenchymal transition (EMT). This may be significant given the epithelioid to sarcomatoid spectrum of mesothelioma. Expression of Δ160 and Δ165 was previously reported in gastric and colorectal cancers and may be associated with oncogenesis. RON has been identified as a therapeutic target in pancreatic cancer and this may also apply in mesothelioma. Functional studies on targeting RON in mesothelioma are ongoing. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 702.

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.