Malignant Pleural Mesothelioma Cell Lines Research Articles

Schedule-Dependent Synergistic Effect of Pemetrexed Combined with Gemcitabine against Malignant Pleural Mesothelioma and Non-Small Cell Lung Cancer Cell Lines

Background: Pemetrexed, a multi-targeted antifolate (MTA), is a promising agent in the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC). With the aim of finding an optimal schedule for the combination therapy of MTA and gemcitabine (GEM), we investigated their interaction against an MPM cell line, 211H, and the NSCLC cell lines A549 and H1299. Methods: Combination index analysis was used in 3 different schedules. Cell cycle analysis by flow cytometry and real-time RT-PCR analysis of thymidylate synthase (TS), folylpolyglutamate synthetase (FPGS) and reduced folate carrier 1 (RFC1) genes were performed to understand the biological consequences of their interaction. Results: MTA showed potent cytotoxicity against 211H cells (IC₅₀, 67 nM for 48 h exposure), compared to NSCLC cell lines. Significantly higher expression of FPGS and RFC1 mRNAs in 211H cells were associated with MTA sensitivity. Simultaneous exposure of MTA and GEM was antagonistic in all cell lines tested. Strong synergism was observed in 211H cells when MTA preceded GEM, but the inverted sequence showed antagonism. Similar results were exhibited in H1299 cells, whereas a moderately synergistic effect was observed in A549 cells when GEM preceded MTA. S phase accumulation by MTA treatment partly supported these results. Conclusion: Sequential administration of MTA and GEM is active, and the schedule of MTA followed by GEM is recommended for treating MPM.

Measles Virus Induces Oncolysis of Mesothelioma Cells and Allows Dendritic Cells to Cross-Prime Tumor-Specific CD8 Response

Despite conventional medical and surgical treatments, malignant pleural mesothelioma (MPM) remains incurable. Oncovirotherapy (i.e., the use of replication-competent virus for cancer treatment) is currently explored in clinical trials. In this study, we investigated the antineoplastic potential of a new oncolytic viral agent, a live-attenuated measles virus (MV) strain derived from the Edmonston vaccine lineage (Schwarz strain). We evaluated both oncolytic activity and immunoadjuvant properties of the MV vaccine strain on mesothelioma tumor cells. Infectivity, syncytium formation, and cytolytic activity of MV were studied on a panel of mesothelioma cells derived from pleural effusions of MPM patients. We observed that MV infected preferentially MPM cell lines in comparison with nontransformed mesothelial cells, leading to an efficient killing of a significant fraction of tumor cells. A cytoreductive activity was also evidenced through formation of multinuclear cellular aggregates (syncytia). The susceptibility of MPM cell lines to measles infection was assessed by the analysis of cell surface expression of the MV vaccine receptor (CD46). We also evaluated whether MV infection of mesothelioma cells could elicit an autologous antitumor immune response. We showed that MV Schwarz strain induced apoptotic cell death of infected mesothelioma cells, which were efficiently phagocytosed by dendritic cells (DC). Loading of DCs with MV-infected MPM cells induced DC spontaneous maturation, as evidenced by the increased expression of MHC and costimulatory molecules along with the production of proinflammatory cytokines. Priming of autologous T cells by DCs loaded with MV-infected MPM cells led to a significant proliferation of tumor-specific CD8 T cells. Altogether, these data strongly support the potential of oncolytic MV as an efficient therapeutic agent for mesothelioma cancer.

Expression of cell adhesion molecule 1 in malignant pleural mesothelioma as a cause of efficient adhesion and growth on mesothelium

Cell adhesion molecule 1 (CADM1), formerly referred to as SgIGSF, TSLC1, or Necl-2, has been characterized as a mast-cell adhesion molecule that mediates efficient interactions with mesothelial cells. Here, we examined whether CADM1 might be involved in the diffuse tumor growth over the pleural surface that characterizes malignant pleural mesothelioma (MPM). Immunohistochemical and western blot analyses revealed that 14 (25%) of 57 MPMs expressed the full-length form of CADM1 on the cell membrane, but non-neoplastic mesothelial cells did not express it at all. The majority of available MPM cell lines also expressed the full-length form of CADM1. We compared CADM1-positive and -negative MPM cells in culture within soft agar and in coculture on mesothelial or fibroblastic monolayers. Within soft agar, CADM1-negative MPM cells were capable of forming colonies, whereas CADM1-positive cells were not, suggesting that CADM1 is a potential tumor suppressor of MPM, consistent with the past characterization of this molecule in other types of tumors. However, in coculture on mesothelial cell monolayers lacking full-length CADM1, CADM1-positive MPM cells spread more widely and grew more quickly, whereas the CADM1-negative cells piled up. Transfection of the CADM1-negative cells with CADM1 cDNA caused them to behave like the CADM1-positive cells, with faster, more widespread growth. These phenotypic differences were not detectable in cocultures on lung fibroblastic monolayers, in which all MPM cells grew much more slowly than on mesothelial cells, irrespective of CADM1 positivity. CADM1 thus appears to mediate efficient adhesion and growth of MPM cells specifically on mesothelial cells, probably via trans-heterophilic binding, and thus may be involved in the manifestation of a considerable subset of MPMs as diffusely growing tumors disseminated over the pleural surface.

Bcl‐xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts

Bcl-xL functions as a dominant regulator of apoptotic cell death and is implicated in chemotherapeutic resistance of malignant pleural mesothelioma (MPM). Mesothelioma cell lines demonstrate increasing levels of Bcl-xL as resistant clones are selected in vitro. Moreover, upon introduction of antisense oligonucleotides specific to Bcl-xL mRNA, MPM cells are sensitized to chemotherapeutic agents. Here we describe the therapeutic effects of a novel combination therapy, Bcl-xL antisense oligonucleotide (ASO 15999) and cisplatin, on mesothelioma cell lines in vitro and in vivo; in addition, efficacy of ASO 15999 in decreasing tumor load as well as its effect on survival in an animal model. Finally, we initiated preliminary toxicity studies involved with intraperitoneal (IP) injections of ASO 15999 into mice. This novel combination, with doses of cisplatin four times below established IC(50) levels, significantly decreased viability of MPM cell lines after 48 hr. The growth of established mouse flank human tumor xenografts was reduced with intra-tumor administration of ASO 15999. Local spread and development of IP xenografts was reduced with treatments of ASO alone, and survival of mice afflicted with these xenografts was prolonged after administration of ASO alone and ASO 15999 + cisplatin combination therapy. These findings suggest that ASO 15999 sensitizes MPM cell lines to the toxic effects of cisplatin. ASO 15999 induced reduction of Bcl-xL is effective in slowing the progression of human mesothelioma cell lines both in vitro and in vivo. More notably, the combination of Bcl-xL ASO and cisplatin extends survival in an orthotopic tumor xenograft model.

Novel dual targeting strategy with vandetanib induces tumor cell apoptosis and inhibits angiogenesis in malignant pleural mesothelioma cells expressing RET oncogenic rearrangement

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a poor prognosis, therefore development of novel effective therapies is urgent. In the present study, we investigated the therapeutic efficacy of vandetanib (ZD6474), an inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, in an orthotopic model of MPM. We found that a human MPM cell line, EHMES-10, expressed RET/PTC3 oncogenic rearrangement and a large amount of VEGF. Vandetanib induced the apoptosis and inhibited the proliferation of EHMES-10 cells in vitro (IC 50 = 0.3 μM). Once-daily oral treatment with vandetanib inhibited tumor angiogenesis, and reduced significantly the growth of thoracic tumors and the production of pleural effusions, resulting in the prolonged survival of mice in EHMES-10 orthograft model. In contrast, the selective EGFR tyrosine kinase inhibitor, gefitinib, had no effect against EHMES-10 cells both in vitro and in vivo. Our results suggest that using vandetanib to target RET-dependent tumor cell proliferation and survival and VEGFR-2-dependent tumor angiogenesis may be promising against MPM expressing RET oncogenic rearrangement and VEGF.

Aberrant splicing and protease involvement in mesothelin release from epithelioid mesothelioma cells

Elevated amounts of soluble mesothelin-related proteins (SMRP) have already been reported in sera and pleural effusions from mesothelioma patients, providing a useful diagnostic marker for malignant pleural mesothelioma (MPM). However, the origin of SMRP is not yet understood. Production of SMRP could be related to abnormal splicing events leading to synthesis of a secreted protein (release) or to an enzymatic cleavage from membrane-bound mesothelin (ectodomain shedding). To test these hypotheses, we used a panel of mesothelioma cells established in culture from pleural effusions of MPM patients. Our in vitro results confirmed specific mesothelin expression and SMRP production in supernatants from epithelioid MPM cell lines, thus providing a relevant cellular model to study soluble mesothelin production mechanisms. The expression of mesothelin-encoding RNA variants was screened by reverse transcription-polymerase chain reaction experiments. Protease involvement in mesothelin cleavage from the cellular surface was investigated by treatment of MPM cells with GM6001, a broad-spectrum MMP- and ADAM-family inhibitor. GM6001 treatment significantly impaired SMRP production by MPM cell lines, in favor of an enzymatic-mediated shedding process. In addition, a splice variant transcript of mesothelin (variant 3) was detected in these MPM cell lines, in accordance with the release of a secreted part of the protein. Our results indicate that both mechanisms could be implicated in soluble mesothelin production by epithelioid mesothelioma cells.

Acquisition of Cisplatin-resistance in Malignant Mesothelioma Cells Abrogates Na+,K+,2Cl-;-cotransport Activity and Cisplatin-induced Early Membrane Blebbing

Aims: Resistance mechanisms are important limiting factors in the treatment of solid malignancies with cis-diamminedichloroplatinum(II) (cisplatin). To gain further understanding of the effects of acquired cisplatin-resistance, we compared a human malignant pleural mesothelioma cell line (P31) to a sub-line (P31res1.2) with acquired cisplatin-resistance. Methods and Results: The role of Na⁺,K⁺,2Cl^--cotransport (NKCC1) activity in cisplatin-induced morphological changes and acquired cisplatin-resistance was investigated in a time-resolved manner. Acquisition of cisplatin-resistance resulted in markedly reduced NKCC1 activity, absence of cisplatin-induced early membrane blebbing, and increased basal caspase-3 activity. At equitoxic cisplatin concentrations, P31res1.2 cells had a faster activation of caspase-3 than P31 cells, but the end-stage cytotoxicity and number of cells with DNA fragmentation was similar. Bumetanide inhibition of NKCC1 activity in P31 cells repressed cisplatin-induced early-phase membrane blebbing but did not increase P31 cell resistance to cisplatin. Conclusions: Together, these results suggest that active NKCC1 was necessary for cisplatin-induced early membrane blebbing of P31 cells, but not for cisplatin-resistance. Thus, acquisition of cisplatin-resistance can affect mechanisms that have profound effects on cisplatin-induced morphological changes but are not necessary for the subsequent progression to apoptosis.

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin

BackgroundThe incidence of malignant pleural mesothelioma (MPM) is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2 has been thought to be a promising cancer therapy.ResultsWe have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab) and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine.ConclusionOur results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM.

The Therapeutic Efficacy of Anti–Vascular Endothelial Growth Factor Antibody, Bevacizumab, and Pemetrexed against Orthotopically Implanted Human Pleural Mesothelioma Cells in Severe Combined Immunodeficient Mice

Malignant pleural mesothelioma (MPM) is an aggressive malignancy, which has a poor prognosis with a median survival of less than 1 year. The vascular endothelial growth factor (VEGF) has been reported to be an ideal therapeutic target, and a multitargeted antifolate, pemetrexed, has been clinically used for the treatment of MPM. We examined the therapeutic efficacy of the antihuman VEGF neutralizing antibody, bevacizumab, in combination with pemetrexed against two different human MPM cells, EHMES-10 and MSTO-211H, orthotopically inoculated into severe combined immunodeficient mice. Bevacizumab inhibited a VEGF-induced proliferation of the human endothelial cells in a dose-dependent manner, but it had no effect on the proliferation of the two MPM cell lines in vitro. The orthotopically inoculated EHMES-10 cells (VEGF high expressing) produced thoracic tumors and a large volume of bloody pleural effusion, whereas the MSTO-211H cells (VEGF low expressing) produced thoracic tumors and a small volume of bloody effusions. Treatment with bevacizumab effectively inhibited the production of thoracic tumors and dramatically prevented the production of pleural effusion by the EHMES-10 cells but not the MSTO-211H cells. Treatment with bevacizumab reduced the number of enlarged tumor-associated vessels and proliferating tumor cells. Moreover, treatment with bevacizumab in combination with pemetrexed more effectively suppressed the formation of the pleural effusion and prolonged the survival compared with the control and monotherapy in the EHMES-10 cell-bearing severe combined immunodeficient mice. These results suggest that the combined use of bevacizumab and pemetrexed may therefore be promising for controlling the progression of MPM highly expressing VEGF.

Activated Src kinase is expressed in malignant pleural mesothelioma tumors; dasatinib inhibition leads to cytotoxicity, cell cycle inhibition, and prevention of invasion and migration

7713 Background: Malignant pleural mesothelioma (MPM) is a lethal disease with few effective therapeutic options. We sought to determine whether Src, a non-receptor tyrosine kinase, could be a new therapeutic target in MPM and to establish the potential therapeutic use of pharmacologic Src inhibitors in this disease. Methods: We analyzed four MPM cell lines (MSTO-211H, NCI-H28, NCI-H2052, and NCI- H2452) for immunohistochemical (IHC) expression of total and phosphorylated Src (Tyr 419, Tyr 530). These cell lines were treated with dasatinib, a Src inhibitor, and evaluated for apoptosis, cell cycle analysis, and migration and invasion. Downstream signaling events were studied by Western blot analysis. We also conducted IHC analyses with total Src, phosphorylated Src Tyr 419 (p-Src Tyr 419), and phosphorylated Src Tyr 530 on 46 archived MPM tumor specimens and correlated the biomarker results with the clinical outcome. Results: All four MPM cell lines expressed total and activated Src (p-Src Tyr 419). Three of the four cell lines were sensitive in vitro to cytotoxicity by dasatinib with inhibition of migration and invasion, cell cycle inhibition, and apoptosis. Treatment with dasatinib inhibited several pathways downstream of Src. In the archived MPM tumor specimens, Src protein was highly expressed on IHC analysis in tumor cells, but that expression did not correlate with overall or progression-free survival. However, expression of activated Src (p-Src Tyr 419) on the tumor cell membrane was higher in patients with stage 4 disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Tyr 419. MPM nodal involvement at N1 was associated with the highest membrane expression of inactive Src (p-Src Tyr 530) (P = 0.02), whereas N2 disease was associated with the lowest expression. No gene mutations in Src exon 12 were found in the cell lines or tumor specimens. Conclusions: Activated Src may have an important role in survival, metastasis, and invasion in MPM, and targeting Src may be an important therapeutic strategy. No significant financial relationships to disclose.

Preclinical studies of the proteasome inhibitor bortezomib in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm that is resistant to chemotherapy. Bortezomib is an FDA-approved proteasome inhibitor that is currently under clinical investigation in multiple neoplasms but has not been studied extensively in MPM. In this report, we determine the biological and molecular response of cultured MPM cells to bortezomib alone and in combination with cisplatin or pemetrexed. We used four MPM cell lines (MS589, H28, H2052, JMN), a normal mesothelial cell line (HM3), and a lung cancer cell line (H23) in survival studies utilizing bortezomib, cisplatin, and pemetrexed alone and in combination by administering concurrently or by varying the order of administration. We determined the effect of bortezomib on the cell cycle, apoptosis, and on the expression of cell cycle proteins p21/WAF1 and p27/KIP1 and on apoptosis-related proteins IAP-1, IAP-2, survivin, and XIAP. Bortezomib was highly cytotoxic to MPM cells and induced both G(2)/M and G(1)/S cell cycle arrest. Apoptosis increased in a concentration- and time-dependent manner in 3 of 4 MPM cell lines. Bortezomib stabilized or increased protein levels of p21/WAF1 and IAP-1 and to a lesser degree p27/KIP1, IAP-2, XIAP, and survivin. In combination studies with cisplatin, bortezomib was generally synergistic at high concentrations and antagonistic at low concentrations. Bortezomib increased the cytotoxicity of cisplatin and pemetrexed in a concentration-dependent manner when administered prior to either. Bortezomib may improve outcome in MPM patients alone or in combination with standard chemotherapy but the order of administration is likely to be important. This study justifies further evaluation of bortezomib in MPM.

Taurolidine and povidone-iodine induce different types of cell death in malignant pleural mesothelioma

Taurolidine and povidone-iodine (PVP-I) are used in every day clinical practice, taurolidine as a broad spectrum antibiotic, and PVP-I as an antiseptic. The type of cell death induced in malignant pleural mesothelioma (MPM) cell lines by these agents was compared, and their ability to sensitize to chemotherapy assessed. Both taurolidine and PVP-I inhibited MPM cell growth after 7.5min incubation, but taurolidine was more effective at later time points and was more specific towards tumour cells than PVP-I. Taurolidine induced death by caspase-dependent and independent mechanisms, whereas in contrast, PVP-I induced a necrotic phenotype that was not caspase-dependent. Interestingly, both taurolidine and PVP-I induced the production of reactive oxygen intermediates and decreased mitochondrial membrane permeability, and cell death was inhibited by the oxygen scavenger N-acetyl cysteine. Taurolidine but not PVP-I treatment resulted in p53 activation in 2/3 MPM cell lines and a decrease in the protein levels of survivin, Bcl-2 and Mcl-1. Survivin also decreased in response to PVP-I whereas Bcl-xL remained unaffected by both treatments. Targeting of Bcl-xL with siRNA sensitized MPM cells to taurolidine and taurolidine treatment sensitized MPM cells to cisplatin-induced apoptosis. In conclusion, taurolidine and PVP-I are both cytotoxic to human MPM cells at early and late time points and induce reactive oxygen intermediate production. Taurolidine induces apoptosis and necrosis, activates p53 and sensitizes cells to cisplatin, whereas PVP-I inhibits cell growth via necrosis. Both agents are promising candidates for use in local treatment within multimodality concepts for MPM.

Expression patterns of inhibitor of apoptosis proteins in malignant pleural mesothelioma

Inhibitor of apoptosis proteins (IAPs) comprise a family of structurally similar proteins, five of which are widely studied in the context of cancer: IAP-1/MIHC/cIAP2, IAP-2/MIHB/cIAP1, livin/ML-IAP/KIAP, survivin, and XIAP/MIHA/hILP. IAPs are overexpressed by most neoplasms, promote tumour cell survival after a wide variety of apoptotic stimuli, and frequently have gene and/or protein expression patterns associated with a relatively poor prognosis. However, many IAPs are also expressed by normal tissues, can facilitate apoptotic cell death, and have expression patterns associated with a relatively favourable prognosis in some cases. The result is that the precise role(s) of IAPs in human tumours is not exactly known. It has been previously reported that IAP-1 is overexpressed in malignant pleural mesothelioma (MPM) and is responsible for a large degree of the resistance of cultured MPM cells to cisplatin. Given the high homology of IAP family members, it is likely that other IAPs will be important in MPM. In the present study, the gene and protein expression patterns of IAP-1, IAP-2, survivin, livin, and XIAP have been determined in MPM cell lines (n=9) and a large number of MPM tumours using high-density oligonucleotide microarrays (n=40) and an MPM tissue array (n=66). Human tumours were linked to clinical data and it was found that IAP-1 and survivin mRNA expression patterns were associated with a relatively shorter patient survival, while those of XIAP and livin were associated with a relatively longer patient survival. Abundant protein for all IAPs was also detected in MPM tumours, where they were expressed primarily in the cytoplasm. Only IAP-1 and livin protein was expressed in the nucleus of MPM tumours. These results provide the rationale for additional study of this gene family in MPM and cancer in general.

Inhibitor of apoptosis proteins are regulated by tumour necrosis factor‐α in malignant pleural mesothelioma

Inhibitor of apoptosis proteins (IAPs) are overexpressed by most neoplasms and promote tumour cell survival after a wide variety of apoptotic stimuli elicited via intrinsic (ie mitochondrial) and extrinsic (ie death receptor) pathways. It has previously been reported that one of these proteins, IAP-1(MIHC/cIAP2), is overexpressed in malignant pleural mesothelioma (MPM) and is responsible for a large degree of the resistance of cultured MPM cells to cisplatin. Subsequent analysis in a larger number of human tumours revealed that additional IAPs (eg IAP-2/MIHB/cIAP1, livin/ML-IAP/KIAP, survivin, and XIAP/MIHA/hILP) are also overexpressed in MPM and, with the exception of IAP-2, have expression patterns that correlate with prognosis. In the present study, potential regulatory mechanisms of IAP genes in MPM were investigated and it was found that tumour necrosis factor-alpha (TNF-alpha) can increase mRNA and protein levels of IAP-1, IAP-2, and XIAP, but not livin or survivin in MPM cell lines (n=4). It was also found that IAP gene expression levels are increased concomitantly with translocation to the nucleus of the TNF-responsive transcription factor NF-kappaB. Co-incubation of MPM cells with TNF-alpha and pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor, prevented TNF-mediated up-regulation of IAP gene expression levels. In survival studies, TNF-alpha was not toxic to MPM cells at any concentration examined. However, MPM cells exposed to TNF-alpha were twice as resistant to cisplatin in dose response survival assays compared with unstimulated controls and were found to have a significantly greater fraction of surviving cells at multiple cisplatin concentrations (p<0.0087). Finally, it was found that levels of circulating TNF-alpha were statistically significantly (p=0.031) (median 312.5 pg/ml) higher in MPM patients (n=6) prior to surgical tumour debulking compared with those after surgery (median 0 pg/ml). These results when combined with previous observations by our laboratory and others strongly suggest that IAPs act synergistically with TNF family members to promote survival of MPM tumour cells after exposure to cisplatin and possibly other chemotherapeutic drugs.

Genomic profiling of malignant pleural mesothelioma with array‐based comparative genomic hybridization shows frequent non‐random chromosomal alteration regions including JUN amplification on 1p32

Genome-wide array-based comparative genomic hybridization analysis of malignant pleural mesotheliomas (MPM) was carried out to identify regions that display DNA copy number alterations. Seventeen primary tumors and nine cell lines derived from 22 individuals were studied, some of them originating from the same patients. Regions of genomic aberrations observed in >20% of individuals were 1q, 5p, 7p, 8q24 and 20p with gains, and 1p36.33, 1p36.1, 1p21.3, 3p21.3, 4q22, 4q34-qter, 6q25, 9p21.3, 10p, 13q33.2, 14q32.13, 18q and 22q with losses. Two regions at 1p32.1 and 11q22 showed a high copy gain. The 1p32.1 region contained a protooncogene, JUN, and we further demonstrated overexpression of JUN with real-time polymerase chain reaction analysis. As MPM cell lines did not overexpress JUN, our findings suggested that induction of JUN expression was involved in the development of MPM cells in vivo, which also might result in gene amplification in a subset of MPM. Meanwhile, the most frequent alteration was the 9p21.3 deletion, which includes the p16(INK4a)/p14(ARF) locus. With polymerase chain reaction analysis, we determined the extent of the homozygous deletion regions of the p16(INK4a)/p14(ARF) locus in MPM cell lines, which indicated that the deletion regions varied among cell lines. Our results with array comparative genomic hybridization analysis provide new insights into the genetic background of MPM, and also give some clues to develop a new molecular target therapy for MPM.

In vivo Loss of Expression of Argininosuccinate Synthetase in Malignant Pleural Mesothelioma Is a Biomarker for Susceptibility to Arginine Depletion

Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production. Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines. In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells. In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.

Radiation-Induced Cellular DNA Damage Repair Response Enhances Viral Gene Therapy Efficacy in the Treatment of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) treated with radiotherapy (RT) has incomplete responses as a result of radiation-induced tumoral stress response that repairs DNA damage. Such stress response is beneficial for oncolytic viral therapy. We hypothesized that a combination of RT and NV1066, an oncolytic herpes virus, might exert an additive or synergistic effect in the treatment of MPM. JMN, a MPM cell line, was infected with NV1066 at multiplicities of infection of .05 to .25 in vitro with and without radiation (1 to 5 Gy). Virus replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 (growth arrest and DNA damage repair 34, a DNA damage-repair protein) by real-time reverse transcriptase-polymerase chain reaction and Western blot test. Synergistic cytotoxicity dependence on GADD34 upregulation was confirmed by GADD34 small inhibitory RNA (siRNA). Synergism was demonstrated between RT and NV1066 across a wide range of doses. As a result of such synergism, a dose-reduction for each agent (up to 5500-fold) can be accomplished over a wide range of therapeutic-effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of siRNA directed against GADD34. RT can be combined with oncolytic herpes simplex virus therapy in the treatment of malignant pleural mesothelioma to achieve synergistic efficacy while minimizing dosage and toxicity.

14 INVITED Vascular targeting

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a poor prognosis, therefore development of novel effective therapies is urgent. In the present study, we investigated the therapeutic efficacy of vandetanib (ZD6474), an inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, in an orthotopic model of MPM. We found that a human MPM cell line, EHMES-10, expressed RET/PTC3 oncogenic rearrangement and a large amount of VEGF. Vandetanib induced the apoptosis and inhibited the proliferation of EHMES-10 cells in vitro (IC50 = 0.3 μM). Once-daily oral treatment with vandetanib inhibited tumor angiogenesis, and reduced significantly the growth of thoracic tumors and the production of pleural effusions, resulting in the prolonged survival of mice in EHMES-10 orthograft model. In contrast, the selective EGFR tyrosine kinase inhibitor, gefitinib, had no effect against EHMES-10 cells both in vitro and in vivo. Our results suggest that using vandetanib to target RET-dependent tumor cell proliferation and survival and VEGFR-2-dependent tumor angiogenesis may be promising against MPM expressing RET oncogenic rearrangement and VEGF.

105 Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

105 Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

P53-Induced Apoptosis Occurs in the Absence of p14ARF in Malignant Pleural Mesothelioma

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14ARF, an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14ARF expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, treatment of p14ARF-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21WAF, PIG3, MDM2, Bax, PUMA increased in p14ARF-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14ARF-deficient cells, treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14ARF; and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14ARF in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.