Malignant Pleural Mesothelioma Cell Growth Research Articles

JSI-124 inhibits cell proliferation and tumor growth by inducing autophagy and apoptosis in murine malignant mesothelioma.

Malignant pleural mesothelioma (MPM), mainly caused by asbestos exposure, has a poor prognosis and lacks effective treatment compared with other cancer types. The intracellular transcription factor signal transducer and activator of transcription 3 (STAT3) is overexpressed and hyperactivated in most human cancers. In this study, the role of STAT3 in murine MPM was examined. Inhibition of the Janus kinase 2 (JAK2)/STAT3 pathway with the selective inhibitor JSI-124 has an antitumor effect in murine MPM. Specifically, we demonstrated that JSI-124 inhibits murine MPM cell growth and induces apoptotic and autophagic cell death. Exposure of RN5 and AB12 cells to JSI-124 resulted in apoptosis via the Bcl-2 family of proteins. JSI-124 triggered autophagosome formation, accumulation, and conversion of LC3I to LC3II. Autophagy inhibitors, Chloroquine (CQ) and Bafilomycin A1 (Baf-A1), inhibited autophagy and sensitized RN5 and AB12 cells to JSI-124-induced apoptosis. Our data indicate that JSI-124 is a promising therapeutic agent for MPM treatment.

Diverse and divergent functions of IL-32β and IL-32γ isoforms in the regulation of malignant pleural mesothelioma cell growth and the production of VEGF-A and CXCL8

In this study, we sought to elucidate the roles of the interleukin (IL)-32β and IL-32γ in mesothelioma cell growth, and vascular endothelial growth factor (VEGF)-A and C-X-C motif chemokine ligand 8 (CXCL8) expression. IL-32 elicited a growth-promoting effect against one of the six mesotheliomas lines and exerted diverse regulatory functions in VEGF-A and CXCL8 secretion from mesotheliomas stimulated with or without IL-17A. Retroviral-mediated transduction of mesothelioma lines with IL-32γ resulted in enhanced IL-32β expression, which facilitated or suppressed the in vitro growth, and VEGF-A and CXCL8 expression. Overexpressed IL-32β-augmented growth and VEGF-A and CXCL8 production were mainly mediated through the phosphatidylinositol-3 kinase (PI3K) signaling pathway. On the other hand, overexpressed IL-32β-deceased growth was mediated through mitogen-activated protein kinase (MAPK) pathway. NCI-H2373IL-32γ tumors grew faster than NCI-H2373Neo tumors in a xenograft model, which was associated with increased vascularity. These findings indicate that IL-32 are involved in the regulation of growth and angiogenic factor production in mesotheliomas.

IL-7 is expressed in malignant mesothelioma and has a prognostic value.

Malignant pleural mesothelioma (MPM) is an aggressive cancer mainly related to asbestos exposure. Despite recent therapeutic advances, notably immunotherapies, the benefit remains limited and restricted to a small percentage of patients. Thus, a better understanding of the disease is needed to identify new therapeutic strategies. Recently, interleukin 7 receptor (IL‐7R) has been described as being expressed by MPM cells and associated with poorer patient survival. Thus, the aim of this work was to study the IL‐7R/IL‐7 pathway in MPM using patient samples. We found that, although more than 40% of MPM cells expressed IL‐7R, IL‐7 had no effect on their intracellular signaling. Accordingly, the addition of IL‐7 to the culture medium did not affect MPM cell growth. Using The Cancer Genome Atlas (TCGA) database, we showed that high IL7 gene expression in MPM tumors was associated with a higher overall patient survival and an induction of genes involved in the immune response. In pleural effusions (PEs), we found that IL‐7 concentration was not a good diagnostic biomarker. However, we observed that high IL‐7 levels in PEs were associated with shorter survival of MPM patients, but not of lung cancer patients. The prognostic value of IL‐7 was also conserved when only patients with epithelioid mesothelioma, the most common histological type of MPM, were analyzed. Taken together, our study suggests that, although the IL‐7R/IL‐7 signaling pathway is not functional in MPM cells, IL‐7 expression in PEs may have prognostic value in MPM patients.

P35.01 YB-1 is a Key Player in Aggressive Behaviour and Chemoresistance in Mesothelioma

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome. YB-1 expression was analysed by qPCR or western blot in cell lines, and in patient material by immunohistochemistry. YB-1 overexpression was achieved by a doxycycline-inducible, retroviral construct, which was stably introduced into MPM cell lines. YB-1 knockdown was performed using YB-1 specific siRNA. For inhibition of YB-1 phosphorylation, the RSK inhibitor BI-D1870 was used. Effects of drug interaction (CI values) were calculated using the CompuSyn software. Cell migration was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. For the zebrafish model, RFP-expressing MPM cells were injected into 48 hour old larvae, imaged after 1 and 2 days and the number of cells in the tail was manually counted. We previously reported that YB-1 is up-regulated in MPM cell lines compared to non-malignant controls. In this study we analysed MPM tissue (n>120) and normal pleura (n=3) specimens for YB-1 expression. While all control samples were negative, tumours showed a heterogeneous YB-1 expression. YB-1 knockdown decreases MPM cell migration and invasion, hence we evaluated the impact of YB-1 overexpression on these phenotypes. MPM cell lines (n=6) which overexpress YB-1 in a doxycycline-inducible manner showed significantly increased cell scattering and migration. Furthermore, YB-1 overexpression lead to significantly higher migratory capacity in vivo using a zebrafish xenograft model. Additionally, when tumour spheroids were co-cultured with endothelial cells, YB-1 overexpression led to a significantly more extensive formation of gaps in the endothelial layer. Finally, since we found that YB-1 knockdown not only decreases cell growth in vitro and in vivo but also decreases the expression of LRP1 and ABCC1, two genes involved in drug resistance, we combined YB-1 knockdown as well as inhibition of YB-1 phosphorylation via an RSK inhibitor with cisplatin chemotherapy. Our data showed highly synergistic combination effects (CI values: 0.1 – 0.5) for several cisplatin doses in combination with either YB-1 siRNA or the RSK inhibitor. In this study we report a deregulated YB-1 expression in MPM tumor specimens compared to non-malignant controls. Our data show an important role of YB-1 in the regulation of cell migration and invasion in vitro and in vivo, which are key characteristics of MPM. Additionally, YB-1 knock down as well as pharmacological inhibition of YB-1 phosphorylation not only reduce MPM cell growth but also sensitise cells to cisplatin chemotherapy. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

The Role of E-Cadherin and microRNA on FAK Inhibitor Response in Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited effective treatment options. Focal adhesion kinase (FAK) inhibitors have been shown to efficiently suppress MPM cell growth initially, with limited utility in the current clinical setting. In this study, we utilised a large collection of MPM cell lines and MPM tissue samples to study the role of E-cadherin (CDH1) and microRNA on the efficacy of FAK inhibitors in MPM. The immunohistochemistry (IHC) results showed that the majority of MPM FFPE samples exhibited either the absence of, or very low, E-cadherin protein expression in MPM tissue. We showed that MPM cells with high CDH1 mRNA levels exhibited resistance to the FAK inhibitor PND-1186. In summary, MPM cells that did not express CDH1 mRNA were sensitive to PND-1186, and MPM cells that retained CDH1 mRNA were resistant. A cell cycle analysis showed that PND-1186 induced cell cycle disruption by inducing the G2/M arrest of MPM cells. A protein−protein interaction study showed that EGFR is linked to the FAK pathway, and a target scan of the microRNAs revealed that microRNAs (miR-17, miR221, miR-222, miR137, and miR148) interact with EGFR 3′UTR. Transfection of MPM cells with these microRNAs sensitised the CHD1-expressing FAK-inhibitor-resistant MPM cells to the FAK inhibitor.

UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma.

Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

MS25.05 Targeting the Micro-RNA: A New Therapeutic Venture

MS25.05 Targeting the Micro-RNA: A New Therapeutic Venture

Nintedanib Is Active in Malignant Pleural Mesothelioma Cell Models and Inhibits Angiogenesis and Tumor Growth In Vivo.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive thoracic tumor type with limited treatment options and poor prognosis. The angiokinase inhibitor nintedanib has shown promising activity in the LUME-Meso phase II MPM trial and thus is currently being evaluated in the confirmatory LUME-Meso phase III trial. However, the anti-MPM potential of nintedanib has not been studied in the preclinical setting.Experimental Design: We have examined the antineoplastic activity of nintedanib in various in vitro and in vivo models of human MPM.Results: Nintedanib's target receptors were (co)expressed in all the 20 investigated human MPM cell lines. Nintedanib inhibited MPM cell growth in both short- and long-term viability assays. Reduced MPM cell proliferation and migration and the inhibition of Erk1/2 phosphorylation were also observed upon nintedanib treatment in vitro Additive effects on cell viability were detected when nintedanib was combined with cisplatin, a drug routinely used for systemic MPM therapy. In an orthotopic mouse model of human MPM, survival of animals receiving nintedanib per os showed a favorable trend, but no significant benefit. Nintedanib significantly reduced tumor burden and vascularization and prolonged the survival of mice when it was administered intraperitoneally. Importantly, unlike bevacizumab, nintedanib demonstrated significant in vivo antivascular and antitumor potential independently of baseline VEGF-A levels.Conclusions: Nintedanib exerts significant antitumor activity in MPM both in vitro and in vivo These data provide preclinical support for the concept of LUME-Meso trials evaluating nintedanib in patients with unresectable MPM. Clin Cancer Res; 24(15); 3729-40. ©2018 AACR.

P3.03-007 miR-137 Acts as a Tumor Suppressor via the Down-Regulation of YB-1 in Malignant Pleural Mesothelioma: Topic: Mesothelioma Transitional

Malignant pleural mesothelioma (MPM) continues to increase in incidence worldwide and has limited therapeutic options. MPM displays characteristic changes in gene expression, including noncoding RNAs such as microRNAs, which have potential therapeutic relevance. One such miRNA is miR-137, a tumor suppressor whose promoter region is frequently methylated in other cancers and lies in in a commonly deleted chromosomal region in MPM (1p21-23). A potential role for miR-137 has yet to be investigated in MPM. One known target of miR-137 is YB-1, a multifunctional protein often up-regulated in other aggressive cancers, where elevated YB-1 levels are linked to poor clinical outcomes. This study investigates the causes of miR-137 suppression, the relationship between miR-137 and YB-1, one of its targets, as well as their roles in MPM cell growth and malignant behavior. Basal expression of miR-137 and YB-1 was determined in 13 MPM cell lines by RT-qPCR and immunoblotting. Cells were treated with 5’Aza-cytidine and RT-qPCR was conducted to link methylation with miR-137 suppression. Copy number variation (CNV) was investigated by ddPCR. Cells were transfected with miR-137 mimic and subsequent YB-1 expression was investigated using RT-qPCR. Proliferation, colony formation and wound-healing assays were conducted after transfection with miR-137 mimics or YB-1-specific siRNAs. miR-137 was absent in 4 MPM cell lines (p<0.01) and was up-regulated in response to 5’Aza-cytidine treatment in these lines, as well as other lines with low basal expression. Copy-number loss was evident in 5 cell lines and gain was present in 2. Increasing levels of miR-137 generally inhibited MPM cell migration, proliferation and colony formation. miR-137 mimics significantly down-regulated YB-1 expression, while YB-1 protein was overexpressed in the majority of MPM cell lines, compared to MeT-5A. YB-1 knock-down resulted in dose-dependent growth inhibition over 120 hours, reduced colony formation and also decreased cell migration. Effects were more pronounced in those cell lines showing high YB-1 protein levels. Our results show that methylation and CNV are likely to play a role in miR-137 down-regulation in MPM and that miR-137 acts as a tumor suppressor in MPM through at least in part the down-regulation of YB-1. We also demonstrated that YB-1 is commonly overexpressed and plays a role in proliferation and migration. These results imply a direct relationship between miR-137 and YB-1 expression, a biological interaction that may prove a useful target in developing future therapeutic approaches in MPM.

SORORIN and PLK1 as potential therapeutic targets in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive type of cancer of the thoracic cavity commonly associated with asbestos exposure and a high mortality rate. There is a need for new molecular targets for the development of more effective therapies for MPM. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and an RNA interference-based screening, we examined the SORORIN gene as potential therapeutic targets for MPM in addition to the PLK1 gene, which is known for kinase of SORORIN. Following invitro investigation of the effects of target silencing on MPM cells, cell cycle analyses were performed. SORORIN expression was analyzed immunohistochemically using a total of 53 MPM samples on tissue microarray. SORORIN was found to be overexpressed in the majority of clinical MPM samples and human MPM cell lines as determined by qRT-PCR. Gene suppression of each SORORIN and PLK1 led to growth inhibition in MPM cell lines. Knockdown of SORORIN showed an increased number of G2M-phase population and a larger nuclear size, suggesting mitotic arrest. High expression of SORORIN (SORORIN-H) was found in 50.9% of all the MPM cases, and there is a tendency towards poorer prognosis for the SORORIN-H group but the difference is not significant. Suppression of SORORIN with PLK1 inhibitor BI 6727 showed a combinational growth suppressive effect on MPM cell growth. Given high-dose PLK1 inhibitor induced drug-related adverse effects in several clinical trials, our results suggest inhibition SORORIN-PLK1 axis may hold promise for the treatment of MPMs.

Tamoxifen Suppresses the Growth of Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is a rare but highly aggressive malignancy most often associated with exposure to asbestos. Recent evidence points to oestrogen receptor (ER)-β having a tumour-suppressor role in MPM progression, and this raises the question of whether selective modulators of ERs could play a role in augmenting MPM therapy. We investigated the action of tamoxifen in inhibiting the growth and modulating the cisplatin sensitivity of four MPM cell lines. Tamoxifen inhibited the growth of MPM cells and also modulated their sensitivity to cisplatin. The MPM cell lines expressed ERβ, but the actions of tamoxifen were not blocked by antagonism of nuclear ERs. Tamoxifen treatment repressed the expression of cyclins by MPM cells, resulting in cell-cycle arrest and caspase-3-coupled apoptosis signaling. The ER-independent actions of tamoxifen on MPM cell proliferation and cell-cycle progression may have clinical benefits for a subset of patients with MPM.

Targeting receptor tyrosine kinases in malignant pleural mesothelioma: Focus on FGF-receptors

Fibroblast growth factor receptors (FGFRs) constitute a subfamily of receptor tyrosine kinases. Four different receptors, FGFR1-4, bind 18 different fibroblast growth factors (FGFs) and signal mainly along the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3 kinase (PI3K) and the phospholipase c gamma (PLCγ) pathway. Physiologically, they are major regulators of embryonic development and metabolism. Deregulation of FGFR signals is increasingly recognized to play important roles in malignant diseases and may constitute a feasible therapeutic target. We recently investigated their role in malignant pleural mesothelioma (MPM), an aggressive malignancy mainly caused by asbestos exposure and with currently limited therapeutic options. We demonstrated high expression of several FGFs/FGFRs, especially FGFR1, FGF2 and FGF18 in cultured tumor cells and tissue specimens and identified FGFR-mediated signals as major driver of MPM cell growth, survival and migration. FGFR blockade by a tyrosine kinase inhibitor or by a dominant-negative receptor construct resulted in reduced MPM growth in vitro and in vivo and, furthermore, enhanced the efficacy of chemo- or radiotherapy. Several other receptor tyrosine kinases, including EGFR, MET and AXL were found to be overexpressed in MPM but translation into clinically successful therapeutic approaches has not yet been achieved. Inhibition of FGF-receptors may have the advantage of targeting both the tumor cells as well as the tumor vasculature and should be further evaluated.

Disulfiram Suppresses Growth of the Malignant Pleural Mesothelioma Cells in Part by Inducing Apoptosis

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.

CARP-1 Functional Mimetics Are a Novel Class of Small Molecule Inhibitors of Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and β, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents.

A Novel Targeting Therapy of Malignant Mesothelioma Using Anti-Podoplanin Antibody

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

84 Tamoxifen induced signalling in the suppression of malignant pleural mesothelioma cell growth

84 Tamoxifen induced signalling in the suppression of malignant pleural mesothelioma cell growth

Antitumor activity of MEK and PI3K inhibitors against malignant pleural mesothelioma cells in vitro and in vivo

Malignant pleural mesothelioma (MPM) is an aggressive malignancy for which there is no approved targeted therapy. We examined the therapeutic efficacy of the mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors against human MPM cell lines both in vitro and orthotopically inoculated into severe combined immunodeficient (SCID) mice. In addition, the molecular mechanisms of these agents were confirmed in vitro and in vivo. The MEK or the PI3K inhibitor suppressed MPM cell growth in vitro in a dose-dependent manner via induction of G1 cell cycle arrest and apoptosis. In addition, combined use of the MEK and PI3K inhibitors showed an additive or synergistic inhibitory effect on MPM cell growth compared to treatment with either individual drug. Treatment with MEK or PI3K inhibitor suppressed the production of thoracic tumors and pleural effusion and prolonged the survival time of EHMES-10 cell-bearing SCID mice. The combination therapy more effectively prolonged the survival time compared to treatment with either individual drug. Immunohistochemical and western blot analysis of thoracic tumors suggested that these agents induced cell cycle arrest, apoptosis and inhibition of tumor angiogenesis. Our results suggest that a combination of MEK and PI3K inhibitors is a promising therapeutic strategy for MPM.

Receptor tyrosine kinase inhibitors and cytotoxic drugs affect pleural mesothelioma cell proliferation: insight into EGFR and ERK1/2 as antitumor targets

Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFR-tyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC 50 values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC 50, while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR B max and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM.

Curcumin suppresses growth of mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis

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 pathways are frequently altered in MPM and thus remain attractive targets for 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. In this study, 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 the 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 effective management of resistant MPM.

In vitro and in vivo therapeutic efficacy of the PPAR‐γ agonist troglitazone in combination with cisplatin against malignant pleural mesothelioma cell growth

Malignant pleural mesothelioma (MPM), an aggressive and refractory tumor type, is increasing in frequency throughout the world. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists have anticancer activity against several cancer cell lines in vitro and in vivo. However, there have been no reports that PPAR-γ agonists induce growth inhibition of MPM cell lines. In this study, we investigated the inhibitory effect of a PPAR-γ agonist in combination with an anticancer agent on MPM cell growth in vitro and in vivo. We examined the therapeutic efficacy of the PPAR-γ agonist troglitazone (TGZ) in combination with cisplatin against a human MPM cell line, both in vitro and orthotopically inoculated into severe combined immunodeficient (SCID) mice. Troglitazone (TGZ) alone inhibited MPM cell growth in vitro in a dose-dependent manner via induction of G1 cell cycle arrest and apoptosis. The combination of TGZ and cisplatin showed an additive inhibitory effect on MPM cell growth compared to treatment with either individual drug. Treatment with 500 mg/kg or 1000 mg/kg TGZ effectively inhibited the production of thoracic tumors and pleural effusion in EHMES-10 cell-bearing SCID mice. Moreover, treatment with 500 mg/kg TGZ in combination with 3 mg/kg cisplatin more effectively prolonged survival compared to treatment with either individual drug. These results suggest that TGZ in combination with cisplatin may become a novel therapy for MPM.