Apoptosis In Malignant Pleural Mesothelioma Cells Research Articles

The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment.To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin.These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

TGFα Promotes Chemoresistance of Malignant Pleural Mesothelioma.

Background: There is no standard chemotherapy for refractory or relapsing malignant pleural mesothelioma (MPM). Our previous reports nevertheless indicated that a combination of an anthracycline (doxorubicin) and a lysine deacetylase inhibitor (valproic acid, VPA) synergize to induce the apoptosis of MPM cells and reduce tumor growth in mouse models. A Phase I/II clinical trial indicated that this regimen is a promising therapeutic option for a proportion of MPM patients. Methods: The transcriptomes of mesothelioma cells were compared after Illumina HiSeq 4000 sequencing. The expression of differentially expressed genes was inhibited by RNA interference. Apoptosis was determined by cell cycle analysis and Annexin V/7-AAD labeling. Protein expression was assessed by immunoblotting. Preclinical efficacy was evaluated in BALB/c and NOD-SCID mice. Results: To understand the mechanisms involved in chemoresistance, the transcriptomes of two MPM cell lines displaying different responses to VPA-doxorubicin were compared. Among the differentially expressed genes, transforming growth factor alpha (TGFα) was associated with resistance to this regimen. The silencing of TGFα by RNA interference correlated with a significant increase in apoptosis, whereas the overexpression of TGFα desensitized MPM cells to the apoptosis induced by VPA and doxorubicin. The multi-targeted inhibition of histone deacetylase (HDAC), HER2 and TGFα receptor (epidermal growth factor receptor/EGFR) improved treatment efficacy in vitro and reduced tumor growth in two MPM mouse models. Finally, TGFα expression but not EGFR correlated with patient survival. Conclusions: Our data show that TGFα but not its receptor EGFR is a key factor in resistance to MPM chemotherapy. This observation may contribute to casting light on the promising but still controversial role of EGFR signaling in MPM therapy.

4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.

Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Endothelial Cell Protein C Receptor Promotes Apoptosis in Malignant Pleural Mesothelioma Cells

Our recent studies showed that endothelial cell protein C receptor (EPCR) functions as a crucial negative regulator of cancer progression in malignant pleural mesothelioma (MPM) (Keshava et al., Cancer Research 73: 3963-73, 2013). In these studies, the introduction of EPCR expression to aggressive MPM cells lacking EPCR completely attenuated their tumorigenicity whereas the knock-down of EPCR expression in non-aggressive MPM cells that constitutively express EPCR increased their tumorigenicity. This study also revealed that EPCR in MPM cells promotes tumor cell apoptosis in vivo. These data are quite intriguing as EPCR-mediated cell signaling typically activates cell survival and anti-apoptotic pathways and is shown to promote tumor growth in breast and lung cancers. The aim of the present study is to understand the mechanism(s) by which EPCR promotes tumor cell apoptosis in MPM. Evaluation of thoracic fluids obtained from nude mice implanted with MPM cells orthotopically showed a significant increase in the levels of mTNFα and mIFNγ in mice implanted with REN (+EPCR) cells compared to mice implanted with parental REN MPM or REN cells transfected with the vector control [REN(z)]. Analysis of tumor cell apoptosis of cultured cells by TUNEL assay showed that REN (+EPCR) cells were highly susceptible to TNFα+IFNγ-induced cell death compared to parental REN or REN(z) cells (80% vs. 20% cell death at 72 h). In agreement with the concept that EPCR in MPM cells promotes the apoptosis, M9K MPM cells that constitutively express EPCR (non-aggressive MPM cell line) were highly susceptible to TNFα+IFNγ-induced cell death; the knock-down of EPCR in these cells rendered them resistant to cytokine-induced cell death. EPCR-induced tumor cell apoptosis in MPM cells was not dependent on activated protein C (APC). Deletion of the cytoplasmic tail of EPCR failed to diminish its cytotoxic effect. Analysis of REN and REN(+EPCR) tumor cell extracts using PathScan intracellular signaling antibody array kit (Cell Signaling Technology, MA) and immunoblot analysis with specific antibodies against proteins involved in apoptotic pathways revealed that EPCR expression in MPM cells, in comparison to MPM cells lacking EPCR, resulted in a sustained cleavage of poly ADP ribose polymerase (PARP) and an enhanced activation of caspase 3 in response to TNFα+ IFNγ treatment. In addition, REN(+EPCR) cells exhibited an increased activation of caspase 8 and a decreased phosphorylation of anti-apoptotic protein BAD. Further signaling studies showed a delayed activation of NF-kB, suppression of activation of STAT3 and AKT in REN(+EPCR) cells compared to REN cells in response to TNFα+ IFNγ treatment. In addition, EPCR expression suppressed the constitutive activation of the proline-rich AKT substrate, 40 kDa (PRAS40). Overall, our studies indicate that EPCR in MPM cells controls the progression of MPM by exerting its effect at multiple levels. First, EPCR in MPM cells elicits elaboration of cytokines TNFα and IFNγ in vivo and then renders the MPM cells susceptible to TNFα+ IFNγ-induced cell death. The EPCR-mediated cytotoxic effect in MPM cells is independent of APC and the cytoplasmic tail and involves the suppression of STAT3 and AKT activation. At present, it is unclear exactly how EPCR initiates the activation of the apoptotic pathway in MPM cells and whether it involves the interaction of EPCR with the endogenous ligand(s) specific to MPM cells. Studies are underway to identify EPCR interacting proteins in MPM cells. DisclosuresNo relevant conflicts of interest to declare.

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.

Preclinical evaluation of microRNA-34b/c delivery for malignant pleural mesothelioma.

The microRNA-34s (miR-34s) have p53 response elements in their 5'-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM.

TRAIL and proteasome inhibitors combination induces a robust apoptosis in human malignant pleural mesothelioma cells through Mcl-1 and Akt protein cleavages

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive malignancy closely associated with asbestos exposure and extremely resistant to current treatments. It exhibits a steady increase in incidence, thus necessitating an urgent development of effective new treatments.MethodsProteasome inhibitors (PIs) and TNFα-Related Apoptosis Inducing Ligand (TRAIL), have emerged as promising new anti-MPM agents. To develop effective new treatments, the proapoptotic effects of PIs, MG132 or Bortezomib, and TRAIL were investigated in MPM cell lines NCI-H2052, NCI-H2452 and NCI-H28, which represent three major histological types of human MPM.ResultsTreatment with 0.5-1 μM MG132 alone or 30 ng/mL Bortezomib alone induced a limited apoptosis in MPM cells associated with the elevated Mcl-1 protein level and hyperactive PI3K/Akt signaling. However, whereas 10–20 ng/ml TRAIL alone induced a limited apoptosis as well, TRAIL and PI combination triggered a robust apoptosis in all three MPM cell lines. The robust proapoptotic activity was found to be the consequence of a positive feedback mechanism-governed amplification of caspase activation and cleavage of both Mcl-1 and Akt proteins, and exhibited a relative selectivity in MPM cells than in non-tumorigenic Met-5A mesothelial cells.ConclusionThe combinatorial treatment using TRAIL and PI may represent an effective new treatment for MPMs.

SOCS-1gene delivery cooperates with cisplatin plus pemetrexed to exhibit preclinical antitumor activity against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO-4, H28 and H226) with adenovirus-expressing SOCS-1 vector to examine the effect of SOCS-1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS-1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF-κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS-1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS-1 regulated NF-κB and STAT3 signaling to induce apoptosis in MESO-4 and H226 cells. Furthermore, SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF-κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS-1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed.

Molecular Changes in Mesothelioma With an Impact on Prognosis and Treatment

In recent decades, research on malignant pleural mesothelioma (MPM) has been developed to improve patients' outcomes by increasing the level of confidence in MPM diagnosis and prognosis. To summarize data on genetic and epigenetic abnormalities in MPM that may be of interest for a better management of patients with MPM. Data were obtained from scientific publications on genetic and epigenetic abnormalities in MPM by studying gene mutations, DNA methylation, and gene and microRNA expression profiling. Molecular changes in MPM consist in altered expression and in activation or inactivation of critical genes in oncogenesis, especially tumor suppressor genes at the INK4 and NF2 loci. Activation of membrane receptor tyrosine kinases and deregulation of signaling pathways related to differentiation, survival, proliferation, apoptosis, cell cycle control, metabolism, migration, and invasion have been demonstrated. Alterations that could be targeted at a global level (methylation) have been recently reported. Experimental research has succeeded especially in abolishing proliferation and triggering apoptosis in MPM cells. So far, targeted clinical approaches focusing on receptor tyrosine kinases have had limited success. Molecular analyses of series of MPM cases have shown that defined alterations are present in MPM subsets, consistent with interindividual variations of molecular alterations, and suggesting that identification of patient subgroups will be essential to develop more specific therapies.

Epigenetic Silencing of MicroRNA-34b/c Plays an Important Role in the Pathogenesis of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM. Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle. Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2'-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c-transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G(1) cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells. We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM.

Proteasome Inhibitor MG132 Induces Apoptosis and Inhibits Invasion of Human Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an aggressive malignancy tightly associated with asbestos exposure. The increasing incidence of MPM and its resistance to all therapeutic modalities necessitate an urgent development of new treatments for MPM. Proteasome inhibitors (PIs) have emerged as promising agents for treating human cancers that are refractory to current chemotherapies. In this study, we characterized MG132, a commonly used PI, for its proapoptotic and anti-invasion activities in NCI-H2452 and NCI-H2052 human thoracic MPM cell lines to determine the therapeutic effect of PIs on MPM. We found that as low as 0.5 µM MG132 caused a significant apoptosis in both cell lines as evidenced by DNA damage, cleavage of poly ADP-ribose polymerase and caspases 3, 7, and 9, and mitochondrial release of Smac/DIABLO and Cytochrome c. Mitochondrial caspase activation was found to be the underlying mechanism of the MG132-induced apoptosis. Mcl-1, among the Bcl-2 and IAP (inhibitor of apoptosis protein) antiapoptotic family proteins tested, was proved to be a major inhibitor of the MG132-induced apoptosis in MPM cells. Meanwhile, subapoptotic doses of MG132 inhibited the invasion of both MPM cell lines through reducing Rac1 activity. These observations demonstrate that MG132 possesses proapoptotic and anti-invasion activities in human MPM cells, therefore encouraging further investigations on the value of PIs for treating MPM.

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.