Malignant Pleural Mesothelioma Cell Lines Research Articles

Identification of Overexpressed Genes in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a fatal tumor lacking effective therapies. The characterization of overexpressed genes could constitute a strategy for identifying drivers of tumor progression as targets for novel therapies. Thus, we performed an integrated gene-expression analysis on RNAseq data of 85 MPM patients from TCGA dataset and reference samples from the GEO. The gene list was further refined by using published studies, a functional enrichment analysis, and the correlation between expression and patients’ overall survival. Three molecular signatures defined by 15 genes were detected. Seven genes were involved in cell adhesion and extracellular matrix organization, with the others in control of the mitotic cell division or apoptosis inhibition. Using Western blot analyses, we found that ADAMTS1, PODXL, CIT, KIF23, MAD2L1, TNNT1, and TRAF2 were overexpressed in a limited number of cell lines. On the other hand, interestingly, CTHRC1, E-selectin, SPARC, UHRF1, PRSS23, BAG2, and MDK were abundantly expressed in over 50% of the six MPM cell lines analyzed. Thus, these proteins are candidates as drivers for sustaining the tumorigenic process. More studies with small-molecule inhibitors or silencing RNAs are fully justified and need to be undertaken to better evaluate the cancer-driving role of the targets herewith identified.

P25.01 Metabolic Characterization of Drug Resistance to Antifolate in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy that develops due to asbestos exposure. MPM is a diffuse disease with a poor prognosis. MPM develops in the pleural cavity and is highly resistant to a number of therapeutics. A combination of pemetrexed (PMX) and cisplatin (CDDP) is the first-line chemotherapy that remains useful today. PMX inhibits the enzymes dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART), which are involved in purine and pyrimidine synthesis. However, the median survival is approximately 12 months after treatment with PMX and CDDP, and the response rate is 41.3%. Therefore, patients with MPM show an approximately 60% incidence of resistance to PMX and CDDP. The mechanism of CDDP resistance has been investigated in several studies; however, the mechanism of PMX resistance is poorly understood. There are reports of TYMS overexpression being involved in PMX resistance; on the other hand, there are also reports of a lack of link to clinical results. In a previous study, I reported that the levels of glycine and IMP may be potential biomarkers of chemotherapy with PMX. For this reason, I hypothesized that MPM cells might acquire PMX resistance via changes in metabolic pathways. I established an MPM cell line of PMX resistance and examined the expression levels of PMX-targeted enzymes and intracellular metabolites of acquired PMX-resistant cell lines. In the first study, I changed medium the concentration of PMX was added to increase by 10% so that two PMX-resistant cell lines. Viable cells by WST-8 assay and the dose response curve. In a second study, I investigated the mRNA expression levels of parental cell lines and PMX-resistant cell lines to investigate whether PMX-resistant cell lines might change the expression levels of PMX-targeted enzymes and other enzymes. In the third study, I examined whether differences in intracellular metabolites between PMX-resistant and parent cell lines in the presence of PMX by metabolome analysis. MPM cells in the presence of PMX for a long time, and then established two PMX-resistant cell lines. These cell lines were extremely resistant to PMX. Next, I confirmed that increased TYMS expression levels by RT-qPCR when MPM cells exposed to PMX for a long time; however, DHFR and GART expression levels not change . Furthermore, expression levels of RFC and multidrug resistance protein (MRP) did not change . Next, I analyzed PMX-resistance and parent cell lines of intracellular metabolites in the presence of PMX, and found that glycine and adenosine changed between parental cell lines and PMX-resistant cell lines in the presence of PMX. In this study, I showed that TYMS expression levels, glycine and adenosine were changed by acquired PMX-resistance. Therefore, we think that finding regulatory genes of TYMS expression and glycine, adenosine production might elucidate the mechanism of drug resistance . I report these results, I would like to discuss.

Near-Infrared Photoimmunotherapy Targeting GPR87: Development of a Humanized Anti-GPR87 Mab and Therapeutic Efficacy on a Lung Cancer Mouse Model

Background: GPR87 is a G protein receptor that is specifically expressed in tumor cells, such as lung cancer, and rarely expressed in normal cells. GPR87 is a promising target for cancer therapy, but its ligand is controversial. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy in which a photosensitizer, IRDye700DX (IR700), binds to antibodies and specifically destroys target cells by irradiating them with near-infrared light. Here, we aimed to develop a NIR-PIT targeting GPR87. Methods: We evaluated the expression of GPR87 in resected specimens of lung cancer and malignant pleural mesothelioma (MPM) resected at Nagoya University Hospital using immunostaining. Humanized anti GPR87 antibody (huGPR87) was generated by introducing VH and VL genes into mouse anti GPR87 antibody which generated by standard hybridoma method. HuGPR87 was conjugated with IR700 and the therapeutic effect of NIR-PIT was evaluated in vitro and in vivo using lung cancer or MPM cell lines. Findings: Among the surgical specimens, 54% of lung cancer and 100% of MPM showed high expression of GPR87. It showed therapeutic effects on lung cancer and MPM cell lines in vitro, and showed therapeutic effects in multiple models in vivo. Interpretation: These results suggest that NIR-PIT targeting GPR87 is a promising therapeutic approach for the treatment of thoracic cancer. Funding: Funding: This research was supported by the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, JSPS), Souhatsu (JST). Declaration of Interest: None to declare Ethical Approval: All in vivo procedures were performed in accordance with the Nagoya University Animal Care and Use Committee's Guide for the Management and Use of Laboratory Animal Resources (approval numbers 2017-29438, #2018-30096, # 2019-31234, #2020-20104). The use of specimens from patients was approved by the Ethics Committee of the Nagoya University Clinical Research Committee (Approval No. 2018-0046).

A comparison between the effects of over-expression of miRNA-16 and miRNA-34a on cell cycle progression of mesothelioma cell lines and on their cisplatin sensitivity

The prognosis of patients affected by malignant pleural mesothelioma (MPM) is presently poor and no therapeutic strategies have improved their survival yet. Introduction of miRNA mimics to restore their reduced or absent functionality in cancer cells is considered an important opportunity and a combination of miR's might be even more effective. In the present study, miR-16 and miR-34a were transfected, singularly and in combination, in MPM cell lines H2052 and H28, and their effects on cell proliferation and sensitivity to cisplatin are reported. Interestingly, the overexpression of both miRs, alone or combined, slows down the cell cycle progression, modulates the p53 and HMGB1 expression and increases the sensitivity of cells to cisplatin, producing a marked impairment of cell proliferation and strengthening the apoptotic effect of the drug. However, the co-overexpression of the two miRs results more effective only in the regulation of the cell cycle, but does not enhance the sensitivity of MPM cells to cisplatin. Consequently, although the potential of miR-16 and miR-34a is confirmed, we must conclude that their combination does not improve the response of MPM to chemotherapy.

Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma

Simple SummaryMalignant pleural mesothelioma (MPM) is an incurable and aggressive malignancy mainly caused by exposure to asbestos fibers. Survival outcomes following the standard of care treatment, including chemotherapy and surgery, remain dismal. Targeted treatments are shortcomings for MPM, as this tumor is driven primarily by a loss of tumor suppressor genes. In this study, we explored the importance of proteins that have been shown to be dysregulated in MPM as a consequence of tumor suppressor gene loss, CUL4A and CUL4B. We assessed their expression levels and identified their correlation with clinical outcomes of MPM. We also aimed to test the efficacy and mechanisms of the available treatments that target these proteins, pevonedistat using in vitro and in vivo models. Our data suggested that CUL4B might serve as a treatment target for MPM and revealed novel mechanism of pevonedistat in the MPM tumor microenvironment. This data may be useful for understanding its efficacy in patients from clinical trials. Neurofibromatosis type 2 (NF2), the tumor suppressor frequently lost in malignant pleural mesothelioma (MPM), suppresses tumorigenesis in part by inhibiting the Cullin4 ubiquitin ligase (CUL4) complex in the nucleus. Here, we evaluated the importance of CUL4 in MPM progression and tested the efficacy of cullin inhibition by pevonedistat, a small molecule inhibiting cullin neddylation. CUL4 paralogs (CUL4A and CUL4B) were upregulated in MPM tumor specimens compared to nonmalignant pleural tissues. High gene and protein expressions of CUL4B was associated with a worse progression-free survival of MPM patients. Among 13 MPM cell lines tested, five (38%) were highly sensitive to pevonedistat (half maximal inhibitory concentration of cell survival IC50 < 0.5 µM). This remained true in a 3D spheroid culture. Pevonedistat treatment caused the accumulation of CDT1 and p21 in both sensitive and resistant cell lines. However, the treatment induced S/G2 cell cycle arrest and DNA rereplication predominantly in the sensitive cell lines. In an in vivo mouse model, the pevonedistat treatment significantly prolonged the survival of mice bearing both sensitive and resistant MPM tumors. Pevonedistat treatment reduced growth in sensitive tumors but increased apoptosis in resistant tumors. The mechanism in the resistant tumor model may be mediated by reduced macrophage infiltration, resulting from the suppression of macrophage chemotactic cytokines, C-C motif chemokine ligand 2 (CCL2), expression in tumor cells.

Polyphenol Extract from Evening Primrose (Oenothera paradoxa) Inhibits Invasion Properties of Human Malignant Pleural Mesothelioma Cells.

Extracts from the defatted evening primrose (Oenothera paradoxa Hudziok) seeds are the source of a range of stable polyphenolic compounds, including ellagic acid, gallic acid, and catechin. Our studies evaluate, for the first time, the influence of evening primrose isopropanol extract (EPE) on malignant pleural mesothelioma (MPM) cells. MPM is rarely diagnosed, its high aggressiveness and frequently noted chemoresistance limit its treatment schemes and it is characterized by low prognostic features. Here, we demonstrate that EPE inhibited MPM growth in a dose-dependent manner in cells with increased invasion properties. Moreover, EPE treatment resulted in cell cycle arrest in the G2/M phase and increased apoptosis in invasive MPM cell lines. Additionally, EPE strongly limited invasion and MMP-7 secretion in MPM cancer cells. Our original data provide evidence about the potential anti-invasive effects of EPE in MPM therapy treatment.

Multicellular contractility contributes to the emergence of mesothelioma nodules

Malignant pleural mesothelioma (MPM) has an overall poor prognosis and unsatisfactory treatment options. MPM nodules, protruding into the pleural cavity may have growth and spreading dynamics distinct that of other solid tumors. We demonstrate that multicellular aggregates can develop spontaneously in the majority of tested MPM cell lines when cultured at high cell density. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Prominent actin cables, spanning several cells, are abundant both in cultured aggregates and in MPM surgical specimens. We propose a computational model for in vitro MPM nodule development. Such a self-tensioned Maxwell fluid exhibits a pattern-forming instability that was studied by analytical tools and computer simulations. Altogether, our findings may underline a rational for targeting the actomyosin system in MPM.

CDKN2A and MTAP Are Useful Biomarkers Detectable by Droplet Digital PCR in Malignant Pleural Mesothelioma: A Potential Alternative Method in Diagnosis Compared to Fluorescence In Situ Hybridisation.

BackgroundThe diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH). The tumor suppressor gene, CDKN2A, is frequently silenced by epigenetic mechanisms in many cancers; in the case of MPM it is mostly silenced via genomic deletion. Co-deletion of the CDKN2A and methylthioadenosine phosphorylase (MTAP) genes has been researched extensively and discovered to be a highly specific characteristic of MPM. Most studies have used FISH to detect the deletion of CDKN2A and IHC for MTAP as a surrogate for this. In this study, we aim to investigate and validate droplet digital PCR (ddPCR) as an emerging alternative and efficient testing method in diagnosing MPM, by particularly emphasizing on the loss of MTAP and CDKN2A.MethodsThis study included 75 formalin fixed paraffin embedded (FFPE) MPM tissue, and 12 normal pleural tissue and 10 RMH as control. Additionally, primary MPM cell lines and normal pleural samples were used as biomarker detection controls, as established in our previous publication. All FFPE specimens were processed to isolate the DNA, that was subsequently used for ddPCR detection of CDKN2A and MTAP. FFPE samples were also analyzed by fluorescence in situ hybridization (FISH) for CDKN2A and MTAP deletion, and for MTAP IHC expression. Concordance of IHC and ddPCR with FISH were studied in these samples.Results95% and 82% of cases showed co-deletion of both MTAP and CDKN2A when determined by FISH and ddPCR respectively. ddPCR has a sensitivity of 72% and specificity of 100% in detecting CDKN2A homozygous loss in MPM. ddPCR also has a concordance rate of 92% with FISH in detecting homozygous loss of CDKN2A. MTAP IHC was 68% sensitive and 100% specific for detecting CDKN2A homozygous loss in MPM when these losses were determined by ddPCR.ConclusionOur study confirms that MTAP is often co-deleted with CDKN2A in MPM. Our in-house designed ddPCR assays for MTAP and CDKN2A are useful in differentiating MPM from RMH, and is highly concordant with FISH that is currently used in diagnosing MPM. ddPCR detection of these genetic losses can potentially be utilized as an alternative method in the diagnosis of MPM and for the future development of a less-invasive MPM-specific detection technique on MPM tumor tissue DNA.

Impact of metallothionein-knockdown on cisplatin resistance in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8–12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.

Effectiveness and Safety of Tumor Treating Fields (150 kHz) plus Cisplatin or Pemetrexed Combination in Mesothelioma: in Vitro and in Vivo Findings

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer linked to asbestos exposure with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, antineoplastic treatment delivered locoregionally to the tumor bed via low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. TTFields are FDA-approved as a first line therapy with pemetrexed plus platinum-based agent for unresectable MPM. TTFields with this combination was evaluated in the phase 2 STELLAR clinical trial (Ceresoli et al, Lancet Oncology 2019) in patients with unresectable MPM, demonstrating a promising median overall survival of ∼18.2 months (vs ∼12 months; historical control) without increases in systemic toxicity. Utilizing in vitro and in vivo models, we investigated the effects of TTFields alone and in combination with cisplatin or pemetrexed in MPM. Using the inovitroTM system, cells from human MPM cell lines (NCI-H2052 and MSTO-211H) were treated at various frequencies of TTFields for 72 hours. The combined treatment of TTFields with cisplatin or pemetrexed was tested by applying TTFields at the 150 kHz optimal frequency with various concentrations of cisplatin (80–5120 nM) or pemetrexed (2–4096 nM). Cell counts, clonogenic potential, and apoptosis induction were determined. TTFields (150 kHz) were applied to Fisher rats with IL-45 intrapleural tumors cells for 8 days and to C57BL/6 mice with RN5 S.C. tumors for 7 days. Tumor volume was determined using MRI. To determine treatment safety, TTFields (2–3 V/cm) were applied to the torsos of Sprague-Dawley rats at 150 kHz for 2 weeks and all major internal organs were histologically compared. The optimal TTFields frequency was 150 kHz in both NCI-H2052 and MSTO-211H human MPM cell lines. TTFields (150 kHz; 1.1 V/cm) application for 72 hours led to a 45-51% reduction in cell counts and a 46-64% additional decrease in clonogenic potential. TTFields plus cisplatin or pemetrexed significantly reduced the cell count, the clonogenic potential and induced apoptosis versus each treatment alone (P <0.0001). Tumor volume was significantly decreased in the TTFields plus pemetrexed plus cisplatin combination group versus the control group in both tumor models. Histological analysis of major internal organs showed no increase in pathological findings when TTFields were applied to the rat torso. These in vitro and in vivo data demonstrate that the optimal TTFields frequency to inhibit MPM is 150 kHz. TTFields alone showed effectiveness and safety, and combined with cisplatin or pemetrexed demonstrated enhanced effectiveness with no increased safety concerns in the torso. Findings were consistent with efficacy and safety findings in the STELLAR trial.

Bex1 significantly contributes to the proliferation and invasiveness of malignant tumor cells.

Invasion has a significant role in cancer progression, including expansion to surrounding tissue and metastasis. Previously, we assessed the invasive ability of cancer cells using an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) method by which cancer cell invasion can be easily visualized. The present study examined multiple lung adenocarcinoma and malignant pleural mesothelioma (MPM) cell lines using the DL-CGH method and identified inherently invasive cell lines. Next, by comparing gene expression between invasive and non-invasive cells by cDNA microarray, the potential candidate gene brain-expressed x-linked protein 1 (Bex1) was identified to be involved in cancer invasion, as it was highly expressed in the invasive cell lines. Downregulation of Bex1 suppressed the invasion and proliferation of the invasive tumor cell lines. The findings of the present study suggested that Bex1 may promote metastasis in vivo and could be a potential oncogene and molecular therapeutic target in lung adenocarcinoma and MPM.

P53-regulated miR-320a targets PDL1 and is downregulated in malignant mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer, related to asbestos exposure, which has a dismal prognosis. MPM diagnosis is late and often challenging, suggesting the need to identify more reliable molecular biomarkers. Here, we set out to identify differentially expressed miRNAs in epithelioid, biphasic, and sarcomatoid MPMs versus normal mesothelium and explored specific miRNA contribution to mesothelial tumorigenesis. We screened an LNA™-based miRNA-microrray with 14 formalin-fixed paraffin-embedded (FFPE) MPMs and 6 normal controls. Through real-time qRT-PCR we extended the analysis of a miRNA subset and further investigated miR-320a role through state-of-the-art techniques. We identified 16 upregulated and 32 downregulated miRNAs in MPMs versus normal tissue, including the previously identified potential biomarkers miR-21, miR-126, miR-143, miR-145. We showed in an extended series that miR-145, miR-10b, and miR-320a levels can discriminate tumor versus controls with high specificity and sensitivity. We focused on miR-320a because other family members were found downregulated in MPMs. However, stable miR-320a ectopic expression induced higher proliferation and migration ability, whereas miR-320a silencing reduced these processes, not supporting a classic tumor-suppressor role in MPM cell lines. Among putative targets, we found that miR-320a binds the 3′-UTR of the immune inhibitory receptor ligand PDL1 and, consistently, miR-320a modulation affects PDL1 levels in MPM cells. Finally, we showed that p53 over-expression induces the upregulation of miR-320a, along with miR-200a and miR-34a, both known to target PDL1, and reduces PDL1 levels in MPM cells. Our data suggest that PDL1 expression might be due to a defective p53-regulated miRNA response, which could contribute to MPM immune evasion or tumorigenesis through tumor-intrinsic roles.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR- may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

EIF4G1 and RAN as Possible Drivers for Malignant Pleural Mesothelioma.

For malignant pleural mesothelioma (MPM) novel therapeutic strategies are urgently needed. In a previous study, we identified 51 putative cancer genes over-expressed in MPM tissues and cell lines. Here, we deepened the study on nine of them (ASS1, EIF4G1, GALNT7, GLUT1, IGF2BP3 (IMP3), ITGA4, RAN, SOD1, and THBS2) to ascertain whether they are truly mesothelial cancer driver genes (CDGs) or genes overexpressed in an adaptive response to the tumoral progression (“passenger genes”). Through a fast siRNA-based screening, we evaluated the consequences of gene depletion on migration, proliferation, colony formation capabilities, and caspase activities of four MPM (Mero-14, Mero-25, IST-Mes2, and NCI-H28) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model. The depletion of EIF4G1 and RAN significantly reduced cell proliferation and colony formation and increased caspase activity. In particular, the findings for RAN resemble those observed for other types of cancer. Thus, we evaluated the in vitro effects of importazole (IPZ), a small molecule inhibitor of the interaction between RAN and importin-β. We showed that IPZ could have effects similar to those observed following RAN gene silencing. We also found that primary cell lines from one out of three MPM patients were sensitive to IPZ. As EIF4G1 and RAN deserve further investigation with additional in vitro and in vivo studies, they emerged as promising CDGs, suggesting that their upregulation could play a role in mesothelial tumorigenesis and aggressiveness. Furthermore, present data propose the molecular pathways dependent on RAN as a putative pharmacological target for MPM patients in the view of a future personalized medicine.

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.

Targeted Phototherapy for Malignant Pleural Mesothelioma: Near-Infrared Photoimmunotherapy Targeting Podoplanin.

Malignant pleural mesothelioma (MPM) has extremely limited treatment despite a poor prognosis. Moreover, molecular targeted therapy for MPM has not yet been implemented; thus, a new targeted therapy is highly desirable. Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer therapy that combines the specificity of antibodies for targeting tumors with toxicity induced by the photoabsorber after exposure to NIR-light. In this study, we developed a new phototherapy targeting podoplanin (PDPN) for MPM with the use of both NIR-PIT and an anti-PDPN antibody, NZ-1. An antibody–photosensitizer conjugate consisting of NZ-1 and phthalocyanine dye was synthesized. In vitro NIR-PIT-induced cytotoxicity was measured with both dead cell staining and luciferase activity on various MPM cell lines. In vivo NIR-PIT was examined in both the flank tumor and orthotopic mouse model with in vivo real-time imaging. In vitro NIR-PIT-induced cytotoxicity was NIR-light dose dependent. In vivo NIR-PIT led to significant reduction in both tumor volume and luciferase activity in a flank model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). The PDPN-targeted NIR-PIT resulted in a significant antitumor effect in an MPM orthotopic mouse model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). This study suggests that PDPN-targeted NIR-PIT could be a new promising treatment for MPM.

Hypoxia Inducible Factor-1α Inhibition in Von Hippel Lindau-mutant Malignant Pleural Mesothelioma Cells.

Molecular targeted agents have been successfully developed against solid tumors and their use is also being investigated for the treatment of malignant pleural mesothelioma (MPM). We have previously reported von Hippel Lindau (VHL) mutations detected by massive parallel sequencing technology in samples of patients with MPM. Here, we conducted an in vitro study to investigate the therapeutic approaches in VHL-mutant MPM. Three MPM cell lines with or without a VHL mutation were used and the effects of molecular-targeted agents on growth inhibition were evaluated. Based on the characteristics of the molecular targeted agents that exhibited growth inhibitory effect, the effects of knockdown by siRNA were also evaluated. NCI-H28 MPM cells harboring the VHL L89H mutation were sensitive to YC-1, known as an inhibitor of hypoxia inducible factor (HIF)-1α, and YC-1treatment induced massive apoptosis in a dose-and-time-dependent manner. Knockdown of HIF-1α by siRNA partially inhibited the growth of NCI-H28 cells, suggesting that an additional blockade may be required to completely inhibit growth signaling. The VHL mutation may predict tumor responses to YC-1, a HIF-1α inhibitor.

Bromodomain Inhibitor JQ1 as a Candidate Therapeutic Agent in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare tumor that develops from the mesothelial linings of the pleural, pericardial, and peritoneal cavities. The actual risk factor for developing the disease is exposure to asbestos in workplace. Bromodomain and extraterminal (BET) domain proteins are epigenetic signaling agents that associate with acetylated histones and expedite the transcription of target genes. This study investigates whether the small molecule BET protein inhibitor JQ1 specifically may be an effective therapy for MPM. Reverse transcriptase polymerase chain reaction methods reveal an inclusive change in gene expression implying that JQ1 is a potential inhibitor which targets the BET proteins. Our results report that JQ1 has tumor-suppressive effects as it significantly ceased cellular activity in MPM cell lines. We predict that JQ1 may be the promising therapy for pleural mesothelioma cancers.

Frequent Homozygous Deletions of Type I Interferon Genes in Pleural Mesothelioma Confer Sensitivity to Oncolytic Measles Virus.

Oncolytic immunotherapy is based on the use of nonpathogenic replicative oncolytic viruses that infect and kill tumor cells exclusively. Recently, we found that the spontaneous oncolytic activity of the Schwarz strain of measles virus (MV) against human malignant pleural mesothelioma (MPM) depends on defects in the antiviral type I interferon (IFN-I) response in tumor cells. In this study, we studied three independent human MPM bio-collections to identify the defects in the IFN-I responses in tumor cells. We show that the most frequent defect is the homozygous deletions (HDs) of all the 14 IFN-I genes (IFN-α and IFN-β) that we found in more than half of MV-sensitive MPM cell lines. These HDs occur together with the HDs of the tumor suppressor gene CDKN2A also located in the 9p21.3 chromosome region. Therefore, the IFN-I-/- MPM cell lines develop a partial and weak IFN-I response when they are exposed to the virus compared with that of normal cells and MV-resistant MPM cell lines. This response consists of the expression of a restricted number of IFN-stimulated genes that do not depend on the presence of IFN-I. In addition, the IFN-I-/- MPM cell lines infected by MV also develop a pro-inflammatory response associated with stress of the endoplasmic reticulum. Our study emphasizes the link between HDs of IFN-I encoding genes and the CDKN2A gene in MPM and sensitivity to MV oncolytic immunotherapy.

Putative Biomarkers for Malignant Pleural Mesothelioma Suggested by Proteomic Analysis of Cell Secretome.

Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiver-operating characteristic analysis predicted a good diagnostic accuracy. A panel of the putative biomarkers represents a promising tool for MPM diagnosis.