Normal Mesothelial Cell Line Research Articles

Small Extracellular Vesicle-Derived Circular RNA hsa_circ_0007386 as a Biomarker for the Diagnosis of Pleural Mesothelioma.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools.

Expression of CD24 gene in human malignant pleural mesothelioma and its relationship with prognosis

Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.

Small RNA-Seq Transcriptome Profiling of Mesothelial and Mesothelioma Cell Lines Revealed microRNA Dysregulation after Exposure to Asbestos-like Fibers

Environmental exposure to fibers of respirable size has been identified as a risk for public health. Experimental evidence has revealed that a variety of fibers, including fluoro-edenite, can develop chronic respiratory diseases and elicit carcinogenic effects in humans. Fluoro-edenite (FE) is a silicate mineral first found in Biancavilla (Sicily, Italy) in 1997. Environmental exposure to its fibers has been correlated with a cluster of malignant pleural mesotheliomas. This neoplasm represents a public health problem due to its long latency and to its aggression not alerted by specific symptoms. Having several biomarkers providing us with data on the health state of those exposed to FE fibers or allowing an early diagnosis on malignant pleural mesothelioma, still asymptomatic patients, would be a remarkable goal. To these purposes, we reported the miRNA transcriptome in human normal mesothelial cell line (MeT-5A) and in the human malignant mesothelioma cell line (JU77) exposed and not exposed to FE fibers. The results showed a difference in the number of deregulated miRNAs between tumor and nontumor samples both exposed and not exposed to FE fibers. As a matter of fact, the effect of exposure to FE fibers is more evident in the expression of miRNA in the tumor samples than in the nontumor samples. In the present paper, several pathways involved in the pathogenesis of malignant pleural mesothelioma have been analyzed. We especially noticed the involvement of pathways that have important functions in inflammatory processes, angiogenesis, apoptosis, and necrosis. Besides this amount of data, further studies will be designed for the selection of the most significant miRNAs to test and validate their diagnostic potential, alone or in combination with other protein biomarkers, in high-risk individuals' liquid biopsy to have a noninvasive tool of diagnosis for this neoplasm.

Dysregulation of microRNAs and tRNA-derived ncRNAs in mesothelial and mesothelioma cell lines after asbestiform fiber exposure

Experimental evidence demonstrated that fluoro-edenite (FE) can develop chronic respiratory diseases and elicit carcinogenic effects. Environmental exposure to FE fibers is correlated with malignant pleural mesothelioma (MPM). An early diagnosis of MPM, and a comprehensive health monitoring of the patients exposed to FE fibers are two clinical issues that may be solved by the identification of specific biomarkers. We reported the microRNA (miRNA) and transfer RNA-derived non coding RNA (tRNA-derived ncRNA) transcriptome in human normal mesothelial and malignant mesothelioma cell lines exposed or not exposed to several concentration FE fibers. Furthermore, an interactive mesothelioma-based network was derived by using NetME tool. In untreated condition, the expression of miRNAs and tRNA-derived ncRNAs in tumor cells was significantly different with respect to non-tumor samples. Moreover, interesting and significant changes were found after the exposure of both cells lines to FE fibers. The network-based pathway analysis showed several signaling and metabolic pathways potentially involved in the pathogenesis of MPM. From papers analyzed by NetME, it is clear that many miRNAs can positively or negatively influence various pathways involved in MPM. For the first time, the analysis of tRNA-derived ncRNAs molecules in the context of mesothelioma has been made by using in vitro systems. Further studies will be designed to test and validate their diagnostic potential in high-risk individuals' liquid biopsies.

ECM Mechanoregulation in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma is a relatively rare, but devastating tumor, because of the difficulties in providing early diagnosis and effective treatments with conventional chemo- and radiotherapies. Patients usually present pleural effusions that can be used for diagnostic purposes by cytological analysis. This effusion cytology may take weeks or months to establish and has a limited sensitivity (30%–60%). Then, it is becoming increasingly urgent to develop alternative investigative methods to support the diagnosis of mesothelioma at an early stage when this cancer can be treated successfully. To this purpose, mechanobiology provides novel perspectives into the study of tumor onset and progression and new diagnostic tools for the mechanical characterization of tumor tissues. Here, we report a mechanical and biophysical characterization of malignant pleural mesothelioma cells as additional support to the diagnosis of pleural effusions. In particular, we examined a normal mesothelial cell line (Met5A) and two epithelioid mesothelioma cell lines (REN and MPP89), investigating how malignant transformation can influence cellular function like proliferation, cell migration, and cell spreading area with respect to the normal ones. These alterations also correlated with variations in cytoskeletal mechanical properties that, in turn, were measured on substrates mimicking the stiffness of patho-physiological ECM.

Selection of microRNAs in extracellular vesicles for diagnosis of malignant pleural mesothelioma by in�vitro analysis

Malignant pleural mesothelioma (MPM) is a malignant tumor which is a challenge for diagnosis and is associated with a poor patient prognosis. Thus, early diagnostic interventions will improve the quality of life and life expectancy of these patients. Recently, cellular microRNAs (miRNAs) have been found to be involved in maintaining homeostasis, and abnormal miRNA expression has often been observed in various diseases including cancer. Extracellular vesicles (EVs) released by many cells contain proteins and nucleic acids. miRNAs are secreted from all cells via EVs and circulate throughout the body. In this study, culture media were passed sequentially through membrane filters 220–50 nm in size, and EVs with diameters of 50 to 220 nm (EVcap50/220) were collected. miRNAs (EV50-miRNAs) in EVcap50/220 were purified, and microarray analysis was performed. EV50-miRNA expression profiles were compared between MPM cells and a normal pleural mesothelial cell line, and six EV50-miRNAs were selected for further investigation. Of these, hsa-miR-193a-5p and hsa-miR-551b-5p demonstrated higher expression in MPM-derived EVcap50/220. These miRNAs reduced the expression of several genes involved in cell-cell interactions and cell-matrix interactions in normal pleural mesothelial cells. Our data suggest that hsa-miR-193a-5p and hsa-miR-551b-5p in EVcap50/220 could be diagnostic markers for MPM.

Normal mesothelial cell lines newly derived from human pleural biopsy explants.

Mesothelial cells are arranged as a monolayer on covering membranes that invest surfaces of body cavities like the pleura and peritoneum. Primary human mesothelial cell (HMC) cultures are needed for studying mesothelial cell homeostasis and developing disease models, such as wound healing or cancers. Remarkably, there is a paucity of useable HMC lines that are currently available that faithfully recapitulate normal in vivo phenotypic characteristics. Here, we present a strategy to recover HMC from human pleural tissue and to immortalize them for extended in vitro culturing. Human pleural membrane was harvested by minimally invasive surgical techniques. HMC were isolated using a two-step process combining explant cellular outgrowth from biopsy tissue and flow cytometry based on cell surface expression of cadherin-1 and CD71. Cell cultures were generated after lentiviral transfection with human telomerase. The new HMC cultures retain the same phenotypic traits and physiologic features as their in vivo counterparts, yet they can be adapted for short-term or long-term culture in large-scale in vitro experimentation. In particular, we generated a new HMC line harboring a germline mutation in breast cancer type-1-associated protein-1 (BAP1), a causal tumor suppressor gene, that could be instrumental to malignant mesothelioma research. Patient-specific, normal HMC may serve as novel discovery tools allowing more powerful research models of both normal physiology and disease processes. Our surgically driven approach leads to a limitless resource of novel mesothelial cell cultures.

Modulation of microRNA expression levels after naturally occurring asbestiform fibers exposure as a diagnostic biomarker of mesothelial neoplastic transformation.

Fluoro-edenite (FE) is a silicate mineral identified in the lava products of Monte Calvario from stone quarries located in the southeast of Biancavilla, a small city of the Etnean volcanic complex (Sicily, Italy). Inhalation of FE fibers has been associated with a higher incidence of Malignant Mesothelioma (MM), a highly aggressive neoplasm of the serosal membranes lining the pleural cavity. Only 5% of MM patients are diagnosed at an early stage and the median survival is approximate 6-12 months. Many diagnostic biomarkers have been proposed for MM. Several studies demonstrated that microRNAs (miRNAs) may be used as good non-invasive diagnostics, as well as prognostic biomarkers for various human diseases, including cancer. On these bases, the aim of the present study was to identify a set of miRNAs involved in the development and progression of MM and potentially used as diagnostic biomarkers. For these purposes, in silico analyses were performed on healthy/exposed to asbestos fibers subjects vs. patients with MM. These analyses revealed a set of miRNAs strictly involved in MM by merging the lists of miRNAs found differentially expressed in the three miRNA expression datasets analyzed. The result of these computational evaluations allowed the execution of functional in vitro experiments performed on normal pleural mesothelial cell line (MeT-5A) and MM cell line (JU77) in order to test the carcinogenetic effects and epigenetic modulation induced by FE exposure. The in vitro results showed that the expression levels of hsa-miR-323a-3p vary significantly in both supernatant- and cell-derived miRNAs derived from treated and untreated cells. Secreted and cellular hsa-miR-101-3p in MeT-5A treated with FE fibers and JU77cells showed different trends of expression. As regard hsa-miR-20b-5p, there was no differential expression between secreted and cellular hsa-miR-20b-5p. This miRNA has been shown a significant up-regulation in JU77cells vs. control and treated MeT-5A. As a future plan, translational analyses will be performed on a subset of patients chronically exposed to FE fibers to further verify the clinical role of such miRNAs in high-risk individuals and their possible use as biomarkers of FE exposure or MM early onset.

PRMT5 silencing selectively affects MTAP-deleted mesothelioma: In vitro evidence of a novel promising approach.

Malignant mesothelioma (MM) is an aggressive asbestos‐related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)‐deficient cancers, in which the accumulation of the substrate 5'‐methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the MTAP gene is frequently codeleted with the adjacent cyclin‐dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock‐down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP‐deleted MM cells. We also observed that PRMT5 knock‐down in MTAP‐deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial‐to‐mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP‐deleted MMs.

EIF4E regulates STEAP1 expression in peritoneal metastasis.

Gastric cancer is the most prominent form of malignancy in China, and the high mortality associated with it is mostly due to peritoneal metastasis. We have previously elucidated that the RNA-binding protein poly r(C) binding protein 1 (PCBP1) and miR-3978 function as repressors of peritoneal metastasis, partially by downregulation of six-transmembrane epithelial antigen of the prostate 1 (STEAP1). We now show that STEAP1 is regulated at the level of cap-dependent translation initiation by phosphorylated eIF4E. Chemically inhibiting phosphorylation of eIF4E or genetic ablation of phosphorylated eIF4E inhibit translational upregulation of STEAP1 in the peritoneal metastasis mimicking cell line MKN45 in comparison to the normal mesothelial cell line HMrSV5. Thus phosphorylation of eIF4E is required for peritoneal metastasis of gastric cancer via translational control of STEAP1. Chemical inhibitors targeting phosphorylation of eIF4E or its interaction with the translation initiation complex thus might prove effective in treating patients with peritoneal metastasis.

Therapeutic potential of targeting S100A11 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with an unfavorable prognosis. The standard therapeutic approaches are limited to surgery, chemotherapy, and radiotherapy. Because the consequent clinical outcome is often unsatisfactory, a different approach in MPM treatment is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancers. Interestingly, it has been found that intracellular and extracellular S100A11 have different functions in cell viability. In this study, we focused on the impact of extracellular S100A11 in MPM and explored the therapeutic potential of an S100A11-targeting strategy. We examined the secretion level of S100A11 in various kinds of cell lines by enzyme-linked immunosorbent assay. Among them, six out of seven MPM cell lines actively secreted S100A11, whereas normal mesothelial cell lines did not secrete it. To investigate the role of secreted S100A11 in MPM, we inhibited its function by neutralizing S100A11 with an anti-S100A11 antibody. Interestingly, the antibody significantly inhibited the proliferation of S100A11-secreting MPM cells in vitro and in vivo. Microarray analysis revealed that several pathways including genes involved in cell proliferation were negatively enriched in the antibody-treated cell lines. In addition, we examined the secretion level of S100A11 in various types of pleural effusions. We found that the secretion of S100A11 was significantly higher in MPM pleural effusions, compared to others, suggesting the possibility for the use of S100A11 as a biomarker. In conclusion, our results indicate that extracellular S100A11 plays important roles in MPM and may be a therapeutic target in S100A11-secreting MPM.

Novel ATP‐competitive Akt inhibitor afuresertib suppresses the proliferation of malignant pleural mesothelioma cells

Malignant pleural mesothelioma (MPM), an asbestos‐related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti‐1/2, AZD5363, GSK690693, ipatasertib, MK‐2206, perifosine, PHT‐427, and TIC10, on six MPM cell lines, namely, ACC‐MESO‐4, Y‐MESO‐8A, MSTO‐211H, NCI‐H28, NCI‐H290, and NCI‐H2052, and a normal mesothelial cell line MeT‐5A. Comparison of IC 50 values of the Akt inhibitors showed that afuresertib, an ATP‐competitive specific Akt inhibitor, exerted tumor‐specific effects on MPM cells. Afuresertib significantly increased caspase‐3 and caspase‐7 activities and apoptotic cell number among ACC‐MESO‐4 and MSTO‐211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G1 phase. Western blotting analysis showed that afuresertib increased the expression of p21WAF 1/ CIP 1 and decreased the phosphorylation of Akt substrates, including GSK‐3β and FOXO family proteins. These results suggest that afuresertib‐induced p21 expression promotes G1 phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin‐induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM.

Abstract 30: S100A11 is a prominent therapeutic target in malignant pleural mesothelioma

Abstract [Background &amp; Purpose] Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells that show a serious malignancy in thoracic cavity with a median survival time of 9-12 months. The therapeutic approaches in clinical standard are limited to surgery and chemotherapy for early and advanced stages, respectively. The strategies unfortunately provide only palliation, thus further advanced approach is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancer tissues. It has been reported that S100A11 plays an important role in progression of many cancers derived from thyroid, ovarian and lung tissues, but its roles and signature in MPM are poorly understood. In this study, we investigated the impact of S100A11 in MPM cell lines and resected tumors. [Material &amp; Methods] We explored the expression of S100A11 in 7 MPM cell lines (HP-1, H28, MSTO-211H, H2052, H290, H2452, and YUMC44) and 2 normal mesothelial cell lines (Met-5A and LP-9). We analyzed the effect of S100A11 on cell proliferation, colony formation, migration, invasion and downstream signaling. The efficacy of anti-S100A11 antibody on cell proliferation and downstream signaling was also determined. [Results] We found that S100A11 protein was consistently upregulated in 7 MPM cell lines at a significant level in comparison to 2 normal mesothelial cells. The same phenomenon was also confirmed in MPM tissue sections by immunohistochemistry, i.e., S100A11 was strongly stained at MPM cells, but not at surrounding normal lung cells. Specific knockdown of S100A11 by small interfering RNA turned the aggressive cells into the attenuated phenotypes for proliferation, invasion and migration. Those were notably observed in both 2 MPM cell lines (H2052 and H2452). Interestingly, we found that MPM cell lines but not normal cells actively secreted the S100A11 protein. To pursue this, we next tried to inhibit the function of the secreted S100A11. Administration of S100A11 neutralizing antibody significantly inhibited the proliferation of 4 MPM cell lines (H2052, H2452, H28, and H290). The antibody had no effect on the proliferation of only one MPM cell line, MSTO-211H, which showed no secretion of S100A11. Taken together, these results suggest that S100A11 secreted from MPM cells can be a prominent target for effective MPM therapy. [Conclusion] Our results suggest that S100A11 is a possible therapeutic target in MPM. Citation Format: Hiroki Sato, Hiromasa Yamamoto, Kei Namba, Hidejiro Torigoe, Takahiro Yoshioka, Kazuhiko Shien, Junichi Soh, Shinichi Toyooka. S100A11 is a prominent therapeutic target in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2017-30

Targeting YAP in malignant pleural mesothelioma.

Malignant mesothelioma is an aggressive cancer that is resistant to current therapy. The poor prognosis of mesothelioma has been associated with elevated Yes‐associated protein (YAP) activity. In this study, we evaluated the effect of targeting YAP in mesothelioma. First, we comprehensively studied YAP activity in five mesothelioma cell lines (211H, H2052, H290, MS‐1 and H2452) and one normal mesothelial cell line (LP9). We found decreased phospho‐YAP to YAP protein ratio and consistently increased GTIIC reporter activity in 211H, H2052 and H290 compared to LP9. The same three cell lines (IC50s < 1 μM) were more sensitive than LP9 (IC50 = 3.5 μM) to the YAP/TEAD inhibitor verteporfin. We also found that verteporfin significantly reduced YAP protein level, mRNA levels of YAP downstream genes and GTIIC reporter activity in the same three cell lines, indicating inhibition of YAP signaling by verteporfin. Verteporfin also impaired invasion and tumoursphere formation ability of H2052 and H290. To validate the effect of specific targeting YAP in mesothelioma cells, we down‐regulated YAP by siRNA. We found siYAP significantly decreased YAP transcriptional activity and impaired invasion and tumoursphere formation ability of H2052 and H290. Furthermore, forced overexpression of YAP rescued GTIIC reporter activity and cell viability after siYAP targeting 3′UTR of YAP. Finally, we found concurrent immunohistochemistry staining of ROCK2 and YAP (P < 0.05). Inhibition of ROCK2 decreased GTIIC reporter activity in H2052 and 211H suggesting that Rho/ROCK signaling also contributed to YAP activation in mesothelioma cells. Our results indicate that YAP may be a potential therapeutic target in mesothelioma.

Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma.

Malignant mesothelioma is an aggressive cancer with limited therapeutic options. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) is a multifunctional protein with anti-cancer activity. The effects of pemetrexed, cisplatin, and cSBL were evaluated in mesothelioma and normal mesothelial cell lines. We evaluated cytotoxicity, apoptosis, caspase-3 cleavage and activation, cell proliferation, cell cycle arrest, and levels of cell cycle proteins in H28 cells treated with pemetrexed, cisplatin, and cSBL alone or in combination. Treatment with cSBL alone was cytotoxic to mesothelioma cells. The anti-cancer effect of cSBL was observed in a broader range of cell lines and exhibited greater cancer cell selectivity than pemetrexed or cisplatin. Combination treatment with pemetrexed + cSBL resulted in greater dose-dependent cytotoxicity than pemetrexed + cisplatin, the standard of care in mesothelioma. The synergistic effect of pemetrexed + cSBL was mediated by the cytostatic effect of pemetrexed and the cytotoxic effect of cSBL. It thus appears that cSBL has therapeutic potential for the treatment of mesothelioma.

Inhibition of NADPH oxidase 4 induces apoptosis in malignant mesothelioma: Role of reactive oxygen species.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is characterized by dysregulated growth and resistance to apoptosis. Reactive oxygen species (ROS)-generating NADPH oxidase (Nox) family enzymes have been suggested to be involved in neoplastic proliferation. Both the antioxidant N-acetylcysteine (NAC) and the inhibitor of flavoprotein-dependent oxidase, diphenylene iodonium (DPI), inhibited the cell viability of MPM cells in a dose-dependent manner. To examine whether Nox-mediated ROS generation confers antiapoptotic activity and thus a growth advantage to MPM cells, we analyzed the mRNA expression of Nox family members using quantitative RT-PCR in 7 MPM cell lines and a normal mesothelial cell line. Nox4 mRNA was expressed in all of the examined MPM cell lines, whereas little or no Nox2, Nox3 and Nox5 mRNA expression was detected. In 2 MPM cell lines, Nox4 mRNA expression was significantly higher than that in a normal mesothelial cell line. siRNAs targeting Nox4 suppressed ROS generation and cell viability in the MPM cell lines. In addition, DPI treatment and knockdown of Nox4 attenuated phosphorylation of AKT and ERK. Taken together, our results indicate that Nox4-mediated ROS, at least in part, transmit cell survival signals and their depletion leads to apoptosis, thus highlighting the Nox4-ROS-AKT signaling pathway as a potential therapeutic target for MPM treatment.

CK2α, over-expressed in human malignant pleural mesothelioma, regulates the Hedgehog signaling pathway in mesothelioma cells

BackgroundThe Hedgehog (Hh) signaling pathway has been implicated in stem cell maintenance and its activation is aberrant in several types of cancer including mesothelioma. Protein kinase CK2 affects several cell signaling pathways through the mechanism of phosphorylation.MethodsProtein and mRNA levels of CK2α and Gli1 were tested by quantitative RT-PCR and immunohistochemistry staining in mesothelioma samples and cell lines. Down-regulated Gli1 expression and transcriptional activity were demonstrated by RT-PCR, Western blot and luciferase reporter assay.ResultsIn this study, we show that CK2α is over-expressed and a positive regulator of Hegdehog/Gli1 signaling in human malignant pleural mesothelioma. First of all, we found that the mRNA levels of CK2α and Gli1 were broadly elevated and correlated (n = 52, r = 0.401, P < 0.05), compared with LP9 (a normal mesothelial cell line). We then investigated their expression at the protein level, and found that all the 7 mesothelioma cell lines tested showed positive staining in CK2α and Gli1 immunohistochemistry. Correlation analysis by Pearson test for CK2α and Gli1 expression in the 75 mesothelioma tumors and the 7 mesothelioma cell lines showed that the two protein expression was significantly correlated (n = 82, r = 0.554, P < 0.01). Furthermore, we demonstrated that Gli1 expression and transcriptional activity were down-regulated after CK2α was silenced in two mesothelioma cell lines (H28 and H2052). CK2α siRNA also down-regulated the expression of Hh target genes in these cell lines. Moreover, treatment with a small-molecule CK2α inhibitor CX-4945 led to dose-dependent inhibition of Gli1 expression and transcriptional activity. Conversely, forced over-expression of CK2α resulted in an increase in Gli1 transcriptional activity in H28 cells.ConclusionsThus, we report for the first time that over-expressed CK2α positively regulate Hh/Gli1 signaling in human mesothelioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0093-6) contains supplementary material, which is available to authorized users.

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

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

A novel microRNA-based treatment approach for malignant pleural mesothelioma.

7586 Background: Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches are needed. The microRNA miR-16 has been implicated as a tumor suppressor in a range of cancer types, and restoration of miR-16 expression has been shown to inhibit tumor cell proliferation. The miR-16 status in MPM is largely unknown. Methods: MicroRNA expression was analysed by TaqMan-based RT-qPCR in 10 MPM cell lines and 60 tumour specimens consisting of archival blocks from patients undergoing surgery. MicroRNA expression was restored in vitro using mimics corresponding to the sequence of the mature microRNA, and effects on proliferation and target genes were assessed with standard methods. Human xenograft-bearing mice were treated with miR-16 mimics packaged in minicells targeted with EGFR-specific antibodies. Results: Expression of miR-16 was consistently down-regulated in MPM cell lines and MPM tumor specimens as were the co-expressed miR-15a and miR-15b. A decrease of 2- to 5-fold in miR-16 expression was found when MPM cell lines were compared with the normal mesothelial cell line MeT-5A. When tumor specimens were compared with normal pleura, the down-regulation of miR-16 was in the order of 10-fold. Using synthetic miR-16 mimics to restore miR-16 expression in MPM cell lines led to time- and dose-dependent growth inhibition in a panel of MPM cell lines but did not affect growth of MeT-5A. Growth inhibition correlated with cell cycle arrest and concordant down-regulation of miR-16 target genes including Bcl-2 and CCND1. In a series of experiments with nude mice bearing MPM (MSTO-H211) xenografts, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of tumor growth. Conclusions: Restoring miR-16 expression represents a novel approach to treatment for MPM. Preparations are being made to test miR-16 packaged in minicells as a new treatment approach for patients with recurrent MPM and NSCLC.

Serum HMGB1 as a prognostic marker for malignant pleural mesothelioma

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