KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

© 2016 Lin et al. Background: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. Methods: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. Results: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. Conclusion: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets

Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma

© 2017 International Association for the Study of Lung Cancer Introduction The upregulation of programmed death ligand 1 (PD-L1) is found in many cancers and contributes to evasion of the host's immune defense. In malignant pleural mesothelioma (MPM), PD-L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD-L1 expression in MPM are poorly understood. To address one possible means of PD-L1 regulation we investigated the relationship between dysregulated microRNA levels and PD-L1 expression. Methods PD-L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse-transcriptase quantitative polymerase chain reaction. Regulation of PD-L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD-L1 was analyzed by using argonaute-2 immunoprecipitation and a luciferase reporter assay. Results In a series of 72 patients with MPM, 18 (25%) had positive PD-L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD-L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD-L1 expression [p < 0.001]), and in multivariate analyses, PD-L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2–4.1, p < 0.01). In the same patient series, PD-L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR-15b, miR-16, miR-193a-3p, miR-195, and miR-200c were significantly lower in the PD-L1–positive samples. Transfecting MPM cell lines with mimics corresponding to miR-15a and miR-16, both of which are predicted to target PD-L1, led to downregulation of PD-L1 mRNA and protein. In addition, miR-193a-3p, with an alternative G-U–containing target site, also caused PD-L1 downregulation. Conclusions Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD-L1 expression in MPM

Dysregulated Expression of the MicroRNA miR-137 and Its Target YBX1 Contribute to the Invasive Characteristics of Malignant Pleural Mesothelioma

© 2017 International Association for the Study of Lung Cancer Introduction: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure. On a genomic level, MPM is characterized by frequent chromosomal deletions of tumor suppressors, including microRNAs. MiR-137 plays a tumor suppressor role in other cancers, so the aim of this study was to characterize it and its target Y-box binding protein 1 (YBX1) in MPM. Methods: Expression, methylation, and copy number status of miR-137 and its host gene MIR137HG were assessed by polymerase chain reaction. Luciferase reporter assays confirmed a direct interaction between miR-137 and Y-box binding protein 1 gene (YBX1). Cells were transfected with a miR-137 inhibitor, miR-137 mimic, and/or YBX1 small interfering RNA, and growth, colony formation, migration and invasion assays were conducted. Results: MiR-137 expression varied among MPM cell lines and tissue specimens, which was associated with copy number variation and promoter hypermethylation. High miR-137 expression was linked to poor patient survival. The miR-137 inhibitor did not affect target levels or growth, but interestingly, it increased miR-137 levels by means of mimic transfection suppressed growth, migration, and invasion, which was linked to direct YBX1 downregulation. YBX1 was overexpressed in MPM cell lines and inversely correlated with miR-137. RNA interference–mediated YBX1 knockdown significantly reduced cell growth, migration, and invasion. Conclusions: MiR-137 can exhibit a tumor-suppressive function in MPM by targeting YBX1. YBX1 knockdown significantly reduces tumor growth, migration, and invasion of MPM cells. Therefore, YBX1 represents a potential target for novel MPM treatment strategies