P3.03-010 Activation of p53 in Malignant Mesothelioma: Topic: Mesothelioma Transitional

Abstract

Certain microRNA (miRNA)-mRNA interactions are associated with critical biologic processes in malignant pleural mesothelioma (MPM). We wondered how miRNA interact with p53 in MPM since this tumor characteristically retains the wild-type allele which is frequently bypassed by deletion of CDKN2A. This interaction among miRNA and p53 in MPM is poorly understood. Study of this interaction could provide insights on disease mechanisms and/ or novel therapeutic strategies. We retrieved several public miRNA expression MPM data sets to perform a broad survey. We combined two meta-analyses approaches to the normalized data to maximize identifying altered miRNA specific to MPM. Then miRNAs were fit into a network where they inhibited MDM2, releasing inhibition of p53, and in turn were themselves induced by p53 (p53 regulation via a reinforcing loop). Significant miRNA of this screening algorithm were confirmed by qPCR analysis in MPM tissues and cell lines. Specific miRNAs were re-expressed in MPM cells by a lentivirus system or by mimic transfection. p53-luciferase reporter system was used to assess p53 activity. MTS assay assessed cell proliferation. Apoptotic cells were detected by Annexin-V assay. Tumorigenic characteristics of MPM cells were evaluated by clonogenicity, soft agar colony formation and 3D sphere assays. Clinical relevance of these miRNA were assessed in the TCGA MPM cohort (cancergenome.nih.gov). Our meta-analysis, revealed significant changes in several p53-regulated miRNAs. For example, miR-145 expression is repressed by 40% at steady state in MPM specimens (n=38) compared to non-malignant controls (n=21). We directly confirmed in MPM tissues a similar trend of this miRNA, while MDM2 mRNA levels were inversely higher. Next, we assessed the functional role of miR-145 in MPM cell lines with wild-type p53. Using a lentiviral expression system to sustain elevations in miR-145 levels, MDM2 transcript and protein levels were repressed, leading to increases in p53 protein and its transcriptional activity. We observed in miR-145-overexpressed MPM cell lines more apoptosis by Annexin V assay, loss of clonogenicity, growth inhibition, and attenuated tumorigenicity. To confirm that p53 can perpetuate a positive reinforcing loop inducing miR-145, we treated a panel of MPM cells with Nutlin-3a and observed coordinated increases in p53 associated with a rise in miR-145 levels. Interestingly, at least one of these miRNA was prognostic in Kaplan-Meier modeling of overall survival. We have identified candidate miRNAs that, in part, regulate p53 activity in MPM cells. These miRNAs function as tumor suppressors. They are candidates for therapeutic validation.