Abstract B37: Investigating the potential of miR-203 as a therapeutic candidate and its role in the pathobiology of malignant pleural mesothelioma (MPM)

Background: MPM is a lethal neoplasm of the pleural layer of cells surrounding lungs and is in dire need of newer therapeutic treatments. microRNA9s (miRs) play a critical role in the pathobiology of many cancers but there are few reports investigating their function in MPM. Recently many studies have highlighted the potential of miRs as therapeutic agents in cancer. We completed a microarray profiling strategy to discover miRs of therapeutic and biological importance in MPM. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Agilent v3 Human miR microarrays. These were scanned and the data was processed using the AgiMicroRna “R” package involving background correction, quantile normalization and summarization. The microarray results were validated by quantitative reverse transcription polymerase chain reaction (qRTPCR) using Taqman assays on the ABI 7300 platform. Also qRT-PCR was employed to determine the levels of miR-203 expression in a panel of 26 mesothelioma cell lines including Met-5A, an SV-40 immortalized pleural mesothelial control cell line. For all qRT-PCR experiments the miR-203 levels were determined relative to endogenous miR-U6 as control using ΔΔCT calculation. Moreover in a previous study RNA from these same tissue specimens were hybridized on Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Results: The bioinformatic analysis of miR microarray data showed that a number of miR9s, including miR-203, are differentially expressed between the tumor and normal samples. A paired T-test conducted on a miRNA-by-miRNA basis and at a highly significant FDR value of 1e-06 showed miR-203 to be down regulated, more than 2 fold, in tumors compared to paired normal tissue. We decided to explore the role of miR-203 in the pathogenesis of MPM since it has been postulated to play a tumor suppressor role in skin and prostate cancer by inhibiting proliferation, metastasis and acting antagonistic to stem cells (1, 2). Using qRT-PCR, we compared levels of expression in 40 pairs of tumor vs. paired normal tissues and demonstrated that miR-203 was down regulated to more than 10 fold in MPM tumors. Also qRT-PCR showed that 70 % of 25 MPM cancer cell lines had lower expression of miR-203 compared to Met-5A cell line. Our previous transcriptomic profiling study had shown differentially expressed transcripts between these same tumors and paired normal specimens. Survivin (BIRC5) message level, which codes for an apoptosis inhibitor protein and reported to be regulated by miR-203 in prostate cancer cell lines (3), was found to be 2.7 fold higher in tumors (p = 2.00e-15) in an expected anti-correlation direction to miR-203 expression levels (i.e. low miR = high target mRNA). Conclusion: Our findings suggest that miR-203 may play a tumor suppressor role in pleural mesothelioma and regulate levels of survivin message. Supported by Grants: DoD W81XWH-07-1-0306 (IW and AT), Fleming Foundation, IASLC Young Investigator Award 2011–2013 (MS).