591. MiRNAs Regulates PVRL4 and Oncolytic Measles Virus Infectivity in Glioblastoma and Breast Cancer

Abstract

Oncolytic measles virus strains are currently being evaluated in several clinical trials including glioblastoma and ovarian cancer, while clinical application in breast cancer is in development. The role of poliovirus receptor-related 4 (PVRL4), as a measles virus receptor, has been well documented. However the regulation of PVRL4 is not well understood. Immunohistochemistry and western blot analysis shows increased PVRL4 protein levels in glioblastoma and breast tumor clinical samples with no significant change in PVRL4 mRNA levels detected by qRT-PCR, suggesting that PVRL4 is likely post-transcriptionally regulated. Genes can be post-transcriptionally regulated by miRNAs by binding to the 3’UTR of the targeted mRNA causing translational repression or degradation. Thus, we sought to investigate the potential role of miRNAs in PVRL4 post-transcriptional regulation. Algorithms from microRNA. org and RegRNA, predicted that, from the miRNAs that are reportedly down-regulated in glioblastoma and breast cancer, miR-31 and miR-128 binds the 3’UTR of PVRL4. Indeed, luciferase binding assays demonstrated miR-31 and-128binding to the 3’UTR of PVRL4, suggesting that PVRL4 is a miRNA targeted gene. QRT-PCR studies validated that miR-31 and miR-128 are down-regulated in glioblastoma and breast tumor clinical samples, further supporting the hypothesis that PVRL4 are post-transcriptionally regulated by miRNAs. In order to understand the effects of the respective miRNAs on PVRL4 and thus on measles virus infectivity, a viral strain (Leonard et. al, 2010, J. Virol.) that exclusively utilizes PVRL4 as its receptor was employed to infect the cancer cell lines followed by evaluation of virus infectivity via measles viral nucleoprotein (N-protein) levels. Subsequent gain -and loss-of-function experiments showed that overexpression of miR-31and miR-128, via lentiviral-vector transduction, down-regulated the endogenous levels of PVRL4 and measles virus infectivity, while transduction of lentiviral-vectors expressing PVRL4 restored measles virus infectivity. The functional affects were reversed or increased upon transduction with the complementary anti-miRs-128 and anti-miR-31. These results are being further validated in ongoing in vivo mouse models where cancer cell lines stably transduced with miR-128, miR-31 or anti-miR-128, anti-miR-31, are transplanted in mice to evaluate tumor size and survival rates upon measles virus infection. These data suggest that PVRL4 is post-transcriptionally regulated by miR-128 and miR-31, and thus, these represent possible miRNA targets that could modulate PVRL4 levels in order to enhance oncolytic measles virus infectivity.This research is supported by P50 CA 108961-7, P50 CA 136393-4, P50 CA 116201-7 and R01 CA 136547-4.