LAUSR

Low serum vitamin D is a risk factor for aggressive prostate cancer. piRNAs and their interacting partners, PIWI-like (PIWIL) proteins, are aberrantly expressed in many cancers, but few studies have focused on piRNAs in the prostate. We used laser-capture microdissected (LCM) human prostate epithelium from frozen samples collected for a clinical trial of high dose vitamin D to examine the small non-coding RNA population by next-generation sequencing. The 8 patients for this study were selected for having either high (avg=48.7pg/ml) or low (avg=21.4pg/ml) prostatic vitamin D (1,25(OH)2D) levels. VDR ChIP-seq was performed as well, using human primary epithelial cells treated for 3hr with EtOH or 50nM vitamin D. In LCM-collected prostate epithelium unexpectedly piRNAs represented ~20% of the mapped reads. Of all the small RNA species investigated, piRNAs were the most affected by vitamin D status, increasing in abundance in tissues wish high vitamin D (p=0.036, t-test). The LCM data were compared to small-RNA sequencing data from benign prostate available from The Cancer Genome Atlas (TCGA), which represent whole prostate tissue and may contain stromal bias. The LCM-collected samples had a distinct, more consistent profile of small RNAs than TCGA samples. Two unambiguous piRNAs that were highly expressed in LCM data, but not in TCGA data, were verified as epithelial-specific in prostate by in situ hybridization. Several piRNA-biogenesis genes lie within 100kb of VDR ChIP peaks, including TDRKH, which was found to be vitamin D-regulated experimentally. The expression of piRNA-interacting PIWIL proteins was assessed by RT-qPCR, immunoblot and immunofluorescence on a tissue microarray containing paired benign and cancer tissues (N=23). Of the 4 human PIWIL proteins, PIWIL1, 2 and 4 were present in normal prostate epithelial cells by PCR. PIWIL1 protein was also detected in PrE and RWPE-1 benign cells, but PIWIL1 was not detected in the PC3 prostate cancer cell line. By immunofluorescence, PIWIL1 protein in prostate epithelium significantly decreased in prostate cancer (p=0.016, paired t-test). In conclusion, vitamin D status associated with the expression of piRNA in prostate epithelium in clinical trial samples. PIWIL1 protein levels were lower in prostate cancer, suggesting a pro-differentiation role for the PIWI pathway that may be affected in vitamin D-deficient patients. Citation Format: Bethany Baumann, Shang Gao, Giovanni Lugli, Larisa Nonn. The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2456.