Understanding the mechanism of warfarin-mediated PC prevention.

Abstract

e635 Background: Multi-institution retrospective trials have shown that prolonged use of warfarin reduces the risk of prostate cancer (PC), an effect that is not observed for other cancers. The anti-coagulant warfarin prevents the gamma-carboxylation (gla) of target proteins by interfering with the vitamin K cycle through its inhibition of the vitamin K epoxide reductase (VKOR). Most known gla proteins are found in the clotting cascade, but here we investigate the role of gla of the androgen receptor (AR) and other warfarin-mediated effects in the prevention of PC by warfarin. Methods: We treated mice with warfarin and performed RNA-seq on prostate tissues. We used immunoprecipitation (IP) followed by mass spec sequencing to identify gla sites on AR. We used cell culture models to investigate the role of AR-gla and other pathways identified by RNA-seq data in the mechanism of action of warfarin. Finally, we sequenced a SNP in VKOR and performed IHC to detect VKOR expression in prostatectomy tissues in an institutional series. Results: RNA-seq data show alteration of several pathways in warfarin-treated mouse prostates, including AR signaling, hormone and lipid synthesis, and genes known to affect PC (e.g. SPINK1). Mass spec shows that AR is gla’d at amino acid 2E, which is known to be mutated in partial androgen insensitivity syndrome patients. Warfarin inhibits AR conformation change and transcriptional activity in benign (non-cancerous) prostate cells. VKOR knock-down recapitulates effects of warfarin treatment, suggesting an on target effect of the drug. Mutation of the AR-gla site has modest effects in benign prostate cells. In a small series of patients, we found that one allele of a VKOR SNP is found at a significantly higher rate in PC patients than expected. We also found that VKOR is highly expressed in normal human prostate epithelia but is lost in cancerous tissue, a pattern of expression that is unique to PC. Conclusions: Warfarin, likely through its ability to control AR-gla, inhibits AR transcriptional activity. The relative contribution of modest AR inhibition and the effect on other pathways must be investigated to determine how warfarin reduces the risk of PC and to identify optimal drug targets for development of chemopreventative therapeutics.