ODP440 Characterization of the Major Single Nucleotide Polymorphic Variants of Aldo-Keto Reductase 1c3 (Type 5 17β-Hydroxysteroid Dehydrogenase)

Abstract

Abstract Background Aldo-keto-reductase (AKR) 1C3, also known as type 517β-hydroxysteroid dehydrogenase and prostaglandin F synthase,is a member of the AKR superfamily that reduces aldehydes and ketones to primary and secondary alcohols. It plays an essential role in the peripheral formation of androgens and is associated with several steroid hormone dependent diseases including prostate cancer (PC), breast cancer, and polycystic ovarian syndrome (PCOS). Furthermore, AKR1C3 is implicated in castration resistant prostate cancer (CRPC) drug resistance on the basis that it is upregulated in CRPC tumors and metastases and overexpressed in cell culture & xenograft models of Abiraterone and Enzalutamide resistance, where resistance is surmounted by AKR1C3 knockdown or treatment with the AKR1C3 inhibitor indomethacin. AKR1C3 has 14 nonsynonymous single nucleotide polymorphisms (nsSNPs) with different global frequencies and ethnic distributions. Association studies support their role in PC and other diseases, but detailed functional genomic studies of these variants are lacking. One study examined5 AKR1C3 nsSNPs for their ability to reduce exemestane, an aromatase inhibitor used to treat breast cancer, to 17β-dihydroexemestane, and reported a 17-250-fold reduction in catalytic efficiency of H5Q, E77G, K104D, and R258C variants compared to WT. This observation provided the impetus to examine impact of these variants on AKR1C3 function in the context of PC. We purified AKR1C3 WT, and the top four most frequently occurring nsSNPs, H5Q, E77G, K104D, and R258C, from E. coli to expand upon their characterization and illuminate functional differences that could affect disease outcome and treatment. METHODS: Kinetic measurements were made using either a continuous spectrophotometric assay monitoring the appearance of the reduced cofactor NADH, or a discontinuous RP UV-HPLC assay based on chromatographic separation of substrate and product. Inhibitor sensitivity studies were conducted with a fluorescence-based plate-reader assay monitoring enzyme activity via oxidation of a standard substrate in the presence of increasing inhibitor. Stability was assessed by monitoring the percent of maximum activity remaining via oxidation of a standard substrate over a range of increasing temperatures for each variant. Results We report no major deviations in kinetics in the top four most frequently occurring AKR1C3 variants compared to WT. Additionally, variants did not display differences in inhibitor sensitivity to competitive inhibitor indomethacin. However, the K104D variant lost activity at a lower temperature than WT, suggesting that this mutation results in a decrease in stability. Conclusions Altogether, these findings contrast with previous claims that AKR1C3 variants, mainly H5Q, give rise to significant advantage or disadvantage over WT in PC and other diseases, and suggests that patients with AKR1C3 dependent diseases would likely be overall unaffected by variant status. Presentation: No date and time listed