Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities.

Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids to drive cellular proliferation. Cytochrome P450 17A1 (CYP17A1), an essential enzyme for sex steroid synthesis, represents a clinically established drug target. Inhibiting CYP17A1 decreases androgen and estrogen biosynthesis and thereby blocks the growth of hormone-dependent cancers. The first part of this dissertation characterizes the previously unreported interaction between the FDA approved CYP17A1 inhibitor, abiraterone, and the estrogen receptor (ER). We show for the first time that abiraterone is a weak ER agonist in preclinical models of ER-positive breast cancer cells. Abiraterone induces cellular growth and expression of the ER response gene, GREB1, by binding to ER, and these effects are inhibited with the ER antagonist fulvestrant (ICI 182,780). To further investigate the impact of CYP17A1 expression in breast cancer, we engineered ER-positive MCF-7 cells to express CYP17A1 (MCF-7/CYP17A1). Progesterone treatment induces cell growth and GREB1 expression in these cells but not in the parental MCF-7 cells, which do not express CYP17A1. Tandem mass spectrometry (LC-MS/MS) analysis confirmed that following progesterone treatment, MCF-7/CYP17A1 cells synthesize downstream steroid products that require CYP17A1 activity including 17OH-progesterone, androstenedione, and testosterone. Treatment of these cells with either abiraterone or a novel CYP17A1 inhibitor decreases progesterone metabolite-induced GREB1 expression in a dose-dependent manner. In addition to studies of CYP17A1 in breast cancer, we further hypothesized that characterization of CYP17A1 genetic variants may lead to insights on enzyme structure and function. We therefore utilized a HEK-293T cell-based expression system to characterize the enzymatic properties of two CYP17A1 gene variants, D216H (rs200063521) and G162R (rs141821705). Our results show that the D216H variant selectively alters 16OH-progesterone production, while no effect on 17OH-progesterone synthesis was observed. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to wild-type. Proteasome inhibition with MG132 indicated that this variant is preferentially ubiquitinated and degraded prematurely. Overall, these studies have broadened our understanding of CYP17A1 enzymatic activity in breast cancer, as well as led to new insights into how CYP17A1 structure relates to enzyme function and stability.PhDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120831/1/ccapper_1.pd