Login to Access Video or Poster Abstract: MP99-02
Sources of Funding: This work is supported in part by grants NIH/NCI CA140468, CA168601, CA179970, DOD PC130062, and US Department of Veterans Affairs, ORD VA Merits I01BX0002653.

Introduction

Most prostate cancer (PCa) patients receiving enzalutamide (Enza) develop drug resistance within 24 months of exposure. This creates a need to better understand the underlying causes of Enza resistance so that improved treatment methods can be developed. Previous studies demonstrate that uncontrolled intraprostatic androgen synthesis promotes Enza resistance. HSD3B1 is a key enzyme contributing to androgen synthesis and its expression is associated with PCa progression. The aim of this study is to determine the contribution of HSD3B1 to Enza resistance in PCa.

Methods

Enza resistant C4-2B PCa cells (C4-2B MDVR) were generated by chronically exposing parental C4-2B cells to increasing Enza concentrations (5-40 ?M) for >12 months and maintained in 20 µM Enza. Differences in gene expression between C4-2B MDVR and parental cells was determined by microarray and RNA-seq. HSD3B1 expression was knocked down in C4-2B MDVR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CD-FBS), or CD-FBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 µM Enza. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay. C4-2B MDVR cells were also treated with apigenin, which has been shown to reduce HSD3B1 expression, and cell number and PSA outcomes were determined under the previously mentioned treatment conditions.

Results

HSD3B1 expression is higher in C4-2B MDVR cells compared to parental C4-2B cells as measured by Microarray and RNA-seq. This correlates to increased intracrine androgens in C4-2B MDVR cells compared to C4-2B cells as examined by LC-MS. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CD-FBS+DHT and CD-FBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or luciferase activity in response to Enza. No response was seen in CD-FBS or CD-FBS+DHEA. Supplementation of parental C4-2B cells with 100 nM P5, but not DHEA, improved resistivity to Enza. Apigenin, a potential inhibitor of HSD3B1 expression, resensitized C4-2B MDVR cells to Enza.

Conclusions

HSD3B1 overexpression in C4-2B MDVR cells contributes to Enza resistance and modulation of this enzyme could be a viable strategy to improve Enza treatment response in PCa cells. HSD3B1 activity appears to be reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating Enza resistance.

Funding

This work is supported in part by grants NIH/NCI CA140468, CA168601, CA179970, DOD PC130062, and US Department of Veterans Affairs, ORD VA Merits I01BX0002653.