Abstract 2898: Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer

Background: Basic-helix-loop-helix proteins (BHLH) are transcription factors that play important roles in critical developmental processes in many organisms by repressing the transcription of various target genes. Previous studies showed that overexpression of the BHLHE40 (also known as DEC1, STRA13 or SHARP2) transcriptional repressor results in growth suppression, cell cycle arrest, and cellular senescence indicating that BHLHE40 may have important tumor suppressor functions. Studies showed that the expression of BHLHE40 is indeed downregulated in some tumors, intriguingly however, it is also overexpressed in many such as lung cancer, breast cancer. Our overall goal is to elucidate the function of BHLHE40 in prostate cancer (CaP) and gain an understanding of how it is regulated. Methods: Initial studies were based on qPCR analysis to compare the expression levels of BHLHE40 in different cell lines ranging from androgen-dependent LNCaP cells to the castration resistant C4, C4-2, C4-2b, R1, Rv1, PC3 and PC3 cells that stably express the androgen receptor (AR) (PC3-AR). Further analysis were done by treating LNCaP and C4-2 cells with 1 nM dihydrotestosterone (DHT) in media containing fetal bovine serum (FBS), or charcoal stripped serum (CSS). Western blot analysis was done to check the level of BHLHE40 protein in these cell lines. Knock-down of AR, or the use of AR inhibitors Enzalutamide and/or Bicalutamide were used to further assess the regulation of BHLHE40 by the AR. Results: qPCR comparison of BHLHE40 transcript levels in LNCaP versus C4, C4-2, R1, Rv1, PC3 cells showed that while LNCaP cells have high transcript levels of BHLHE40, the CRPC cells all had very low transcript levels of this transcription factor. When comparing LNCaP to PC3 and PC3-AR cells, it was interesting to note that PC3-AR cells showed significantly higher transcript levels of BHLHE40 than PC3 implying that BHLHE40 may be regulated by the AR pathway. Treatment with 1 nM DHT on these cells showed that BHLHE40 increases over time following DHT treatment. In contrast, inhibition of AR by Enzalutamide (2 μM) showed reduced BHLHE40 levels, although Bicalutamide (10 μM) did not significantly altered the expression level of BHLHE40 in LNCaP cells. Conclusions: Our results indicate that BHLHE40 is regulated by the AR pathway, although whether this is direct or indirect is yet to be determined. This observation is significant, since it indicates that the AR may be utilizing this transcription factor to suppress the transcription of other genes. Future studies will attempt to determine if BHLHE40 may have an androgen-responsive-element (ARE) in its promoter and via ChIP assay to identify possible binding sites for AR in conjunction with luciferase assay to assess androgen-receptor mediated regulation. Citation Format: Zsofia Kiss, Paramita Ghosh. Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2898.