Presentation Authors: David Greenwald*, Dan-Ping Hu, Wen-Yang Hu, Gail Prins, Chicago, IL
Introduction: Speckle-type POZ Protein (SPOP) is the most frequently mutated gene in primary prostate cancer (PCa). The overall risk of PCa has been reported up to 3.8-fold for men with BRCA1 mutations. Both SPOP and BRCA1 act as E3 ubiquitin ligases, each targeting several proteins, including estrogen receptor-Î± (ER-Î±), for proteasome-mediated degradation. Previously our laboratory identified ER-Î± and estrogen receptor-Î² (ER-Î²) in prostate stem cells (PSC). On PSC, estrogen activation of ER-Î± stimulates PSC to self-renew, whereas estrogen activation of ER-Î² inhibits PSC self-renewal, acting as a â€œbrakeâ€ for PSC homeostasis. Further work found that ER-Î± down-regulates ER-Î² in PSC thus forming a tight regulatory loop for controlling PSC self-renewal by estrogens. As such, increased PSC ER-Î± levels reduce ER-Î² levels. This effectively removes the â€œbrakeâ€ to enable an increase in PSC self-renewal. Importantly, estrogen exposure has also been shown to promote prostate carcinogenesis. The objective of this study was to determine if SPOP & BRCA1, via ER-Î± selective E3 ligase activity, regulate PSC ER-Î± levels and whether this affects PSC proliferation.
Methods: Primary cultures of normal human prostate epithelial cells (PrEC) were grown in a prostasphere-based, bromodeoyuridine (BrdU) label-retention assay that permits identification of initiating stem cells within the prostasphere (PS). PrEC were subjected to ER-Î± selective E3 ligase knockdown with siRNA transfection (SPOP and/or BRCA1), confirmed by RT qPCR. Fluorescence immunocytochemistry (ICC) was used to visualize ER-Î± & ER-Î² protein levels in BrdU labeled stem cells, and quantified with Image J software.
Results: SPOP & BRCA-1 knockdowns in PrEC and prostaspheres were achieved with a mean 29% (8-54%) efficiency and mean 21% (9-36%) efficiency, respectively. ICC revealed a statistically significantly elevated level of ER-Î± in BrdU label-retaining stem cells of both SPOP and BRCA1 knockdowns compared to scramble siRNA controls. ICC also revealed a statistically significantly suppressed level of ER-Î² in BrdU label-retaining stem cells of both SPOP and BRCA1 knockdowns compared to scramble siRNA controls.
Conclusions: The results indicate that both SPOP & BRCA1, via ER-Î± selective E3 ligaseÂ¬ activity, regulate ER-Î± levels which in turn affects ER-Î² levels in PSC. Results also indicate that these alterations lead to changes in the ability of PrEC to form PS, indicating changes in PSC self-renewal capacity. We propose that functional SPOP & BRCA1 mutations may lead to higher stem cell ER-Î± expression, which may be pro-oncogenic in primary PCa.