Androgen Receptor-Mediated Nuclear Transport of NRDP1 in Prostate Cancer Cells Is Associated with Worse Patient Outcomes.

Abstract

Simple SummaryNRDP1 is an E3 ubiquitin ligase that has been shown by our group and others to target ErbB3 for proteasomal degradation in prostate and breast cancer cells and thereby decrease the likelihood cancer progression. Our group has found that NRDP1 can be located in the nucleus as well as the cytoplasm of prostate cancer (CaP) cells, which is unexpected as NRDP1 lacks a nuclear localization signal. Here we elucidate the mechanism by which nuclear translocation of NRDP1 can occur and demonstrate that nuclear NRDP1 retains its ubiquitin ligase activity. Our patient data and cell line studies indicate that increased levels of nuclear NRDP1 contributes CaP progression, thereby underscoring the clinical relevance of our findings and supporting continued investigation and elucidation of the specific role(s) played by NRDP1 in the nucleus of CaP cells.To our knowledge, our group is the first to demonstrate that NRDP1 is located in the nucleus as well as the cytoplasm of CaP cells. Subcellular fractionation, immunohistochemistry, and immunofluorescence analysis combined with confocal microscopy were used to validate this finding. Subcellular fractionation followed by western blot analysis revealed a strong association between AR and NRDP1 localization when AR expression and/or cellular localization was manipulated via treatment with R1881, AR-specific siRNA, or enzalutamide. Transfection of LNCaP with various NRDP1 and AR constructs followed by immunoprecipitation confirmed binding of NRDP1 to AR is possible and determined that binding requires the hinge region of AR. Co-transfection with NRDP1 constructs and HA-ubiquitin followed by subcellular fractionation confirmed that nuclear NRDP1 retains its ubiquitin ligase activity. We also show that increased nuclear NRDP1 is associated with PSA recurrence in CaP patients (n = 162, odds ratio; 1.238, p = 0.007) and that higher levels of nuclear NRDP1 are found in castration resistant cell lines (CWR22Rv1 and PC3) compared to androgen sensitive cell lines (LNCaP and MDA-PCa-3B). The combined data indicate that NRDP1 plays a role in mediating CaP progression and supports further investigation of both the mechanism by which nuclear transport occurs and the identification of specific nuclear targets.