Enzalutamide (MDV3100) is a second generation Androgen Receptor (AR) antagonist with proven efficacy in the treatment of castration resistant prostate cancer (CRPC). The majority of treated patients, however, develop resistance and disease progression and there is a critical need to identify novel targetable pathways mediating resistance. The purpose of this study was to develop and extensively characterize a series of enzalutamide-resistant prostate cancer cell lines. Four genetically distinct AR-positive and AR-pathway dependent prostate cancer cell lines (CWR-R1, LAPC-4, LNCaP, VCaP) were made resistant to enzalutamide by long-term culture (> 6 months) in enzalutamide. Extensive characterization of these lines documented divergent in vitro growth characteristics and AR pathway modulation. Enzalutamide-resistant LNCaP and CWR-R1 cells, but not LAPC-4 and VCAP cells, demonstrated increased castration-resistant and metastatic growth in vivo. Global gene expression analyses between short-term enzalutamide treated vs. enzalutamide-resistant cells identified both AR pathway and non-AR pathway associated changes that were restored upon acquisition of enzalutamide resistance. Further analyses revealed very few common gene expression changes between the four resistant cell lines. Thus, while AR-mediated pathways contribute in part to enzalutamide resistance, an unbiased approach across several cell lines demonstrates a greater contribution toward resistance via pleiotropic, non-AR mediated mechanisms.

The function and clinical utility of stem cell markers in metastatic castration-resistant prostate cancer (mCRPC) remains unresolved, and their expression may confer important therapeutic opportunities for staging and therapy. In the adult human prostate, CD133 (PROM1) expression identifies infrequent prostate epithelial progenitor cells and putative cancer stem cells. Previous work demonstrated an association with CD133 and cancer cell proliferation using in vitro model systems. The primary objective here was to investigate the expression of CD133 in circulating tumor cells (CTCs) from patients with mCRPC and to test the hypothesis that patients with mCRPC had CD133-positive CTCs associated with increased cell proliferation, changes in the androgen receptor (AR) protein expression, or AR nuclear co-localization. We utilized ImageStreamX technology, which combines flow cytometry and fluorescence microscopy, to capture and analyze CD45-negative/EpCAM-positive CTCs for CD133, Ki-67, and AR. All patient samples (20/20) contained CD133-positive populations of CTCs, and on average 50.9 ± 28.2% (range of 18.2% to 100%) of CTCs were CD133-positive. CD133-positive CTCs have increased Ki-67 protein expression compared to CD133-negative CTCs, implying that CD133-positive CTCs may have greater proliferative potential when compared to their CD133-negative counterparts. CD133-positive and CD133-negative CTCs have similar levels of AR protein expression and cellular co-localization with nuclear markers, implying that CD133 expression is independent of AR pathway activity and an AR-independent marker of mCRPC proliferation. These studies demonstrate the presence of CD133-positive populations in CTCs from mCRPC with increased proliferative potential.