Presentation Authors: Michael Fiandalo*, Buffalo, NY, Vitaliy Sviripa, Lexington, KY, John Stocking, Buffalo, NY, Danny Holbert, Morgantown, WV, John Wilton, Krystin Mantione, Kristopher Attwood, Hans Minderman, Yue Wu, Buffalo, NY, David Watt, Lexington, KY, James Mohler, Buffalo, NY
Introduction: Men with advanced prostate cancer (CaP) receive androgen deprivation therapy (ADT), which lowers circulating testosterone (T) levels, impairs androgen receptor (AR) activation and results in CaP regression. ADT is palliative and CaP recurs as lethal castration-recurrent/resistant CaP CRPC). The small molecule anti-androgen, enzalutamide, is used to treat CRPC. However, expression of the AR-V7 splice variant impairs enzalutamide activity. A fluorescent-DHT surrogate, DHT-coumarin-1 (DHT-C-1), was one of many fluorescent-androgen surrogates synthesized to track androgen metabolism in CaP models. DHT-C-1, unexpectedly, inhibited growth of AR positive and AR-V7 positive CaP cell lines. The objective of these studies was to characterize DHT-C-1 activity in AR and AR-V7 positive and AR negative CaP cell lines.
Methods: Androgen sensitive and AR-positive LAPC-4, VCaP and LNCaP, castration-recurrent AR and AR-V7 positive CWR-R1 and 22rv1, and androgen-independent and AR negative PC-3 and DU145 cell lines were treated with serum-free complete media (to simulate androgen deprivation), coumarin alone, to show the fluorophore did not impair CaP cell growth or AR signaling, DHT or DHT-C-1. PC-3 cells that stably expressed AR-green fluorescent protein (AR-GFP) were used for ImageStream analysis. ImageStream, a hybrid technique of flow cytometry and fluorescent-microscopy, was used to assess CaP cell uptake of DHT-C-1. MTT was used to assess cell growth and quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess kallikrein-related peptidase 2 (KLK2) and prostate-specific antigen (PSA) transcript abundance levels.
Results: ImageStream revealed DHT-C-1 was taken up by all 7 CaP cell lines. DHT-C-1 demonstrated nuclear co-localization with AR-GFP and nuclear marker DRAQ5 occurred only in AR positive CaP cell lines. ImageStream revealed that DHT-C-1 co-localized with the AR-ligand binding domain. DHT-C-1 treatment impaired growth of all 5 of the AR positive and AR-V7 positive CaP cell lines during androgen deprivation. VCaP cell growth inhibition required 1000 times lower concentration of DHT-C-1 than enzalutamide. qRT-PCR revealed that DHT-C-1 treatment impaired induction of PSA and KLK2 transcription, which suggested DHT-C-1 interfered with AR-regulated signaling pathways.
Conclusions: DHT-C-1 demonstrated activity against CaP cells that express AR or AR-V7. DHT-C-1 may be effective alone or in combination to improve enzalutamide response. DHT-C-1 in vivo testing is in progress using CaP xenografts.
Source of Funding: Acknowledgement. P01-CA77739, P20-RR020171, DoD Prostate Cancer Research Program Award No. W81XWH-16-1-0635 and Post-doctoral Training Award W81XWH-15-1-0409 and, in part, by the NCI Cancer Center Support Grant to Roswell Park (P30-CA016056) for the Bioan