Generation 2.5 Antisense Oligonucleotides Targeting the Androgen Receptor and Its Splice Variants Suppress Enzalutamide-Resistant Prostate Cancer Cell Growth

Abstract

Purpose: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (AR FL ) or variants (AR-Vs) in disease progression.Experimental Design: To define functional roles of AR FL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown AR FL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts.Results: ENZ-R-LNCaP cells express high levels of both AR FL and AR-V7 compared with CRPC-LNCaP; in particular, AR FL levels were approximately 12-fold higher than AR-V7. Both AR FL and AR-V7 are highly expressed in the nuclear fractions of ENZ-RLNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of AR FL alone, or AR FL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and ARregulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both AR FL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of AR FL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts.Conclusions: These data identify the AR as an important driver of ENZ resistance, and while the contributions of AR FL and AR-Vs can vary across cell systems, AR FL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both AR FL and AR-Vs is a rational approach for AR-dependent CRPC.

Critical Role of Androgen Receptor Level in Prostate Cancer Cell Resistance to New Generation Antiandrogen Enzalutamide

“…In the present study, we were able to show significantly increased AR mRNA and protein levels in 2 out of 3 enzalutamide resistant sub-lines, suggesting AR overexpression as a common mechanism for enzalutamide resistance. This is in line with findings from Yamamoto et al who detected high levels of fulllength AR and AR variants in enzalutamide resistant LNCaP [22]. However, the underlying mechanisms causing AR overexpression seem to be diverse among different cell lines.…”

Androgen receptor signaling in castration-resistant prostate cancer: a lesson in persistence

“…Unfortunately, EZN-4176 failed phase I trials in CRPC due to poor AR knockdown in the clinical setting and minimal antitumor activity (Bianchini et al 2013). More recently, an exon 1-targeting ASO with improved tissue half-life and activity against enzalutamide-resistant models and CRPC patient-derived xenografts was developed (Yamamoto et al 2015). One advantage of this latter ASO is that targeting exon 1 will theoretically yield activity against all C-terminally truncated forms of the AR and LBD mutants.…”

RNA Therapeutics in Oncology: Advances, Challenges, and Future Directions

“…AZD5312 is an exciting molecule as it targets both the full-length androgen receptor RNA as well as several splice variants of AR that are associated with resistance to commonly used antiandrogen therapies in prostate cancer. 136,137 …”