Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients

Abstract

BackgroundThe androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.ResultsWe previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.ConclusionsThis work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users.

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

“…The other major phenotypic output of common LBD mutations is antagonist-agonist switching (Supplementary Table 1), which likely explains the withdrawal syndrome observed after cessation of firstgeneration antagonists seen in 15-30% of patients (Small et al 2004). T878A confers agonist properties to flutamide and nilutamide, H875Y to nilutamide and W742C/L to bicalutamide (Veldscholte et al 1990, Suzuki et al 1996, Tan et al 1997, Hara et al 2003, Azad et al 2015, O'Neill et al 2015, Lallous et al 2016. Interestingly, O'Neill and coworkers recently demonstrated that T878A inhibited bicalutamide-activated W742L (O'Neill et al 2015) in what may represent the first evidence of antagonism arising from the heterodimerization of distinct AR mutants.…”

“…The advent of the omics era, and resultant integrative genomic analyses of metastatic samples, has further highlighted the extent and frequency of AR pathway alterations in CRPC that may contribute to inappropriate activation or reactivation of this pathway (Grasso et al 2012, Beltran et al 2013, Azad et al 2015, Robinson et al 2015, Lallous et al 2016. Although these alterations have been the subject of other recent excellent articles (for example Penning 2014, Joshi et al 2015, Wyatt & Gleave 2015, this review will specifically focus on ADT-resistance mechanisms driven by direct structural changes to the AR and altered interplay between the AR and its coregulators.…”

“…These alterations have been reported to facilitate AR signaling in CRPC by conferring: (i) ligand promiscuity, thereby allowing AR to be activated even in the presence of low/ absent levels of androgens and (ii) agonist properties to AR antagonists (Supplementary Table 1). Recurrently occurring LBD missense mutations in CRPC are L702H, W742C, H875Y and T878A (Grasso et al 2012, Robinson et al 2015, Lallous et al 2016. With the exception of W742C, these AR mutants are represented in commonly used PCa cell lines (T878A in LNCaP, C42B and MDAPCa-2B; L702H in MDA-MB-2B and H875Y in 22Rv1 and CWR-R1), which has facilitated our understanding of their function.…”

“…With the exception of W742C, these AR mutants are represented in commonly used PCa cell lines (T878A in LNCaP, C42B and MDAPCa-2B; L702H in MDA-MB-2B and H875Y in 22Rv1 and CWR-R1), which has facilitated our understanding of their function. The T878A mutant, first identified in the LNCaP cell line (Veldscholte et al 1990), is the archetypal promiscuous receptor, being activated by progesterone, estrogen and glucocorticoids (Veldscholte et al 1990, Berrevoets et al 1993, Suzuki et al 1996, Culig et al 1999, Steketee et al 2002, Lallous et al 2016. H875Y and L702H also broaden ligand specificity by enabling AR activation by glucocorticoids (Suzuki et al 1993, Taplin et al 1995, Steketee et al 2002.…”

“…AR gene mutations In CRPC, AR mutations have been reported to occur at a frequency of 5-30% in pretreated tumors (Taplin et al 1999, Wallen et al 1999, Grasso et al 2012, Beltran et al 2013, Robinson et al 2015, Kumar et al 2016, CTCs (Jiang et al 2010) and ctDNA (Azad et al 2015, Lallous et al 2016. Although infrequent, AR mutations have also been detected in primary PCa prior to ADT or arising during treatment, and there is evidence that therapy-mediated selection of such mutations can underlie resistance in some patients (Tilley et al 1996, Taplin et al 1999, Thompson et al 2003, Steinkamp et al 2009, Carreira et al 2014, Cancer Genome Atlas Research 2015.…”