Mechanisms of androgen receptor activation in advanced prostate cancer: differential co-activator recruitment and gene expression

Abstract

Prostate tumour growth depends on androgens; hence treatment includes androgen ablation and anti-androgens. Eventually tumours progress and in approximately 30% of patients this is associated with mutation of the androgen receptor. Several receptor variants associated with advanced disease show promiscuous activation by other hormones and anti-androgens. Such loss of specificity could promote receptor activation, hence tumour growth, in the absence of conventional ligands, explaining therapy failure. We aimed to elucidate mechanisms by which alternative ligands promote receptor activation. The three most commonly identified variants in tumours (with amino-acid substitutions H874Y, T877A and T877S) and wild-type receptor showed differences in co-activator recruitment dependent upon ligand and the interaction motif utilized. Co-expression and knockdown of co-activators that bind via leucine or phenylalanine motifs, combined with chromatin immunoprecipitation and quantitative PCR, revealed these preferences extend to co-activator recruitment in vivo and affect receptor activity at the transcriptional level, with subsequent effects on target gene regulation. The findings suggest that mutant receptors, activated by alternative ligands, drive growth via different mechanisms to androgenactivated wild-type receptor. Tumours may hence behave differently dependent upon any androgen receptor mutation present and what ligand is driving growth, as distinct subsets of genes may be regulated.

Microarray coupled to quantitative RT–PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells

“…However, the AR is also activated by E2, as well as by progestins such as medroxyprogesterone acetate (MPA) (Brooke et al, 2008). In order to determine whether the different AR agonists act in a similar manner to regulate the expression of androgen-responsive genes, a Q-RT-PCR was carried out for the 347 androgen-regulated genes described in the section above, using total RNA prepared in triplicate from LNCaP cells following addition of DHT, E2 or MPA for 24 h. In addition, LNCaP cells were treated for 24 h with the selective androgen-receptor modulators (SARMs) CPA and OHF, which show mixed agonist/antagonist activities, and with the anti-androgen bicalutamide (BIC), also known as casodex, which has little or no agonist activity for AR (Brooke et al, 2008).…”

“…Finally, some AR mutations, such as the AR-T877A mutation in LNCaP cells, increase the agonist activity of some of these weak AR agonists, as well as the antiandrogens CPA and hydroxyflutamide (OHF) (Steketee et al, 2002). However, detailed analysis of the expression of androgen-regulated genes in response to the weak agonists and AR antagonists !been confined, by and large, to reporter gene studies (Brooke et al, 2008).…”

Repression of Androgen Receptor Activity by HEYL, a Third Member of the Hairy/Enhancer-of-split-related Family of Notch Effectors

“…HEYL Depletion Assays-HEYL levels were depleted using an On-Target Dharmacon siRNA pool (L-008690-00-0005, ThermoScientific) as reported previously (25,27). Briefly, MCF7 cells were plated in serum-rich medium and grown to 70% confluence before being washed several times in starvation medium and transfected with siRNA.…”

“…Plasmids-The plasmids pSG5-FLAG-HEY1 (25), pSG5-FLAG-HEY2 (25), GST-NTD (25), GST-LBD (25), GST-HEY1 (25), pVP16-LEXA (27), pSVARo (28), Probasin-LUC (7), pSG5-AR⌬LBD (29), pSG5-SRC1e (30), LexA (7) -Gal4 (4) -LUC (25), TAT-GRE-E1B-LUC (29), AR⌬1 (also known as AR123) (31), and AR⌬5 (also known as AR131) (31) have been described previously. HEYL was amplified from MCF7 cDNA with primers (forward, 5Ј-GCA GCC TGC GGA ATT CAT GAA GCG A-3Ј; and reverse, 5Ј-GAA GGG GAT CCT CAG AAA GCC CC-3Ј), digested with EcoRI and BamHI, and cloned into pSG5 to create pSG5-HEYL.…”

“…trans-Repression Assays-trans-Repression assays were carried out essentially as reported previously (25,27).…”