ERG/AKR1C3/AR Constitutes a Feed-Forward Loop for AR Signaling in Prostate Cancer Cells

Abstract

Purpose Intratumoral androgen synthesis in prostate cancer (PCa) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus contributing to CRPC in a castrated environment. The TMPRSS2-ERG transcription factor has been shown to be present in primary PCa tumors as well as CRPC tumors. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. Experimental design We used a panel of assays including lentivirus transduction, gene expression, chromatin immunoprecipitation and sequencing, Liquid chromatography-Mass spectrometric quantitation, immunocytochemistry, immunohistochemistry and bio-informatics analysis of gene microarray data bases to determine ERG regulation of androgen synthesis. Results We found that ERG regulated the expression of the ABE AKR1C3 in PCa cells via direct binding to the AKR1C3 gene. Knockdown of ERG resulted in reduced AKR1C3 expression, which caused a reduction in both DHT synthesis and PSA expression in VCaP PCa cells treated with 5α-androstanedione, a DHT precursor metabolite. Immunohistochemical staining revealed that ERG was co-expressed with AKR1C3 in PCa tissue samples. Conclusions These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Androstanedione to DHT in PCa cells, and that ERG regulates this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive PCa patients in the clinic for anti-AR driven therapies; and AKR1C3 may serve as a valuable therapeutic target in the treatment of CRPC.

Therapy escape mechanisms in the malignant prostate

“…Interestingly, this intra-tumoural conversion of adrenal steroids is not only a mechanism of resistance against ADT; upregulation of AKR1C3 was also found in cells that are resistant to the second-line antiandrogen enzalutamide [89]. A recent study has shown that the transcription factor ERG, Page 12 of 46 A c c e p t e d M a n u s c r i p t 12 / 12 which is fused to the androgen-responsive TMPRSS2 in about 50% of PCa patients [90], is a direct positive regulator on the AKR1C3 promoter [91]. ERG is co-expressed with AKR1C3 in PCa tissue samples.…”

Mechanisms of drug resistance that target the androgen axis in castration resistant prostate cancer (CRPC)

“…In this manner TMPRSS2-ERG overrides the repressive effect of the AR on the AKR1C3 gene promoter. TMPRSS2-ERG k/d in VCaP cells led to a reduction in AKR1C3 expression and a decrease in the ability to convert Adione to DHT supporting this mechanism [45]. It is noteworthy that analysis of gene expression in 25 mCRPC tumors obtained from the Gene Expression Omnibus revealed a significant correlation (p <0.0001 r = 0.69) between AKR1C3 and ERG co-expression where AKR1C3 increased expression ranged from 0- 100-fold and ERG expression ranged from 0-250-fold over non-expressing tumors [45].…”

“…These estimates are likely to be conservative since whether patients had received prior second-line ADT therapy was not reported when measuring transcript levels. Data obtained from the Oncomine database for 363 prostate cancer tumor specimens when used to construct Kaplan-Meier survival plots showed that the median survival time for patients with tumors that were low AKR1C3 expressors and low TMPRSS2-ERG expressors was 12-13 years, but patients whose tumors were high AKR1C3 expressors and high TMPRSS2-ERG expressors had median survival times of only 7-10 years [45]. In this study high and low expressors fell either side of the median value, however, it is not clear as to whether the prostate cancer specimens were derived from radical prostatectomy samples, needle biopsies, or transurethral prostate tissue specimens.…”

“…These results suggest that 3α/β-diol also represent potential precursors of DHT, and the back conversion to DHT from androgen derivatives catalyzed by HSD17B6 may be a promising target to counter ADT resistance. HSD17B6 is also upregulated by TMPRSS2-ERG and when this transcription factor is silenced in VCaP cells there is a dramatic reduction in HSD17B6 expression but only a modest effect on the conversion of 3α-diol to DHT suggesting that AKR1C3 may be the major source of potent androgens in these cells [45]. …”

Direct Metabolic Interrogation of Dihydrotestosterone Biosynthesis from Adrenal Precursors in Primary Prostatectomy Tissues

“…This results in generation of a 5α-reduced metabolite, 5α-androstanedione (5α-dione), which can be 17-keto reduced to DHT to bypass T via a different metabolic route (Figure 1a, right ). Surprisingly, this “5α-dione pathway” (DHEA→AD→5α-dione→DHT) appears to be the predominant directionality of DHEA metabolism in the majority of metastatic CRPC cell lines, as well as in sampled metastatic CRPC patient tissues (11–14). We therefore sought to determine whether the same metabolic phenotype is operational in the eugonadal setting of clinically localized prostate cancer, where both gonadal T and adrenal steroids are potential precursors of DHT.…”