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

Abstract

Purpose: Intratumoral androgen synthesis in prostate cancer 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 prostate cancer 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 bioinformatics analysis of gene microarray databases, to determine ERG regulation of androgen synthesis.

Results: We found that ERG regulated the expression of the ABE AKR1C3 in prostate cancer 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 prostate cancer cells treated with 5α-androstanedione (5α-Adione), a DHT precursor metabolite. Immunohistochemical staining revealed that ERG was coexpressed with AKR1C3 in prostate cancer tissue samples.

Conclusions: These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Adione to DHT in prostate cancer 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 prostate cancer patients in the clinic for anti-androgen receptor-driven therapies; and AKR1C3 may serve as a valuable therapeutic target in the treatment of CRPC.

Figure 1

TMPRSS2-ERG regulates androgen biosynthetic enzyme (ABE) expression in PCa cells. A.) RT-qPCR analysis of ERG in VCaP shScrambled (shScr) and VCaP shERG lentiviral cells. Western blot analysis of ERG in VCaP shScr and shERG cells. MOI = multiplicity of infection for lentiviral transduction. B.) RT-PCR analysis of ABE expression in VCaP shScr and shERG cells. PCR cycle numbers are indicated above each DNA gel. C.) RT-qPCR analysis of HSD17B6, HSD17B4, and AKR1C3 in VCaP shScr and VCaP shERG cells. D.) Western blot analysis of AKR1C3 in VCaP, VCaP shScr and shERG cells. E.) Western blot analysis of AKR1C3 in LNCaP ERG40 and BPH-1 ERG40 lentiviral cells. LNCaP Ctrl and BPH-1 Ctrl cells represent empty vector controls. For statistical analysis a two-tailed, paired, student's t-test was performed (N=3) **P<0.01, ***P<0.001. Numbers above western blots represent fold changes determined by densitometry. Representative of three independent experiments.

Figure 2

ERG binds directly to the AKR1C3 gene. A.) Diagram illustrating the AKR1C3 gene located on chromosome 10. Black tick marks indicate ERG binding sites (5’-GGAA/T-3’) located in the region of −2 Kb to +2 Kb relative to the transcription start site. B.) ChIP-PCR analysis of putative ERG binding sites in the AKR1C3 gene. ChIP was performed in VCaP cells using anti-ERG antibody; IgG antibody was used as a control for non-specific binding. NTC is non-template control. C.) ChIP-PCR analysis of a known ERG binding site in PLAU promoter region using VCaP cells. Negative control primers were used in VCaP cells to demonstrate specificity of the ChIP-PCR technique. D.) ChIP-Seq analysis performed in duplicate using anti-ERG antibody in VCaP cells.

Figure 3

Androstanedione is a precursor metabolite for DHT in TMPRSS2-ERG fusion-positive cells. A.) Diagram illustrating the DHT biosynthesis pathway and the enzymes that synthesize DHT. B.) HPLC/MS/MS analysis in VCaP shSrc cells; DHT levels were quantified as pmol per one million cells. C.) HPLC/MS/MS analysis in VCaP shScr and shERG cells; DHT levels were quantified as pmol per one million cells. Vehicle controls represent 0.1% EtOH treated samples. For statistical analysis a two-tailed, unpaired, student's t-test was performed (N=3) *P<0.05, **P<0.01, ***P< 0.001.

Figure 4

Androstanedione induces AR activation through ERG regulated AKR1C3 expression. Immunocytochemistry in VCaP cells treated with vehicle or (100 nM) 5α-Adione for 24 hours. Confocal images were shown in panels A and B; cells were stained with anti-AR, Texas Red and DAPI as a green pseudo color. PCR amplification of PSA expression is shown in panel C-D. C.) RT-qPCR of PSA expression in VCaP shScr and VCaP shERG lentivirus infected cells, D.) RTPCR analysis of PSA expression in VCaP shScr and shERG lentivirus infected cells. NTC is non-template control. E.) RT-qPCR analysis of PSA expression in VCaP shScr and shAKR1C3 lentivirus infected cells. For statistical analysis a two-tailed, unpaired, student's t-test was performed C.) (N=5) and E.) (N=2) *P<0.05, ***P<0.001.

Figure 5

ERG and AKR1C3 are co-expressed in human prostate tumor tissue specimens, and predict lower survival probability. A.) Immunohistochemical analysis of AKR1C3 and ERG in human prostate tumor tissue specimens. B.) Distribution of 64 patients by presence or absence of AKR1C3 and ERG in prostate tumor tissues, P<0.0001. C.) Immunohistochemical analysis of AKR1C3 and ERG in LuCaP xenograft specimens. D.) Correlation plot of AKR1C3 and ERG expression using microarray data from 25 metastatic PCa samples. Data was extracted from Gene Expression Omnibus database E.) Kaplan-Meier survival plot of (Left) AKR1C3 low tumors vs. AKR1C3 high tumors,(Middle) ERG fusion-negative tumors vs. ERG fusion-positive tumors and (Right) AKR1C3 low and TMPRSS2-ERG negative vs. AKR1C3 high and TMPRSS2-ERG positive tumors. Data was extracted from Oncomine database. For statistical analyses of Kaplan-Meier Survival a Mantel-Cox Test and a Gehan-Breslow-Wilcoxon Test were performed..

Figure 6

Schematic representation of proposed role of TMPRSS2-ERG fusions in androgen biosynthesis and AR activation via feed-forward activation.