ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer
Changmeng Cai, … , Steven P. Balk, Xin Yuan
Changmeng Cai, … , Steven P. Balk, Xin Yuan
Published February 15, 2013
Citation Information: J Clin Invest. 2013;123(3):1109-1122. https://doi.org/10.1172/JCI66666.
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Research Article Oncology

ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer

Abstract

Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion–positive PCa.

Authors

Changmeng Cai, Hongyun Wang, Housheng Hansen He, Sen Chen, Lingfeng He, Fen Ma, Lorelei Mucci, Qianben Wang, Christopher Fiore, Adam G. Sowalsky, Massimo Loda, X. Shirley Liu, Myles Brown, Steven P. Balk, Xin Yuan

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Figure 2

SOX9 expression is androgen and ERG regulated in TMPRSS2:ERG-positive VCaP PCa cells.

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SOX9 expression is androgen and ERG regulated in TMPRSS2:ERG-positive VC...
(A) VCaP; LNCaP; LNCaP-CSS3 (derived from LNCaP cells adapted to hormone-depleted conditions); C4-2; LNCaP-ABL; LAPC4-PR; LAPC4-CR (derived from androgen-dependent or castration-resistant LAPC4 xenografts); and CWR22-Rv1 cells were treated with or without DHT for 24 hours followed by qRT-PCR. (B) VCaP and LNCaP cells were treated with 0, 0.01, 0.1, 1, or 10 nM DHT for 24 hours and SOX9 mRNA was measured by qRT-PCR. (C) LNCaP and VCaP cells were stimulated with or without DHT for 4 hours, and binding of activated phospho-RNA polymerase II (phospho-Ser5 on CTD) to the SOX9 transcriptional start site (TSS) was assessed by ChIP. (D) VCaP cells were treated with 0–10 nM (0, 0.1, 1, or 10) DHT and with vehicle (DMSO) or 10 μM Bic for 24 hours and immunoblotted. (E) LNCaP cells were treated with 0, 0.01, 0.1, 1, or 10 nM DHT for 24 hours, followed by immunoblotting. (F) Expression of SOX9, ERG, or AR was examined by IHC in VCaP xenografts prior to castration (Androgen-dependent), at 4 days after castration, or at relapse (Castration-resistant). Original magnification, ×400; Scale bars: 20 μm. (G and H) VCaP cells stably infected with nontarget control (NTC) or ERG shRNA-1 were treated with or without DHT for 24 hours, and SOX9 or ERG mRNA was then measured by qRT-PCR, or SOX9, ERG, and β-tubulin protein were assessed by immunoblotting. Data in bar graphs represent means ± SD of at least 3 biological repeats. Significant differences from DHT-negative controls are indicated (*).