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 4

SOX9 is a major downstream effector of ERG in TMPRSS2:ERG fusion–positive PCa cells.

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SOX9 is a major downstream effector of ERG in TMPRSS2:ERG fusion–positiv...
(A) LN-toSOX9 cells treated with DHT and/or doxycycline (inducing SOX9 expression) were assessed for Matrigel invasion. Cells invading through membrane were stained and quantified. Images are from a representative experiment, and graph represents the mean and SD from 3 independent experiments. (B) VCaP-toSOX9 cells (grown in DHT-supplemented medium) treated with NTC siRNA, ERG siRNA, or ERG siRNA plus doxycycline were immunoblotted as indicated and (C) assessed for invasion. (D) VCaP cells with SOX9 shRNA or control shRNA (shNTC) were treated with DHT and immunoblotted or (E) assessed for invasion. (F) Effect of SOX9 siRNA on PLAT mRNA expression in VCaP cells cultured in FBS medium. (G) VCaP-toSOX9 cells (grown in DHT-supplemented tetracycline-free medium) treated with nontarget control siRNA, ERG siRNA, or ERG siRNA plus doxycycline were analyzed by qRT-PCR. (H) VCaP-shSOX9-1 versus control VCaP-shNTC cells were treated with DHT for 0, 2, or 5 days, and cell recovery was measured by MTT assay. (I) The same number of VCaP-shNTC or VCaP-shSOX9-2 cells were injected into the left or right flank of the same mouse. When the VCaP-shNTC xenografts reached approximately 1 cm in diameter (~8 weeks), the mice were sacrificed and tumor volumes were compared. Graph shows volume of each VCaP-shSOX9 tumor relative to the corresponding control on the opposite flank. (J) IHC for Ki67 in xenograft set numbers 1 and 2 (%Ki67+ cells based on 4 random areas). Original magnification, ×400. Scale bars: 20 μm. Data in bar graphs represent means ± SD of at least 3 biological repeats. dox, doxycycline.