Kardooni Hoda, Gonzalez-Gualda Estela, Stylianakis Emmanouil, Saffaran Sina, Waxman Jonathan, Kypta Robert M
Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK.
School of Engineering, University of Warwick, Coventry CV4 7AL, UK.
Cancers (Basel). 2018 May 28;10(6):165. doi: 10.3390/cancers10060165.
The gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-β/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the promoter, induces DKK3 expression, and inhibits TGF-β/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-β/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-β. TGF-β signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-β found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-β signaling.
该基因编码一种分泌蛋白Dkk-3，它可抑制前列腺肿瘤的生长和转移。在包括前列腺癌在内的多种癌症中，其因启动子甲基化而下调。基因沉默研究表明，Dkk-3通过限制TGF-β/Smad信号传导来维持前列腺上皮细胞的正常稳态。虽然Dkk-3的异位表达会导致前列腺癌细胞凋亡，但尚不清楚Dkk-3在癌细胞中是否具有生理作用。在此，我们表明用DNA甲基转移酶（DNMT）抑制剂地西他滨处理PC3前列腺癌细胞可使启动子去甲基化，诱导DKK3表达，并抑制TGF-β/Smad依赖的转录活性。使用CRISPR-dCas9-VPR直接诱导DKK3表达也抑制了TGF-β/Smad依赖的转录，并减弱了PC3细胞的迁移和增殖。在对TGF-β无反应的C4-2B细胞中未观察到这些效应。TGF-β信号可通过SMAD蛋白直接调节基因表达，并通过增加DNMT表达间接调节，从而导致启动子甲基化。对因启动子甲基化而下调且预计受TGF-β调节的基因进行分析发现，DKK3的诱导增加了编码环氧化酶-2的PTGS2的表达。总之，这些观察结果为使用CRISPR介导的DKK3诱导作为前列腺癌的潜在治疗方法提供了支持，并突出了Dkk-3对TGF-β信号传导调节的复杂性。
