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CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-? Signaling in Prostate Cancer.

ABSTRACT: The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 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-?/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 DKK3 promoter, induces DKK3 expression, and inhibits TGF-?/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-?/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-?. TGF-? 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-? 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-? signaling.

SUBMITTER: Kardooni H

PROVIDER: S-EPMC6025141 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-β Signaling in Prostate Cancer.

Kardooni Hoda H Gonzalez-Gualda Estela E Stylianakis Emmanouil E Saffaran Sina S Waxman Jonathan J Kypta Robert M RM

Cancers 20180528 6

The <i>DKK3</i> gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. <i>DKK3</i> 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 ...[more]

PMID: 29843383

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Project description:The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-beta signaling directed the prostatic induction. UGM from TGF-beta type II receptor stromal conditional knockout mice (Tgfbr2(fspKO)) or control mice (Tgfbr2(floxE2/floxE2)) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2(floxE2/floxE2) UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2(fspKO) UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2(fspKO) UGM compared with Tgfbr2(floxE2/floxE2) UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-beta activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-beta signaling to mediate epithelial Wnt activity.

Dataset Information

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-? Signaling in Prostate Cancer.

Publications

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-β Signaling in Prostate Cancer.

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