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

Hoda Kardooni,Estela Gonzalez-Gualda,Emmanouil Stylianakis,Jonathan Waxman,Robert Kypta,Sina Saffaran

doi:10.3390/cancers10060165

Hoda Kardooni, Estela Gonzalez-Gualda + Show 4 more

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https://doi.org/10.3390/cancers10060165

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Journal: Cancers	Publication Date: May 28, 2018
Citations: 29	License type: CC BY 4.0

Affiliation: Imperial College London, CIC bioGUNE, University of Warwick

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-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 DKK3 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.

Highlights

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second cause of cancer-related deaths among men in the U.S [1]
Reporter cells wereInanalyzed using Combined Bisulfite Restriction Analysis (CoBRA) to determine the extent of order to confirm that Dickkopf-related protein 3 (DKK3) mRNA expression is repressed by promoter methylation, PC3
The results showed a remarkable increase of DKK3 mRNA levels, combination were co-transfected with dCas9-VPR plasmid and DKK3 mRNA expression was measured the extent of which depended on the guide RNA (gRNA) used and the cell line transfected

Summary

Introduction

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second cause of cancer-related deaths among men in the U.S [1]. First line treatment includes androgen receptor (AR)-directed therapies, since most tumors express AR and depend on its signaling. Many patient tumors become resistant after hormone-therapy and relapse within 24 months, developing castration-resistant PCa (CRPC) [2], highlighting the need for novel therapies and treatments targeting alternative signaling pathways involved in PCa progression. Recent studies have demonstrated that epigenetic alterations, changes in gene promoter methylation, are frequent during PCa tumorigenesis [3]. Cancer genomics studies have identified recurrently mutated genes and mutation hotspots in a number of cancer types. Such studies in prostate adenocarcinomas have identified no genes recurrently mutated in more than a seventh of cases [4].

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