CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-β 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-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.

Long noncoding RNA LINC00261 suppresses prostate cancer tumorigenesis through upregulation of GATA6-mediated DKK3

“…Furthermore, a recent study has already established a relationship between DKK3 and prostate cancer, DKK3 was down-regulated in prostate cancer cells where it inhibited tumor growth, proliferation and migration of prostate cancer [8]. In this study, we proved that DKK3 expression was reduced in prostate cancer cells compared with adjacent normal tissues.…”

Dihydrotestosterone inhibits arylsulfatase B and Dickkopf Wnt signaling pathway inhibitor (DKK)‐3 leading to enhanced Wnt signaling in prostate epithelium in response to stromal Wnt3A

“…Other reports suggest that DKK3 may affect Wnt signaling due to interaction with β‐TrCP . Also, effects of decline in DKK3 on TGF‐β/Smad signaling and on matrix metalloproteases in invasive prostate cancer have been reported . Attenuation of TGF‐β signaling in prostate cancer following CRISPR‐mediated reactivation of DKK3 expression has been reported .…”

“…Also, effects of decline in DKK3 on TGF‐β/Smad signaling and on matrix metalloproteases in invasive prostate cancer have been reported . Attenuation of TGF‐β signaling in prostate cancer following CRISPR‐mediated reactivation of DKK3 expression has been reported . Gene therapy with DKK3 has been proposed for treatment of prostate cancer (NCT01931046) …”

Vitamin D Sufficiency Enhances Differentiation of Patient-Derived Prostate Epithelial Organoids

“…However, DKK3 is the most structurally divergent member of the Dickkopf family (Krupnik et al, 1999) and has varied effects on Wnt, ranging from no effect (Krupnik et al, 1999;Pinho Christof Niehrs, 2007;Romero et al, 2013) to promoting (Ferrari et al, 2019;Nakamura and Hackam, 2010;Yin et al, 2018) or inhibiting Wnt (Bhattacharyya et al, 2017;Leonard et al, 2017;Liu et al, 2019;Sharma Das et al, 2013). DKK3 also functions as both a positive and negative regulator of TGFβ signaling, depending on the model (Al Shareef et al, 2018;Busceti et al, 2017;Kardooni et al, 2018;Li et al, 2017;Pinho Christof Niehrs, 2007;Romero et al, 2013;Wang Z et al, 2015), but DKK3 did not impact TGFβ in this study (data not shown). Despite conflicting reports of DKK3's signaling targets, it has consistently been shown to restrain cell proliferation (Kawano et al, 2006;Leonard et al, 2017).…”