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

Cancers
Hoda KardooniRobert Kypta

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,...Continue Reading

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Citations

Dec 6, 2019·International Journal of Molecular Sciences·Sergey BrezginVladimir Chulanov
Jul 2, 2019·Molecular Medicine Reports·Chenyao HuangLinfeng Lai
Aug 9, 2020·Aging and Disease·Allen CaobiNazira El-Hage
Dec 11, 2019·Scientific Reports·Li-Yuan Yu-LeeSue-Hwa Lin
Jan 10, 2021·Seminars in Cancer Biology·Imran AnsariSaurabh Singh
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Nov 17, 2020·Nucleic Acids Research·Agustin Sgro, Pilar Blancafort
Feb 28, 2021·Nature Reviews. Urology·Justin R Gregg, Timothy C Thompson
Jun 8, 2021·Molecular Therapy. Methods & Clinical Development·Eric AlvesPilar Blancafort

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Methods Mentioned

BETA
transfection
FCS
RNA-Seq
PCR
X-ray

Software Mentioned

sgRNA Designer
ToppGene
cBioPortal
Image J
MethPrimer
Oncomine
QCapture
UCSC genome browser
Excel

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