EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines

Abstract

Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities.

Assessment of MicroRNA (96) and MicroRNA (298) as Biomarkers for Diagnosis and Prognosis of Rheumatoid Arthritis in Egyptian Patients

“…As it was proven from previous studies that miR-298 correlates negatively with its target gene polycomb protein enhancer of zeste2 (EZH2) in ovarian cancer 12,49 . As well, the authors identified secreted frizzled-related protein 1 (SFRP1) which is a wnt signaling antagonist as a target gene of EZH2 in rheumatoid arthritis synovial fibroblasts and found that EZH2 silencing results in a significant increase in SFRP1 mRNA 50,51 . Besides wnt signaling inhibition, SFRP overexpression may also cause a marked increase of Th17 cells in RA synovial fluid 40,52 .…”

The Interaction Between Epigenetic Changes, EMT, and Exosomes in Predicting Metastasis of Colorectal Cancers (CRC)

“…They are diagnostic and prognostic biomarkers and suppress the growth and metastasis of CRC tumor cells. SFRP4 and SFRP5 may also inhibit invasion in CRC, but the underlying mechanisms in regulating the signaling pathways are more complex by comparison ( 32 , 33 , 44 ). Additional studies focusing on the SFRPs family as it relates to CRC should be undertaken to clarify their overall role and potentially derive a novel therapeutic regimen.…”

CBX7 is Dualistic in Cancer Progression Based on its Function and Molecular Interactions

“…The different regulatory effects on the same protein between the two reveal that CBX7 may be independent of PRC1 composed of BMI-1 to achieve EMT inhibition. Not surprisingly, like CBX7 in breast cancer, abnormal activation of the Wnt signaling pathway is found in 90% of colon cancer, and one of the reasons may be the absence of secreted frizzled-related protein 4(sFRP4) (Liu et al, 2020). The sFRP4 could compete with Wnt proteins via binding their receptor (Frizzled) to act as an antagonist of the Wnt signaling pathway in colonic cancer (Liu et al, 2006;Liu et al, 2020).…”

“…Not surprisingly, like CBX7 in breast cancer, abnormal activation of the Wnt signaling pathway is found in 90% of colon cancer, and one of the reasons may be the absence of secreted frizzled-related protein 4(sFRP4) (Liu et al, 2020). The sFRP4 could compete with Wnt proteins via binding their receptor (Frizzled) to act as an antagonist of the Wnt signaling pathway in colonic cancer (Liu et al, 2006;Liu et al, 2020). Interestingly, both DKK-1 and sFRP4 are antagonists of the Wnt signaling pathway, but the effect of CBX7 in regulating sFRP4 in colon cancer is quite different from the effect of regulating DKK-1 in breast cancer.…”

“…Interestingly, both DKK-1 and sFRP4 are antagonists of the Wnt signaling pathway, but the effect of CBX7 in regulating sFRP4 in colon cancer is quite different from the effect of regulating DKK-1 in breast cancer. CBX7 and other PcG proteins, such as EZH2 and Jumonji, AT rich interactive domain 2 (JARID2) are found to be enriched in the SFRP4 gene promoter region to regulate gene expression without DNA methylation (Liu et al, 2020). Perhaps it is the synergistic effect of CBX7, EZH2, and JARID2 on histone methylation modification that leads to epigenetic gene silencing (Walters et al, 2014;Pallante et al, 2015).…”