Objectives: Cyclin Dependent Kinase 2-Associating Protein 1 (CDK2AP1) is a putative tumor suppressor gene that is downregulated in 70% of human oral cancer. It has been shown that CDK2AP1 is a key component in Nucleosome Remodeling and Histone Deacetylase (NuRD) complex. Our recent data demonstrates that deletion of Cdk2ap1 alters epigenetic regulation of oncogenic pathways: Wnt signaling pathway, colorectal cancer, hematopoietic cell lineage, and basal cell carcinoma. We hypothesize that Cdk2ap1 plays an essential role in DNA methylation and an altered expression of Cdk2ap1 has a significant effect on the DNA methylation profile of Wnt pathway genes. This hypothesis was tested by examining the DNA methylation affected by deletion of Cdk2ap1 in mESCs.
Methods: Bisulfite sequencing was performed to validate the potential target genes found by Methyl-DNA immunoprecipitation (meDIP) assay combined with CpG / promoter microarray. The level of phospho-β-catenin was measured by Western blotting and Immunofluorescence (IF) analysis. β-catenin activity was measured by TCF/LEF-reporter assay with luciferase construct. WT and Cdk2ap1-/- mESCs were treated with Wnt pathway inhibitors: PKI, PI3K and GSK3β.
Results: We validated three Wnt genes (Axin2, Apc2 and Nfat5) from DNA methylation array. We found that deletion of Cdk2ap1 leads to significant activation of Wnt pathway by measuring phospho-β-catenin. The TCF/LEF reporter assay showed that there is an increase of 1.75 fold induction in Cdk2ap1-/- mESCs. Mechanistically, we were able to revert the level of phospho-β-catenin of Cdk2ap1-/- back to normal by using PI3K inhibitor, LY294002.
Conclusion: Our data demonstrates that Cdk2ap1 plays a significant role in epigenetic regulation of Wnt pathway genes. Future study will be devoted to address the functional significance of CDK2AP1-mediated epigenetic modifications in specific disease such as oral cancer and in global context such as human embryonic stem cell self-renewal and differentiation.
Funding agency: NIDCR/NIH, CIRM
Funding number: 5F30DE020003-02(JJK), R01-DE014857(DTW), RB2-01562(YK)
Keywords: Biochemistry, Carcinogenesis, Cell biology, Oral biology and Stem cell