Histone Demethylase JMJD2D Interacts With β-Catenin to Induce Transcription and Activate Colorectal Cancer Cell Proliferation and Tumor Growth in Mice

Abstract Background & Aims Wnt signaling contributes to development of colorectal cancer (CRC). We studied interactions between lysine demethylase 4D (KDM4D or JMJD2D) and beta-catenin, a mediator of Wnt signaling, in CRC cell lines and the effects on tumor formation in mice. Methods We obtained colorectal tumor specimens and surrounding non-tumor colon tissues (controls) from patients undergoing surgery in China; levels of JMJD2D were measured by immunohistochemical or immunoblot analysis. JMJD2D expression was knocked down in CRC (CT26, HCT116, and SW480 cells) using small hairpin RNAs and cells were analyzed in viability, flow cytometry, colony formation, and transwell migration and invasion assays. Cells were also grown as tumor xenografts in nude mice or injected into tail veins or spleens of mice and metastases were measured. We also performed promoter activity, co-immunoprecipitation, and chromatin immunoprecipitation assays. We also performed studies with Apc min/+ and JMJD2D-knockout mice; these mice were crossed and colorectal tumor formation in offspring ( Apc min/+ ; Jmjd2d +/+ and Apc min/+ ; Jmjd2d –/– ) was analyzed. JMJD2D-knockout and wild-type (control) mice were given azoxymethane followed by dextran sodium sulfate (DSS) to induce colitis-associated colorectal cancer; some mice were given the JMJD2D inhibitor 5-c-8HQ or vehicle to examine the effects of 5-c-8HQ on intestinal tumor formation. Results Levels of JMJD2D were significantly higher in human colorectal tumors than in control tissues and correlated with levels of proliferating cell nuclear antigen. JMJD2D knockdown reduced CRC cell proliferation, migration, and invasion as well as growth of xenograft tumors and formation of metastases in mice. JMJD2D was required for expression of beta-catenin in CRC cell lines; ectopic expression of JMJD2D increased the promoter activities of genes regulated by beta-catenin ( MYC , cyclin D1 , MMP2 , and MMP9 ). We found that JMJD2D and beta-catenin interact physically, and JMJD2D demethylated H3K9me3 at promoters of beta-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice and their tumor tissues had lower levels of beta-catenin, MYC, cyclin D1, and proliferating cell nuclear antigen than tumors from control mice. Apc min/+ ; Jmjd2d –/– mice developed fewer and smaller colon tumors than Apc min/+ mice. Mice given 5-c-8HQ developed smaller and fewer colitis-associated tumors, with lower levels of cell proliferation, than mice given vehicle. Apc min/+ mice given 5-c-8HQ also developed fewer tumors in intestines and colons than mice given vehicle. Conclusions Levels of the histone demethylase JMJD2D are increased in human colorectal tumors, compared to non-tumor colon tissues. JMJD2D interacts with beta-catenin to activate transcription of its target genes and promote CRC cell proliferation, migration, and invasion as well as formation of colorectal tumors in mice.