O-Linked N-Acetylglucosamine (O-GlcNAc) Expression Levels Epigenetically Regulate Colon Cancer Tumorigenesis by Affecting the Cancer Stem Cell Compartment via Modulating Expression of Transcriptional Factor MYBL1.

To study the regulation of colorectal adenocarcinoma progression by O-GlcNAc, we have focused on the O-GlcNAc-mediated epigenetic regulation of human colon cancer stem cells (CCSC). Xenograft tumors from colon tumor cells with O-linked N-acetylglucosamine transferase (OGT) knockdown grew significantly slower than those formed from control cells, indicating a reduced proliferation of tumor cells due to inhibition of OGT expression. Significant reduction of the CCSC population was observed in the tumor cells after OGT knockdown, whereas tumor cells treated with the O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-sequencing experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAc-modified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-sequencing analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the transcription start site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels is therefore likely to result, at least in part, from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression.