Effect of ASXL1 on the stemness of colorectal cancer initiating cells.

Abstract

683 Background: Impaired cellular differentiation causes uncontrolled proliferation of stem cells and can be a cause of oncogenesis. The epigenome regulates the expression of genes that are essential for cellular differentiation. On the other hand, chromosomal abnormalities are common in colorectal cancer cells. We hypothesize that epigenetic disorders caused by chromosomal abnormalities may result in oncogenesis. Methods: The Cancer Genome Atlas (TCGA) database was investigated to identify epigenetic regulating genes affected by chromosomal abnormalities. Patient-derived colorectal cancer cells were obtained from consented patients to validate these findings. Results: The amplification of Chr20q11 is the most frequent chromosomal alteration in left-sided colonic and rectal cancers. ASXL1, a recruiter of PRC2, is coded in Chr20q11 and has upregulated expression in many cases. Excessive expression of ASXL1 may cause oncogenesis as PRC2 trimethylates histone H3 on lysine 27 (H3K27me3), an epigenetic mark of transcriptional silencing. Immunofluorescence revealed that the ASXL1 protein was located in the nuclei of colorectal cancer cells that did not express differentiation markers. This finding was consistent with the finding that ASXL1 highly expressed at the bottom of crypts where undifferentiated cells exist in normal colonic epithelium. Knockdown of ASXL1 in colorectal cancer cells reduced the levels of H3K27me3 and diminished organoid formation in vitro. Knockdown of ASXL1 in patient-derived cancer cells obtained from clinical specimens impaired the tumorigenicity of cancer initiating cells in vivo and increased the expression of differentiation markers. Murine colonic tumor cells from ApcMin mice highly expressed ASXL1, while homozygous Asxl 1 knockout reduced the occurrence of tumors in vivo. Many putative genes regulated by ASXL1 are identified by gene expression analysis and ChIP-sequencing. Conclusions: High expression of ASXL1 initiates oncogenesis and maintains the stemeness of colorectal cancer cells.