The role of histone methyltransferase DOT1L in regulation of Breast cancer stem cells

Abstract

DOT1L (DOT1-Like), a histone methyltransferase to histone H3 Lys 79 (H3K79), plays an important role in regulation of transcription, cell cycle and the DNA damage response. Although the oncogenic role of DOT1L in leukemia has been well established, it is still unknown whether DOT1L is implicated in breast cacinogenesis. Here, I reported that DOT1L has a critical role in regulating both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties in human breast cancer. In MCF10A nontransformed epithelial cells and non-invasive T47D human breast cancer cells, overexpression of DOT1L led to morphological transformation, downregulation of epithelial markers, upregulation of mesenchymal markers, and enhanced cell migration and invasion. Consistently, shRNA-mediated DOT1L knockdown in invasive MDA-MB-231 cells suppressed the EMT and invasive properties. Moreover, I showed that DOT1L cooperates with HRAS oncogene to promote tumorigenesis in MCF10A cells. Using chromatin immunoprecipitation, I also found that DOT1L increased the expression of EMT transcription factors (EMT-TFs) including Snail1, ZEB1, and ZEB2 through enhancing H3K79 methylation and H3 acetylation, and inhibiting DNA Methylation at their promoter regions, thereby downregulating the transcription of their target gene, E-cadherin. Furthermore, DOT1L facilitated recruitment of c-Myc and CBP/p300 coactivator complex by directly interacting with this complex and leading to dissociation of c-Myc from HDAC1 and DNMT1 proteins to these promoters. These effects were reversed by knockdown of c-Myc using siRNA, indicating that regulation of EMT-TFs by DOT1L was dependent on c-Myc. Furthermore, I demonstrated that DOT1L increases the EMT-associated stem cell properties in MCF10A and breast cancer cell lines. DOT1L led to expansion of CD44+/CD24-/ESA+ breast CSC population. The self-renewal of CSCs was also enhanced by DOT1L as measured by the abilities for tumorsphere and anchorage-independent growth in vitro. These effects were reversed by knockdown of EMT-TFs using siRNA, implying that the enhanced stemness of CSC by DOT1L overexpression is mediated by EMT-TFs. Taken together, these findings suggest that DOT1L promotes EMT and EMT-associated stem cell activity through epigenetic transcriptional activation of the EMT-TFs in human breast cancer.