The miR-200 family is controlled by epigenetic-based mechanisms and mediates transition between non-stem and stem-like cell phenotypes.

Abstract

MicroRNAs (miRNAs) are ~22 nucleotide (nt) single-stranded non-coding RNAs which are important regulators of gene expression in many biological processes including controlling cellular phenotype. The epithelial to mesenchymal transition (EMT) and the reverse process termed mesenchymal to epithelial transition (MET) are key programs that control the transition of cells between stem-like and non-stem phenotypes which are collectively termed epithelial plasticity. The miR-200 family is a key regulator of EMT however its role in controlling the transition between stem-like and non-stem phenotypes has not been well characterized. I utilized immortalized human mammary epithelial cells (HMLE) to investigate the function and regulation of the miR-200s during their conversion from a non-stem to a stem-like phenotype. HMLE cells were found to spontaneously convert from a non-stem to a stem-like phenotype. Isolation and comparison of the miR-200 expression between the spontaneously derived stem- like cells (sl-HMLE) and non-stem HMLE cells (nsl-HMLE) showed that the spontaneous conversion to a stem-like phenotype was accompanied by the loss of miR-200 expression. Likewise, miR-200 expression was also found to be down-regulated in prospective breast cancer stem cells (bCSCs) from metastatic pleural or ascites effusions and SUM159PT breast cancer cell line compared to non-CSC cells. This phenotypic change from a non-stem to a stem-like phenotype was directly controlled by the miR-200s as restoration of its expression partially converted the sl-HMLE cells to a non-stem phenotype with decrease stem-like properties and induction of an MET-like phenotype, although restoration of the miR-200 expression in SUM159PT prospective bCSCs did not have this effect. Next, using bioinformatic approaches and cell-based assays, I aimed to identify new miR-200 targets that are responsible for regulating the stem-like properties in both sl-HMLE cells and SUM159PT prospective bCSCs. Although the predicted genes (WNT5A, PKCα and PKCε) were not direct miR-200 targets, preliminary data suggest those genes may be involve in the survival or anoikis-resistance of stem-like cells and bCSCs. Investigation of the mechanism(s) controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b/a/429 cluster was silenced primarily through polycomb group-mediated silencing whereas the miR-200c/141 cluster was repressed by DNA methylation. Furthermore, slight increase in EZH2 expression was observed in the stem-like phenotype and this might potentially contribute to the polycomb group-mediated silencing of the miR-200b/a/429 cluster. Lastly, preliminary co-immunoprecipitation results suggest that the targeting of polycomb group proteins to the miR-200b/a/429 promoter is not dependent on the ZEB1 transcription factor which is a repressor of the miR-200 transcription. Collectively, these results indicate that the miR-200 family plays a critical role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therefore, combination of chemotherapy with therapies targeted against the miR-200 family members and epigenetic modifications would be beneficial towards treatment of breast cancer.