Title

Molecular mechanisms of microRNA regulation in breast cancer cells

Date of Completion

January 2010

Keywords

Biology, Molecular|Chemistry, Biochemistry|Health Sciences, Oncology

Degree

Ph.D.

Abstract

MicroRNAs are key regulators of gene expression that are involved in the maintenance of many biological processes including cell proliferation, and the apoptotic response, pathways which are aberrant in cancer. MicroRNAs repress the translation of target mRNAs, and microRNA expression is commonly dysregulated in cancers. Little is known about the role of microRNAs in tumor development, maintenance, and recurrence. The FOXO1 transcription factor is a putative tumor suppressor that orchestrates the regulation of genes involved in apoptosis and cell cycle checkpoints. Though the expression of this gene is dysregulated in many cancers, the mechanism resulting in aberrant expression of human FOXO1 in cancer cells is poorly understood. We show that FOXO1 mRNA is downregulated in breast tumor samples as compared to normal breast tissue. We subsequently identified functional and specific microRNA target sites in the FOXO1 3' UTR for miR-27a, miR-96, and miR-182. These three microRNAs are highly expressed in MCF-7 breast cancer cells and Ishikawa endometrial carcinoma cells, which have low levels of FOXO1 protein. Antisense to these microRNAs led to a significant increase in endogenous FOXO1 expression, as well as FOXO1-dependent cell death. We have identified a novel mechanism of FOXO1 regulation, and targeting of FOXO1 by microRNAs may contribute to transformation or maintenance of an oncogenic state in breast and endometrial cancer cells. ^ The role microRNA-mediated regulation was also examined in the mammosphere culture system, which enriches for breast tumor-initiating cells. Under these conditions MCF-7 cells underwent an epithelial to mesenchymal transition (EMT), characterized by the loss of epithelial markers, induction of mesenchymal markers, and morphological phenotype (termed LMS cells). MicroRNA expression was analyzed in cells undergoing mammosphere-associated EMT. Three microRNAs, miR-130a, miR-221, and miR-222 were chosen for further study because they are known to target ERα and maintain high expression levels in LMS cells. We hypothesized that the induction of EMT and the mesenchymal phenotype was due to the suppression of ERα by these microRNAs. Inhibition of miR-130a, miR-221, or miR-222 led to re-expression of ERα in LMS cells, providing evidence that these microRNAs have a direct functional role in mammosphere-associated EMT and tumor-initiating cell plasticity. ^