Title

Elucidating microRNA-mediated regulation of estrogen signaling and response in human breast cancer cells

Date of Completion

January 2009

Abstract

Basal-like breast cancers are more invasive, of a higher grade, and confer poorer clinical prognosis than ERα-positive, Luminal-A cancers. Unfortunately, mechanisms by which this ERα-negative tumor subtype arises remain obscure. MicroRNAs are small noncoding RNAs that diminish the stability and/or translation of mRNAs. To determine whether microRNAs are involved in generating an ERα-negative Basal-like tumor subtype, we first investigated if miR-206, previously shown to be elevated in ERα-negative breast tumors, could regulate ERα expression. We demonstrated that two miR-206 binding sites located within the 3'-UTR of human ERα mRNA were functionally active in ERα-positive, MCF-7 breast cancer cells. We also demonstrated that miR-206 coordinately targets mRNAs encoding the co-activator proteins SRC-1 and SRC-3, and the transcription factor GATA-3, in turn abrogating global ERα/estradiol signaling in MCF-7 cells. Moreover, overexpression of miR-206 in MCF-7 cells resulted in decreased cell proliferation, enhanced apoptosis, diminished expression of numerous estrogen-responsive genes, and a loss of a Luminal-A phenotype. We then investigated whether the signaling pathways known to be disrupted in breast cancer were potential regulators of miR-206. While miR-206 levels were inhibited by estradiol and ERα agonists in MCF-7 cells, EGFR siRNA treatments and MEK inhibitors indicated EGFR/MAPK signaling promotes miR-206 levels in ERα-negative, EGFR-positive MDA-MB-231 cells. Furthermore, blocking EGF-induced enhancement of miR-206 abrogated the known EGF inhibitory effects on estrogenic responses in MCF-7 cells. While these findings elucidate the involvement of microRNAs in the induction of a Luminal-A to Basal-like phenotypic switch in breast cancer cells, little is known regarding the dysregulation of Argonaute-2 (the catalytic engine of microRNA effector complexes) that also occurs within breast cancer. We demonstrated that high levels of Argonaute-2 in ERα-negative vs. ERα-positive breast cancer cell lines, was due to hyperactive EGFR/MAPK signaling. Also, the stable overexpression of Argonaute-2 in MCF-7 cells resulted in enhanced proliferation, reduced cell-cell adhesion, and increased migratory ability, indicating that Argonaute-2 may induce tumorigenic progression in ERα-positive breast cancer cells. Overall, miR-206 and Argonaute-2 are highly dysregulated in breast cancer due to gain of ancillary oncogenic signaling pathways, can actively modulate breast tumorigenic processes, and could become novel biomarkers for this disease. ^