Roles of the MicroRNA miR-31 in tumor metastasis and an experimental system for the unbiased discovery of genes relevant for breast cancer metastasis

Abstract

In these studies, the microRNA miR-31 was identified as a potent inhibitor of breast cancer metastasis. miR-31 expression levels were inversely associated with the propensity to develop metastatic disease in human breast cancer patients. Additionally, various functional analysis revealed that miR-31 expression was both necessary and sufficient to impede breast cancer metastasis. These effects did not involve confounding influences on primary tumor development; instead, miR-31 exerted its anti-metastatic activities by impinging upon at least three distinct steps of the invasion-metastasis cascade: local invasion, one or more early post intravasation events, and metastatic colonization. At a mechanistic level, miR-31 impaired metastasis via the pleiotropic suppression of a cohort of target genes that otherwise operate to promote metastasis, including integrin a5, radixin, and RhoA. Significantly, the concomitant re-expression of integrin a5, radixin, and RhoA sufficed to override the full spectrum of miR-31'7s anti-metastatic activities. Moreover, the concurrent short hairpin RNA-conferred knockdown of endogenous integrin a5, radixin, and RhoA levels closely phenocopied the known consequences of ectopic miR-31 expression on metastasis. Integrin a5, radixin, and RhoA were found to act during at least partially unique steps of the invasion-metastasis cascade downstream of miR-31. Notably, the temporally controlled re-activation of miR-31 in already-established metastases elicited metastatic regression. These anti-metastatic therapeutic responses were attributable to the capacity of acutely re-expressed miR-31 to induce both cell cycle arrest and apoptosis; such effects arose specifically within the context of the foreign microenvironment present at a metastatic locus. When taken together, these findings provide mechanistic insights concerning the regulation of breast cancer metastasis and suggest that miR-31 may represent a clinically useful prognostic biomarker and/or therapeutic target in certain aggressive human carcinomas. In addition, a novel experimental system for the unbiased identification of metastasisrelevant genes was described. The utility of this system was demonstrated in an initial proof-of-concept screen, which implicated RhoJ as a previously unappreciated modulator of cell motility. Collectively, these observations imply that the single-cell clone-based screening methodology outlined herein may represent a generally useful means by which to enumerate novel regulators of various metastasis-relevant processes.