SUMMARY MicroRNAs are well-suited to regulate tumor metastasis due to their capacity to coordinately repress numerous target genes, thereby potentially enabling their intervention at multiple steps of the invasion-metastasis cascade. We identify a microRNA exemplifying these attributes, miR-31, whose expression correlates inversely with metastasis in human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive breast tumor cells suppresses metastasis. We deploy a stable microRNA sponge strategy to stably inhibit miR-31 in vivo; this allows otherwise-non-aggressive breast cancer cells to metastasize. These phenotypes do not involve confounding influences on primary tumor development and are specifically attributable to miR-31-mediated inhibition of several steps of metastasis, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization. Such pleiotropy is achieved via coordinate repression of a cohort of metastasis-promoting genes, including RhoA. Indeed, RhoA re-expression partially reverses miR-31-imposed metastasis-suppression. These findings indicate that miR-31 uses multiple mechanisms to oppose metastasis.

It remains unclear whether a microRNA (miRNA) affects a given phenotype via concomitant down-regulation of its entire repertoire of targets or instead by suppression of only a modest subset of effectors. We demonstrate that inhibition of breast cancer metastasis by miR-31-a miRNA predicted to modulate >200 mRNAs-can be entirely explained by miR-31's pleiotropic regulation of three targets. Thus, concurrent re-expression of integrina5, radixin, and RhoA abrogates miR-31-imposed metastasis suppression. These effectors influence distinct steps of the metastatic process. Our findings have implications concerning the importance of pleiotropy for the biological actions of miRNAs and provide mechanistic insights into metastasis.Supplemental material is available at http://www.genesdev.org.Received June 15, 2009; revised version accepted September 24, 2009. MicroRNAs (miRNAs) are an evolutionarily conserved family of regulatory RNAs that inhibit their mRNA targets post-transcriptionally, leading to modulation of diverse biological processes, including the development and progression of cancer (Ambros 2004;Bartel 2009;Ventura and Jacks 2009). An individual miRNA is capable of regulating dozens of distinct mRNAs (Baek et al. 2008;Selbach et al. 2008), and it is thought that pleiotropic suppression of multiple downstream effectors may underlie the phenotypic changes observed upon perturbing the levels of certain miRNAs (Rodriguez et al. 2007;Thai et al. 2007;van Rooij et al. 2007;Zhao et al. 2007;Johnnidis et al. 2008;Ventura et al. 2008). It remains unclear, however, whether these consequences depend on simultaneous deregulation of the entire repertoire of targets of a given miRNA or instead on the altered activity of only a small subset of effectors.Metastases, which are responsible for 90% of human cancer deaths, arise via a complex series of events, collectively termed the invasion-metastasis cascade (Fidler 2003;Gupta and Massagué 2006). In order to metastasize, cells in a primary tumor must become motile, degrade surrounding extracellular matrix (local invasion), intravasate into the vasculature, retain viability during transit through the circulation, extravasate into the parenchyma of a distant tissue, survive in this foreign microenvironment to form micrometastases, and, finally, thrive in their new milieu and establish macroscopic secondary tumors (colonization) (Fidler 2003). Colonization is the rate-limiting step of the invasion-metastasis cascade, yet the molecular underpinnings of this process are poorly understood (Gupta and Massagué 2006).We determined recently that expression of the miRNA miR-31 was both necessary and sufficient to inhibit the metastasis of human breast cancer xenografts, and that miR-31 levels correlated inversely with metastatic relapse in breast carcinoma patients ). We attributed these effects to miR-31's ability to pleiotropically suppress a cohort of prometastatic targets; however, we did not identify a minimal set of downstream effectors whose concomitant re-expression is suffic...

miR-31 inhibits breast cancer metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin α 5 (ITGA5), radixin (RDX), and RhoA. We previously showed that the concomitant overexpression of ITGA5, RDX, and RhoA was capable of overriding the antimetastatic effects of ectopically expressed miR-31 in vivo. However, these prior studies failed to investigate whether the combined suppression of the endogenous mRNAs encoding these three proteins recapitulated the in vivo consequences of miR-31 expression on metastasis. We show here that short hairpin RNA-mediated concurrent downregulation of ITGA5, RDX, and RhoA is sufficient to phenocopy the full spectrum of described influences of miR-31 on metastasis in vivo, including the effects of this microRNA (miRNA) on local invasion, early post-intravasation events, and metastatic colonization. These findings provide mechanistic insights into the metastatic process and have implications about the importance of pleiotropy for the biological actions of miRNAs. Cancer Res; 70(12); 5147-54. ©2010 AACR.