Abstract A20: The Six1-regulated miR-106b-25 cluster is a mediator of the tumor promotional effects of TGF-β signaling in human breast cancer

Abstract Recent studies have highlighted the developmental transcription factor Six1 as an important mediator of breast cancer progression and metastasis. Six1 is overexpressed in a striking 90% of metastatic breast cancer lesions, and patients whose tumors overexpress Six1 have a decreased time to metastasis and relapse, as well as an overall decrease in survival. Six1 mediates metastasis via multiple mechanisms, including its ability to induce an epithelial-to-mesenchymal transition (EMT) and tumor initiating cell (TIC) characteristics, both of which are dependent on an upregulation of TGF-β signaling. Indeed, the Six1-induced increase in TGF-β signaling is critical for the ability of Six1 to induce late stage metastasis. Interestingly, Six1 not only activates TGF-β signaling, but it also enables the switch of TGF-β signaling from tumor suppressive to tumor promotional, a phenomenon of considerable importance in cancer pathogenesis. This event, coined the “TGF-β paradox,” has been an area of extensive research, but remains largely elusive. To further investigate the mechanism by which Six1 mediates the switch in TGF-β signaling, we performed a miRNA microarray screen and identified a cluster of miRNAs, the miR-106b-25 cluster, that is upregulated in response to Six1 overexpression. The miR-106b-25 cluster consists of three miRNAs, miR-106b, miR-93, and miR-25, which reside together in the intron of the MCM7 gene. Importantly, overexpression and knockdown experiments demonstrate that Six1 regulates all three miRNAs within the cluster. Interestingly, these miRNA have previously been implicated in the impairment of TGF-β-mediated growth suppression through repression of the cell cycle inhibitor, p21, and pro-apoptotic factor, Bim. These data suggest that Six1-induced upregulation of these miRNA may mediate the switch in TGF-β signaling from tumor suppressive to tumor promotional. Surprinsingly, bioinformatic analysis revealed that the miR-106b-25 cluster may also contribute to the activation of TGFβ signaling through repression of the TGF-β signaling inhibitor, Smad7, which mediates the degradation of TβRI. Indeed, overexpression of the miR-106b-25 cluster results in repression of the Smad7 protein, with concominant upregulation of TβRI. Furthermore, activation of TGF-β signaling is observed with miR-106b-25 overexpression, as demonstrated by an increase in phosphorylation of the downstream effector of the TGF-β pathway, Smad3, and by an upregulation of downstream TGF-β transcriptional targets. miRNA inhibition also demonstrates that miR-106b and miR-93 are necessary for Six1 induced TGF-β activation. Additionally, like Six1, the miR-106b-25 cluster is sufficient to induce features of EMT, including a redistribution of E-cadherin, and increased β-catenin transcriptional activation. Furthermore, the miR-106b-25 cluster is also sufficient to increase the TIC population as measured by flow cytometry, mammosphere formation, and in vivo serial dilution assays. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-β signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promotional. Citation Format: Anna L. Smith, Ritsuko Iwanaga, David J. Drasin, Douglas S. Micalizzi, Rebecca L. Vartuli, Heide L. Ford. The Six1-regulated miR-106b-25 cluster is a mediator of the tumor promotional effects of TGF-β signaling in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A20.