Abstract 4597: MAPK-mediated gene expression in ER-negative breast cancer: Role of miRNAs and Ago2

Abstract

Abstract Background: We have previously shown that the ERα-negative phenotype in breast cancer can be driven by hyperactive MAPK signaling resulting from up-regulation of ErbB family members. We generated a hyperactive MAPK gene signature that is indicative of ERα negativity, contains many genes related to invasion, motility, metastasis, and EMT, and that shows inverse regulation of many genes known to be estrogen dependent. Recent studies have identified differential micro-RNA (miRNA) expression and activity between ERα+ and ERα- breast tumors, implying that miRNAs and their protein targets may play critical roles in both the development and maintenance of these breast cancers. Argonaute-2 (Ago2, EIF2C2), a member of the Argonaute family of proteins, associates with other proteins and miRNAs to form the RNA Induced Silencing Complex (RISC). It is also known that MAPK signaling enhances the expression and stability of Ago2, and in the absence of MAPK signaling Ago2 levels are depleted. Objective: In this study, we sought to confirm the role of MAPK signaling in Ago2 expression and stability, to determine the role of miRNAs in MAPK mediated down-regulation of ERα expression, to identify MAPK-regulated miRNAs and miRNAs whose activity is mediated through Ago2, and to investigate the roles of these miRNAs in the expression of genes in the hyperactive MAPK gene signature. Results: Inhibiting MAPK signaling in our hyperactive MAPK cell lines using the MEK inhibitor U0126 resulted in significantly decreased Ago2 expression. Conversely, hyperactivation of MAPK signaling significantly increased Ago2 expression. Using an ERα, 3′UTR-luc reporter containing the sequence known to be targeted by several miRNAs upregulated in ERα- vs ERα+ breast cancer, we determined that hyperactive MAPK drives miRNA targeting of ERα. We then generated an miRNA profile associated with hyperactive MAPK. Notably among the miRNAs whose expression is increased by MAPK are miRNA 221/222, which have been previously shown to be upregulated in ERα- breast cancer and target ERα directly. Using our MAPK gene signature, we determined the miRNAs predicted to regulate those genes, and the activities of these miRNAs in the presence and absence of hyperactive MAPK signaling. Similarly, we are establishing the subset of miRNAs whose activity is altered upon siRNA knockdown of Ago2, indicating that their activities are mediated specifically through Ago2, and then correlating these miRNAs with those predicted to target the MAPK gene signature. Conclusions: Hyperactive MAPK signaling is directly involved in the generation and maintenance of the ERα negative phenotype. We have shown that specific miRNAs play an important role in this biological process. Ultimately, we will determine if Ago2 plays an important role as a direct mediator of signal transduction events and global gene expression through its integral role in the biological activity of these specific miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4597.