Abstract P4-07-01: Upregulation of miR-224 in breast cancer exhibiting hyperactivation of ERK1/2 MAPK signaling associates with repression of Raf kinase inhibitory protein and poor clinical outcome

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks expression of estrogen (ER) and progesterone receptors, and which does not have amplification of HER2. One particular characteristic of TNBC is overexpression of EGFR, a growth factor receptor, and hyperactivation of downstream signaling pathways. Women with breast cancers that overexpress EGFR and exhibit hyperactive growth factor signaling pathways tend to suffer higher incidence of disease recurrence, metastasis, and disease-related death, suggesting that targeting EGFR signaling or the pathways and genes that it activates may present an opportunity to identify genes that can be utilized as targeted therapy in TNBC. Our lab has recently identified a microRNA signature that is associated with hyperactive ERK1/2 MAPK signaling, a key pathway activated downstream of EGFR. Our hyperactive MAPK microRNA signature (hMAPK-miRNA) is found in the substantial majority of ER-negative breast tumors as well as in a subset of ER+ breast tumors with low expression of ER, and is significantly associated with poor survival and increased disease recurrence. One of these upregulated microRNAs, miR-224, has recently been shown to act as an oncogene in hepatocellular carcinoma by repressing RAF-kinase inhibitory protein (RKIP), a negative regulator of ERK1/2 MAPK signaling through repression of RAF1. RKIP is also involved in repressing a number of genes associated with breast cancer metastasis including OPN, CXCR4, and MMP1. Building on these observations and the fact that many TNBC exhibit activated growth factor signaling through the ERK1/2 MAPK axis, we hypothesize that miR-224 may act as an oncogene in breast cancer by repressing RKIP, leading to a feed-forward loop resulting in sustained activation of MAPK signaling. Here we show that breast tumors classified as hMAPK-miRNA by our hMAPK-miRNA signature display significantly lower expression of RKIP, elevated expression of miR-224, and show a significant inverse correlation between expression of RKIP and miR-224. We also observe significantly higher expression of RKIP repressed genes OPN, CXCR4, and MMP1 in hMAPK-miRNA tumors. Using MCF-7 cell lines stably transfected to have hyperactivation of ERK1/2 MAPK signaling, we investigate the effects that alterations of MAPK signaling has on expression of miR-224, RKIP, and RKIP regulated genes. We further demonstrate the effects of knockdown of miR-224 expression in our primary tumor cultures from TNBC on RKIP-regulated gene expression, hMAPK activity and miRNAs, and in vitro and in vivo measures of tumorigenesis and metastasis. miR-224 targeting of RKIP leading to sustained hMAPK activation then would be expected to result to aberrant expression of MAPK-mediated miRNAs, repressing ER, CCND1, and other genes involved in luminal identity, activating CCNB1 and other basal-associated genes, contributing to an ER-negative phenotype and driving breast cancer cell proliferation, survival, EMT, and metastasis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-01.