Abstract B39: Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer

Over the past decade an accumulating body of evidence has demonstrated the pivotal role of stromal cells in promoting breast cancer progression. In breast cancer, the stroma is mainly comprised of carcinoma-associated fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that are known to activate MAPK pathway. We have previously established that hyperactivation of MAPK in breast cancer cells leads to repression of estrogen receptor (ER) expression. Aberrant mitogenic signaling also affects expression of microRNAs (miRNAs). We have recently identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK-miRNAs). Most ER- breast cancers and a population of ER+ breast cancers are identified by this signature, and these hMAPK-miRNA tumors exhibit significantly decreased disease-free survival and overall survival compared to tumors that are not hMAPK-miRNA. Some of these hMAPK-miRNAs have been established as targeting ER. To further investigate the contribution of CAFs in activation of MAPK signaling in cancer cells, we isolated CAF cell cultures from three different primary breast tumors: CAF19 (from a luminal A tumor), CAF21 (from a Her-2 like tumor) and CAF23 (from a basal like tumor) and tumor cell cultures (DTs) from 5 different primary breast tumors. There is differential expression of hMAPK miRNAs between these CAFs and DTs, including those that target ER. Thus, we propose that interactions between cancer and stromal cells lead to activation of MAPK signaling in the cancer cell and aberrant miRNA expression within the TME that contribute to the biology of ER-negative tumors. To further investigate the connection between hMAPK-miRNAs and stroma, we analysed TCGA breast cancer datasets and found that hMAPK-miRNAs are significantly associated with tumors that possess high stromal scores. Co-culture of CAFs with ER+ breast cancer cells, as well as treatment of ER+ breast cancer cells with conditioned media (CM) from CAFs, induces both MAPK activation and altered expression of miRNAs, specifically MAPK miRNAs within the breast cancer cells. Treatment of ER+ breast cancer cells with CM from CAFs represses both ER protein and mRNA expression, and importantly, this ER repression is specific to CAFs isolated from ER-negative breast tumors (CAF21 and CAF23) compared to the CAFs obtained from ER-positive tumors (CAF19). Additionally, CM from CAF21 and CAF23 caused repression of an ER 39UTR reporter construct transfected into ER+ breast cancer cells, indicating a role for miRNAs either secreted into the CM or induced in the cancer cell in repressing ER. To investigate a role for hMAPK-miRNAs secreted by CAFs in mediating ER repression, nanostring analysis of miRNAs isolated from ER+ breast cancer cells treated with CAF CM was performed and differentially expressed miRNAs between CM from CAFs obtained from ER-positive (DT19) vs ER-negative tumors (DT21 and DT23) were identified. qPCR analysis of conditioned media also confirmed the presence of hMAPK-miRNAs in CAF CM. Collectively, these data indicate a role for CAFs in regulating hMAPK-miRNAs, and thus in establishing an ER-negative breast cancer phenotype, and importantly, differences between CAFs from ER-negative as compared to ER-positive tumors in this role. They further suggest that hMAPK-miRNAs secreted from CAFs participate in the induction of the ER-negative phenotype induced by MAPK. Citation Format: Sanket H. Shah, Phil Miller, Emilio Issa, Katherine Drews-Elger, Dorraya El-Ashry. Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B39. doi:10.1158/1538-7445.CHTME14-B39