Abstract B108: Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs
Authors: Shah Sanket H. , Miller Philip , Drews-Elger Katherine , Brinkman Joeli , Lairet Stefania , Steinberg Alana , El-Ashry Dorraya 
Published: May 2015

Abstract
The tumor microenvironment (TME) has been shown to play a vital role in tumor development and progression. Carcinoma associated fibroblasts (CAFs) form a very essential component of the TME. In fact, the TME in different types of carcinomas, particularly carcinomas of the breast, is comprised mainly of fibroblasts. CAFs in breast cancer TME secrete cytokines and growth factors that are known to activate a multitude of signaling pathways in breast cancer cells. One of these pathways is the MAPK pathway, a signaling pathway activated downstream of ERBB receptor tyrosine kinase family members. We have previously demonstrated that activation of the MAPK pathway represses estrogen receptor (ER) expression leading to an ER-negative phenotype in breast cancer.
We have generated dissociated CAF cell cultures from ER+, ER-/Her2+, and triple negative (TN) primary breast tumors. We have characterized these CAFs on the basis of fibroblast markers, epithelial markers, microarray analysis, soft agar assay and in vivo tumorigenecity studies. Our results show that these CAF populations from primary breast tumors are pure populations of CAF cell cultures. These CAFs provide a model that allows us to examine the role of CAF interaction with ER+ breast cancer cells regarding activation of MAPK and subsequent repression of ER expression. We demonstrate that treatment of the ER+ MCF-7 cell line with conditioned media (CM) from the CAFs results in transient activation of MAPK signaling and subsequent repression of ER. Continuous exposure of ER+ breast cancer cells to soluble factors from CAFs also results in activation of MAPK and down-regulation of ER expression. Importantly, CM from human mammary fibroblasts (HMFs) and CAFs generated from an ER+ tumor do not down-regulate ER expression. Gene expression analysis and cytokine arrays indicate key differences in gene and cytokine expression between HMFs and the ER+ tumor CAFs compared to the two CAFs generated from ER- tumors.
We have previously identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK). This signature identifies the majority of ER- breast cancers as well as a population of ER+ breast cancers that express lower levels of ER. Like the ER- breast cancers identified by this signature, ER+ cancers identified by this signature exhibit gene expression patterns indicative of activated MAPK signaling, and exhibit significantly increased disease recurrence and significantly reduced disease survival. Here we show that breast tumors cancers bearing this hMAPK-miRNA signature have elevated expression of numerous stromal markers associated with poor clinical outcome, as well as a number of microRNAs that are differentially expressed between normal human mammary fibroblasts (HMFs) and mammary tumor carcinoma CAFs. Some of these miRNAs have been established as targeting ER. Using reporter constructs to investigate microRNA regulation of ER, we have observed that treatment of ER+ breast cancer cells with CM from ER- CAFs results in enhanced microRNA-dependent repression of ER.
These data are suggestive of a role for CAF secreted factors in activating MAPK in breast cancer cells leading to repression of ER. In addition, CAFs are able to increase the expression of hMAPK-miRNAs that can down-regulate ER expression either via the activation of MAPK in the cancer cell or secreted directly from the CAFs. We hypothesize that CAFs may contribute to an ER-negative phenotype in breast cancer cells not only by activating MAPK signaling in the tumor cells via secretion of growth factors, but also potentially by transfer of MAPK-regulated miRNAs to the breast cancer cells as well.
Citation Format: Sanket H. Shah, Philip Miller, Katherine Drews-Elger, Joeli Brinkman, Stefania Lairet, Alana Steinberg, Dorraya El-Ashry. Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B108.