Abstract
Cancer Associated Fibroblasts (CAFs) are an integral component of the TME, and the majority of breast tumor stroma is comprised of CAFs. Multiple juxtacrine and paracrine interactions occur between cancer cells and CAFs that direct tumor progression. These interactions occur via soluble agents, including cytokines, hormones, growth factors, and secreted microRNAs. Some of these soluble agents induce MAPK activation in tumor cells. Our lab has established that MAPK activation in tumor cells is one of the key mechanisms involved in repression of ER. MAPK activation in tumor cells leads to alteration in miRNA expression. We previously identified a microRNA signature indicative of hyperactive MAPK signaling (hMAPK-miRNA signature) that significantly associated with reduced recurrence-free and overall survival. Here we report that the hMAPK-miRNA signature associates with a high metric of stromal cell infiltrate, and we investigate the role of microRNAs, particularly hMAPK-microRNAs, secreted by CAFs on estrogen receptor (ER) expression in breast cancer cells. We found that CAF conditioned media isolated specifically from ER-negative primary breast tumors led to ER repression. Nanoparticle tracking analysis and transmission electron microscopy confirmed the role of CAF-secreted exosomes in CAF mediated ER-repression. Differentially expressed hMAPK-microRNA 221/222 in CAF CM as well as in MCF-7/ltE2- cells treated with this CM were identified. Knockdown of miR-221/222 in CAFBAS rescued ER repression in ER-positive cell lines treated with CAFBAS-conditioned media demonstrating that CAF-secreted hMAPK-miR221/222 is directly involved in ER-repression. CAFs also play an important role in multiple steps of tumor metastasis, and we investigated its association with the metastatic process. Utilizing our sized based microfilter technology we demonstrate, for the first time, that circulating CAFs (cCAFs, identified by FAP and alpha-SMA co-expression) are present in peripheral blood of patients with metastatic breast cancer. In a pilot patient cohort, we detected the presence of cCAFs in 30 of 34 (88.2%) patients with metastatic breast cancer (MET group) and in 3 of 13 (23.1%) patients with localized breast cancer (LOC group) with long-term disease free survival (Fisher’s exact test p-value = 4.02 x10^-7); importantly, no cCAFs were detected from healthy donors. We also report that counts of cCAFs and circulating tumor cells (CTCs) are significantly greater in breast cancer patients from the MET group than the LOC group (Wilcoxon Rank Sum Test p-values of 0.000017 and 0.002, respectively). Collectively, our results demonstrate that CAF-secreted hMAPK-miRNA contributes to the MAPK-induced ER repression in breast cancer cells and the presence of cCAFs is significantly associated with clinical metastasis.