Epithelium/stroma cross-talk in breast cancer

During tumorigenesis stroma initially plays a suppressing role, but overtime evolves to promote tumor growth, as demonstrated in many experimental studies. Normal tissue architecture becomes disordered; the extracellular matrix (ECM) is remodeled by mesenchymal cells and the relationships between epithelium and basement membrane are disrupted. Tumor stroma is the microenvironment which surrounds the malignant cells and makes up about a half of malignant tumors. The cells found in the stroma include blood and lymph endothelial cells, pericytes, leukocytes, macrophages and fibroblasts. In particular, fibroblasts termed “cancer associated fibroblasts” (CAFs) have received increased attention because of their involvement in tumor development, including invasion and metastasis. In the tumor stroma, a great number of CAFs and myofibroblasts, high capillary density, deposition of mature type I collagen and other ECM components are observed. The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. To verify if this cross-talk may affect some aspects of cell biology (i.e. proliferation, adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (CAFs) in monolayer or in a three-dimensional system (nodules). In terms of proliferation, we demonstrated that CAFs exerted a mitogenic effect on both ER+ and ER- breast cancer cells, while mammary cancer cells had no influence on the growth of both kinds of fibroblasts. NFs and CAFs respectively induced or inhibited E-cadherin expression in MCF-7 cells; CAFs promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. An increase in N-cadherin was observed in CAFs but not in NFs, as a result of the interaction with both kinds of cancer cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity and even a marked increase in the directness induced by CAFs. A dramatic increase in Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating the fatty acid membrane composition, has been observed in both MCF-7 and MDA-MB-231 cells as the result of the interaction with CAFs. In addition, the presence of fibroblasts, in particular CAFs, stimulated binding of SREBP1, a transcription factor which specifically binds to the SREBP response element on the SCD1 promoter, in tumor cells. Finally, performing co-culture assays in the presence neutralizing antibodies against hepatocyte growth factor, transforming growth factor-beta and fibroblast growth factor-2, which are released by CAFs, we demonstrated that both the speed and the directness increases induced by CAFs in cancer cell migration were strongly reduced or completely abolished. All these results provide new insights in the understanding the role of CAFs in promoting the tumor cell invasiveness and may help in defining new therapeutic targets in breast cancer.