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The mammary gland is one of the best-studied examples of an organ whose structure and function are influenced by reciprocal signaling and communication between cells and their microenvironment. The mammary epithelial cell (MEC) microenvironment includes stromal cells and extracellular matrix (ECM). Abundant evidence shows that the ECM and growth factors co-operate to regulate cell cycle progression, and that the ECM is altered in breast tumors. In particular, mammographically dense breast tissue is a significant risk factor for developing breast carcinomas. Dense breast tissue is associated with increased stromal collagen and epithelial cell content. In this article, we overview recent studies addressing the effects of ECM composition on the breast cancer cell cycle. Although the normal breast ECM keeps the MEC cycle in check, the ECM remodeling associated with breast cancer positively regulates the MEC cycle. ECM effects on the downstream biochemical and mechanosignaling pathways in both normal and tumorigenic MECs will be reviewed., (Copyright © 2012 Wiley Periodicals, Inc.)

The behavior of breast epithelial cells is influenced by their microenvironment which includes stromal cells and extracellular matrix (ECM). During cancer progression, the tissue microenvironment fails to control proliferation and differentiation, resulting in uncontrolled growth and invasion. Upon invasion, the ECM encountered by breast cancer cells changes from primarily laminin and collagen IV to primarily collagen I. We show here that culturing invasive breast cancer cells in 3-dimensional (3D) collagen I inhibits proliferation through direct regulation of cyclin E1, a G(1)/S regulator that is overexpressed in breast cancer. When the breast cancer cell line MDA-MB-231 was cultured within 3D collagen I gels, the G(1)/S transition was inhibited as compared to cells cultured on conventional 2D collagen or plastic dishes. Cells in 3D collagen downregulated cyclin E1 protein and mRNA, with no change in cyclin D1 level. Cyclin D1 was primarily cytoplasmic in 3D cultures, and this was accompanied by decreased phosphorylation of Rb, a nuclear target for both cyclin E1- and cyclin D1-associated kinases. Positive regulators of cyclin E1 expression, the transcription factor c-Myc and cold-inducible RNA binding protein (CIRP), were decreased in 3D collagen cultures, while the collagen I receptor β1 integrin was greatly increased. Inhibition of β1 integrin function rescued proliferation and cyclin E1 expression as well as c-Myc expression and Rb phosphorylation, but cyclin D1 remained cytoplasmic. We conclude that cyclin E1 is repressed independent of effects on cyclin D1 in a 3D collagen environment and dependent on β1 integrin interaction with collagen I, reducing proliferation of invasive breast cancer cells.

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress-induced RNA-binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF-7 breast cancer cells by stabilizing its mRNA. We show here that cold-inducible RNA-binding protein (CIRP) co-regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co-regulate cyclin E1, HuR and CIRP levels were altered in MCF-7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co-precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co-localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR-containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets.

MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development,normal cell function and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis.In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.