Regulation of cyclin E1 by the breast cancer microenvironment.

摘要

The behavior of breast epithelial cells is influenced by their microenvironment, which includes stromal cells and extracellular matrix. During breast cancer progression, the tissue microenvironment fails to control proliferation, resulting in uncontrolled growth and invasion. Upon invasion, the extracellular matrix encountered by breast cancer cells changes from primarily laminin and collagen IV to primarily collagen I. The current study used a three-dimensional (3D) collagen I culture model to address how the microenvironment controls the breast cancer cell cycle after invasion through the basement membrane. We show that culturing breast cancer cells in 3D collagen I inhibits proliferation through direct regulation of cyclin E1, a G1/S regulator that is overexpressed in breast cancer and implicated in its etiology. 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 regulates progression through early G1, where it is upregulated by signals from the extracellular environment. Cyclin D1 relocalized to the cytoplasm in 3D cultures and phosphorylation of Rb, a nuclear target for both cyclin E1- and cyclin D1-associated kinases, was decreased. Positive regulators of cyclin E1, the transcription factor c-Myc and cold-inducible RNA binding protein (CIRP), were decreased in 3D collagen cultures, while the collagen I receptor beta1 integrin was greatly increased. Inhibition of beta1 integrin function rescued proliferation, cyclin E1 and c-Myc expression, and Rb phosphorylation, but had no effect on cyclin D1 localization. We conclude that cyclin E1 is repressed independent of effects on cyclin D1 in a 3D collagen environment and dependent on beta1 integrin interaction with collagen I, reducing proliferation of invasive breast cancer cells. These results differ from studies of breast epithelial and cancer cells in 2D collagen or 3D Matrigel in which disruption of beta1 integrin function decreased proliferation. We also show that matrix metalloproteinases (MMPs) MMP-2 and MMP-9 increased dramatically in 3D collagen. Their inhibition reversed cyclin E1 reduction, indicating their upregulation may be key to this process. In addition to studying MDA-MB-231 cells, we show that 3D collagen has similar effects on the nontumorigenic breast epithelial cell line MCF10A and the breast cancer cell line HMT-3522 T4-2 cells. Proliferation and cyclin E1 were downregulated in both of these cell lines, with MCF10A cells undergoing global G1 arrest and subsequent apoptosis. Our data suggest that one way breast cancer cells adapt themselves to a collagen environment is by upregulating a collagen I receptor to decrease proliferation through cyclin E1 repression. Cyclin E1 therefore appears to link the ECM and cell cycle machinery, arguing the importance of considering the role of ECM in breast cancer etiology, valuing cyclin E1 as a prognostic factor, as well as targeting cyclin E1 overexpression in clinical therapy.