Microenvironmental Regulation of Epithelial-Mesenchymal Transition

Abstract

Epithelial-mesenchymal transition (EMT) is a phenotypic alteration that endows epithelial cells with mesenchymal characteristics including loss of cell-cell contact and acquisition of motility and invasiveness. An essential process in development, EMT is implicated in cancer progression. The cellular microenvironment plays an important role in regulating cellular processes. Here we examine the effects of the biochemical and mechanical properties of the microenvironment on EMT.

Matrix metalloproteinase-3 (MMP3), an enzyme that degrades the extracellular matrix (ECM), is a potent EMT inducer and causes genomic instability. Mammary epithelial cells exposed to MMP3 undergo EMT via an upregulation of Rac1b, a constitutively active splice variant of Rac1, and production of reactive oxygen species (ROS). We examine the role of the ECM in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. alpha 6-integrin is required for inhibition of EMT by laminin whereas alpha 5-integrin is required for the promotion of EMT by fibronectin.

We also explore the role of the stiffness of the matrix in MMP3-induced EMT. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against EMT whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Rac1b localizes to the plasma membrane in cells cultured on stiff substrata. At the membrane, Rac1b forms a complex with NADPH oxidase and promotes the production of reactive oxygen species, expression of Snail, and activation of the EMT program. In contrast, soft microenvironments inhibit the membrane localization of Rac1b and subsequent redox changes.

Finally we investigate how the gradients of mechanical stress that arise from tissue geometry affect EMT. When treated with transforming growth factor-beta (TGF-beta), cells at the corners and edges of square mammary epithelial sheets express EMT markers, whereas those in the center do not. The EMT-permissive regions experience the highest mechanical stress, as predicted computationally and confirmed experimentally. Myocardin-related transcription factor (MRTF)-A is localized to the nuclei of cells located in high-stress regions, and inhibiting cytoskeletal tension or MRTF-A expression abrogates the spatial patterning of EMT.

Overall, our work demonstrates that EMT is regulated by various microenvironmental factors. Understanding the mechanisms of these regulations will provide insights into new therapeutic strategies to treat breast cancer.