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Notch Regulation of Epithelial-Mesenchymal Transition During Wound Healing and Migration of Mammary Epithelial Cells
The development and remodeling of epithelial tissues require extensive changes in cell morphology and migration; a process termed Epithelial Mesenchymal Transition (EMT). Although EMT occurs under physiological conditions such as wound healing, it has also been implicated in malignant transformation. Epithelial-derived tumor cells often exhibit mesenchymal properties, such as poor differentiation state and increased motility. Specifically in breast tumors, increasing tumor grades correlate with increased mesenchymal-like properties. Signals leading to cell proliferation, differentiation, apoptosis and fate determination are conveyed between cells to coordinate tissue development and homeostasis, and the Notch family of transmembrane proteins has been implicated in this process. Notch is required for differentiation and fate determination during development, but also in adult tissues where it is required for maintenance of cell identity. Mutations leading to aberrant activation of Notch signaling have been identified in several types of carcinomas; in particular Notch-4 mutations are found in mouse mammary tumors. Since Notch is required for differentiation and the maintenance of epithelial cell identity and since EMT represents loss of differentiation, we hypothesize that Notch signaling might play a central role during EMT. We propose to establish a cell-based wound healing assay to study EMT in human mammary epithelial cells. We hypothesize that loss of cell-cell contacts after introduction of a wound in a monolayer leads to altered Notch signaling in the leading edge of migrating cells, inducing local EMT and cell migration. Pharmacological and molecular reagents will be used to alter Notch signaling and evaluate the role of the Notch pathway in cell-cell adhesion, migration and EMT. Immunofluorescence, time-lapse and confocal microscopy, in combination with biochemical analysis and single-cell based studies will allow us to directly assess the role of Notch signaling during wound healing. This study will be later extended to 3D-cultures and xenograft models to address the role of Notch signaling in mammary tissue development and remodeling, as well as mammary carcinogenesis in vivo. To our knowledge, this is the first study that directly examines the role of Notch signaling in human mammary biology and tumorigenesis. Understanding the molecular mechanisms underlying Notch signaling and EMT is essential for the establishment of successful in vivo studies and the development of potential anticancer drugs.
Most tissues, including the breast, are made of tightly packed and highly organized sheets of cells called epithelia. To maintain tissue structure, epithelial cells make use of strong cell-cell adhesion contacts. However, various physiological situations require changes in tissue organization, for example during embryonic development, and in the adult during breast development in puberty or in pregnancy. At these times, epithelial cells can undergo a process termed Epithelial Mesenchymal Transition (EMT), which involves changes in cell shape, a decrease in cell-cell adhesion and an increase in cell motility. It is believed that inappropriate activation of this EMT pathway, due to genetic mutations for instance, may also lead to cancer. Indeed, many cancer cells exhibit EMT properties, particularly in advanced malignancies and metastases. Specifically in breast tumors, increasing tumor grades correlate with increased EMT. Notch is a cell-surface protein that is essential for the development of various tissues. Upon cell-cell contact the Notch protein activates a genetic program that determines epithelial cell fate. Furthermore, mutations in Notch proteins have been identified in various cancers including breast cancer. The fact that Notch is required for cell specification and that abnormal Notch proteins are found in cancer implies that Notch may be a key player in tumor development. We propose to establish a cell-based tissue culture assay to study EMT and cell migration and to look at the role of Notch in this system. Various cell lines derived from human breast tissues ranging from normal to highly metastatic will be used to analyze the role of Notch signaling. Specific pharmacological and molecular reagents that directly activate or inactivate the Notch pathway will be used. This study is vital to the understanding of the molecular mechanisms regulating EMT and will address directly the potential involvement of Notch in this process. To our knowledge, this is the first study that directly examines the role of Notch signaling in human mammary biology and carcinogenesis. Understanding the mechanisms underlying Notch signaling and EMT is essential for the establishment of successful in vivo studies and the development of potential anticancer drugs.