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Role of Rab25 GTPase in breast epithelial cell proliferation
Rab GTPases represent a family of almost 70 Ras-like enzymes that regulate receptor trafficking. Rabs catalyze collection of receptors into nascent transport vesicles, motility of transport vesicles, and their docking and fusion with target membranes. Rab25 is strongly implicated as a determinant of breast cancer aggressiveness. In a recent study, genomic amplification of Rab25 was detected in half of breast cancers examined, and increased expression decreased overall patient survival. The goal of this application is to investigate the role of Rab25 in regulating breast epithelial cell proliferation. Rab25 is primarily, epithelial cell-specific and it is localized to a compartment termed the apical recycling endosome. There, Rab25 is somehow involved in the sorting of receptors to the apical plasma membrane. The goal of this proposal will be to test the hypothesis that overexpression of Rab25 enhances receptor tyrosine kinase (RTK) signaling by altering receptor down regulation and enhancing PI3P signaling events. We will explore the role of Rab25 in signal transduction in three cell lines: AU565 cells (a HER2 overexpressing mammary carcinoma); MCF7 cells (estrogen responsive, differentiated mammary epithelium); and MCF-12A non-tumorigenic mammary epithelial cells. Aim 1. We will (A) determine the endogenous levels of Rab25 in these cells, (B) explore ligand-induced RTK phosphorylation after treatment with siRNA to deplete Rab25 or introduction of a plasmid to induce Rab25 expression, with specific focus on EGF-, HER2-, and IGF-1 receptors. These experiments will test the hypothesis that activated RTK steady state levels are influenced by Rab25. If so, we will determine the rate of ligand-induced receptor degradation to test a role for Rab25 in receptor down regulation and/or recyling. Aim 2. Explore the ability of Rab25 to hydrolyze GTP. Like Ras, Rab25 is expected to use cellular factors to stimulate both nucleotide hydrolysis and GDP release. Rab25 is unusual: residues key to GTP hydrolysis resemble Ras mutants that are poor at GTP hydrolysis. Previous studies concluded that Rab25 is an active GTPase but the assay employed has a fatal flaw. We will determine if Rab25 can convert GTP to GDP. If not, Rab25 would represent a constitutively active protein, explaining its potency in transformation of normal breast epithelia, upon up-regulation. In summary, these experiments seek to determine precisely what Rab25 does to speed the progression of breast cancer. Future work would include identification of effector proteins that sense Rab25's activation state and mediate its activities.
This application is stimulated by the recent finding that a protein named Rab25 is strongly implicated as a determinant of the aggressiveness of breast cancer. In one study, the tumor cells were found to contain extra copies of the Rab25 gene; moreover, higher than normal levels of Rab25 decreased overall patient survival and was an independent indicator of disease-free survival. The goal of this application to learn what Rab25 does, and why it is so strongly linked to breast cancer. Cell surface receptors signal cells to proliferate upon binding to extracellular hormones and growth factors. They are then removed from the cell surface to stop the signaling process. Rab proteins control the removal of receptors from the surface and their delivery to compartments inside the cell that degrade the hormones and the receptors, and thereby turn off the growth signals. This lab has studied Rab GTPases for twelve years and we have significant expertise to contribute to the study of Rab25. We will explore the role of Rab25 in cell signaling in three cell lines: a HER2-overexpressing mammary carcinoma; estrogen responsive, differentiated mammary epithelial cells; and non-tumorigenic mammary epithelial cells. We seek to determine how Rab25 influences receptor signaling and trafficking in normal breast cells and in cancerous breast cells. Our goal is to determine precisely what Rab25 does to speed the progression of breast cancer.