A role of proepithelin in tumor invasion: Promotion of cell migration and invasion of bladder cancer cells through the activation of ERK1/2 and the formation of a paxillin/FAK/ERK complex

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The growth factor proepithelin (also known as progranulin or PC-derived growth factor) has emerged in recent years as an important regulator of cell growth, migration and transformation in several epithelial cells. In this study we have investigated whether proepithelin plays any role in the migration and invasion of bladder cancer cells using an in vitro system. In our earlier experiments, using conditioned medium from proepithelin-overexpressing fibroblasts as source of proepithelin, we established that proepithelin promotes migration of normal and transformed bladder cells. Here we demonstrate a direct activity of human recombinant proepithelin purified to homogeneity from 293-EBNA cells. Specifically, we show that nanomolar amounts of proepithelin promote the migration and invasion through a three dimensional extracellular matrix of 5637 transitional carcinoma-derived cells. Moreover, proepithelin stimulates in vitro closure of a wound. These effects require the activation of the Erk1/2 kinases pathway, as determined by Western blot using phospho-specific antibodies for the activated proteins. The role Erk1/2 in proepithelin-stimulated cell signaling of 5637 cells was further confirmed using specific inhibitors of the Erk1/2 kinase pathway (U0126) and siRNA strategies. We also demonstrate that proepithelin promotes tyrosine-phosphorylation of the scaffolding protein paxillin and the formation of a paxillin/FAK/phosphor-ERK in vivo complex. The critical role of paxillin in proepithelin-promoted migration of 5637 cells was confirmed using siRNA strategies and we demonstrate that depletion of endogenous paxillin abolishes proepithelin-mediated migration and wound healing. Our results provide the first evidence for a role of proepithelin in stimulating migration and invasion of bladder cancer cells, and support the hypothesis that this growth factor may play a critical role in the establishment of the invasive phenotype. This work has been supported by a grant from the Commonwealth of Pennsylvania,, the Benjamin Perkins Bladder Cancer Fund, the Martin Greitzer Fund and National Institutes of Health Grants RO1 DK 068419 (A.M.) and RO1 CA39481 and RO1 CA047282 (R.V.I.). Michelle Hellman has been supported through the Medical Student Summer General Medicine Research Program by the generosity of the Thomas Jefferson University’s Office of the Dean.