DU145 human prostate carcinoma invasiveness is modulated by urokinase receptor (uPAR) downstream of epidermal growth factor receptor (EGFR) signaling Asmaa Mamoune, a,b,1 Jareer Kassis, a,b,1,2 Sourabh Kharait, a,b Susanne Kloeker, c Elisabeth Manos, c David A. Jones, c and Alan Wells a,b, * a Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA b Pittsburgh VAMC, Pittsburgh, PA 15261, USA c Division of Molecular Pharmacology, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA Received 16 January 2004, revised version recieved 6 May 2004 Available online 17 June 2004 Abstract Tumor cell motility and invasion have been linked to upregulated signaling from both the epidermal growth factor receptor (EGFR) and that for urokinase-type plasminogen activator (uPAR). However, we do not know whether these events are interdependent or unrelated, despite the obvious diagnostic and therapeutic implications. Gene microarray analyses have suggested that EGFR signaling via phospholipase C-g (PLCg) induces uPAR transcription. We utilized two sublines of the DU145 human prostate carcinoma cell line that are genetically engineered to differentially activate the EGFR/PLCg cascade and are variously invasive in vitro and in vivo. uPAR protein levels in these cells were found to be dependent on PLC signaling, pharmacologic inhibition of PLC signaling reduced uPAR expression. To determine whether uPAR was a required element in EGFR-mediated invasion, we stably expressed uPAR cDNA in either sense or antisense orientation in the two DU145 sublines. Interestingly, uPA production was modulated in parallel, although to a lesser degree, with uPAR in these sublines. Antisense to uPAR significantly restricted invasion of the highly invasive DU145 WT cells through Matrigel and reduced aggressiveness of tumors in nude mice. Up-regulation of uPAR significantly increased the invasiveness of the moderately invasive DU145 parental (DU145 P) cells through Matrigel, but this increased invasiveness was not seen in mice. uPA activity appears to contribute to invasiveness at least through Matrigel, as antibody to uPA or amiloride limited the transmigration. These results support a model of tumor invasion promoted by autocrine EGFR signaling involving reinforcing altered gene expression, of uPAR at least, that further induces cell motility. Herein, a number of key molecules whose expression levels are interrelated, including both EGFR and uPAR, are required but none are sufficient in the absence of other keys molecules in promoting tumor progression. D 2004 Elsevier Inc. All rights reserved. Keywords: Tumor progression; EGF receptor; Phospholipase C-g; Motility; Migration Introduction One of the rate-limiting steps of prostate tumor cell invasion is its ability to breach an extracellular matrix (ECM) [1]. An increasing amount of recent data not only have elucidated a variety of cellular pathways and mecha- nisms involved, but also have alluded to the interdepen- dence and cross-communication of many of these pathways. However, one relatively common aspect of cellular invasion mechanisms is their mediation by surface receptors which, in transformed cell, are often overexpressed and upregu- lated. Of these receptors, the epidermal growth factor receptor (EGFR) is the most frequently upregulated in tumors including prostate carcinomas [2,3]. EGFR signaling promotes tumor progression via both epigenetic events such as de-adhesion and cytoskeletal reorganization [4], as well as altering the transcriptional profile of the cancer cell [5,6]. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.05.008 * Corresponding author. Department of Pathology, University of Pittsburgh, 713 Scaife Hall, Pittsburgh, PA 15261. Fax: +1-412-647-8567. E-mail address: wellsa@msx.upmc.edu (A. Wells). 1 These authors contributed equally to this publication. 2 Current address: Building 10, Room B1B40, National Cancer Institute, NIH, Bethesda, MD 20892. www.elsevier.com/locate/yexcr Experimental Cell Research 299 (2004) 91 – 100