Molecular Inhibition of Phospholipase Cy Signaling Abrogates DU-145 Prostate Tumor Cell Invasion’

Up-regulated signaling from the epidermal growth factor receptor (EGFR) has been correlated with tumor invasion and metastasis in numerous human neoplasias. Recently, we have demonstrated that increased levels of EGFR promote the invasiveness of human prostate carcinoma DU145 cells However, the intracellular signaling pathway responsible for this enhanced tumor invasiveness has not been identified. We postulated that increased cell motility signaled via phospholipase C y (PLCy) activation was critical for tumor invasiveness. Highly invasive DU-145 cells engineered to overexpress the EGFR were stably transfected with a dominant-negative fragment of PLOy from the Zregion (PLCz) or with irrelevant peptide minigenes. PLCz was expressed only in the appropriate transfectant lines, with a concomitant decrease in inositol phosphate generation. The transfectant cell lines all formed tumors when inoculated into the peritoneal cavity of athymic mice. Tumors from the cells expressing PLCz fragment were significantly less invasive than the transfectants containing the control minigenes, as assessed by the diaphragm invasion model and invasion into abdominal soft Organs. The cells expressing PLCz grew and formed colonies in soft agar at rates comparable to the cells expressing the control minigenes. These data suggest that up-regulated signaling by EGFR promotes prostate tumor invasiveness secondary to increased cell motility. Furthermore, PLCy represents a potential therapeutic target to limit tumor progression promoted by up-regulated signaling from the EGFR and related receptors with intrinsic tyrosine kinase activity. Received 5/27/97: revised 8/1/97; accepted 8/12/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This study was supported by a Merit Award from the Veterans Administration. 2 Present address: Department of Biology, Carver Research Foundation, Tuskegee University. Tuskegee. AL 36088. 3 To whom requests for reprints should be addressed. at Department of Pathology, LHRB53 I , University of Alabama at Birmingham. Birmingham. AL 35294-0007. Phone: (205) 934-0357; Fax: (205) 975-9927: E-mail: [email protected]. INTRODUCTION Prostate cancer-related deaths are due mainly to tumor invasion and metastasis ( 1 , 2). Curative therapies are available only if the cancer is organ confined. Once the tumor breaches the prostate capsule to invade locally or metastasize to distant sites, none of the prevailing treatments dramatically enhance patient survival (2-6). Hence, a more in-depth understanding of the basic biology of prostate cancer progression to the invasive and metastatic stages would increase the knowledge required for developing novel therapeutic approaches. Cell properties such as proliferation. motility. and proteolysis are principal in tumor cell progression to the invasive and metastatic stage. Disruption of any one of these processes may, in turn, interfere significantly with tumor progression. Tumor cell motility is critical in this process of tumor invasiveness and metastasis. Therefore, interventions have been designed to inhibit enhanced tumor motility and prevent tumor progression (7-10). One potential target would be the motility signaling pathway induced by growth factors acting via up-regulated growth factor receptors ( I 1 ). The growth factor receptor most often found up-regulated in human tumors that have progressed to the invasive and metastatic state is the EGFR4 ( I 1). Upregulated EGFR has been correlated with tumor invasion and metastasis in glioblastoma multiformes (12), gastric (13). bladden (14), non-small cell lung (15), and breast carcinomas (16). These clinical correlations are supported by experimental model systems in which metastatic potential of human colon carcinoma cells correlated with EGFR level (17, 18). Thus, EGFR upregulation has been correlated not with simple proliferation but with tumor progression. Cancers of the prostate present autocrine stimulatory loops in which both the EGFR and its activating ligand, transforming growth factor a. are produced by the carcinoma cells (19-23). We have demonstrated previously that increased levels of fulllength EGFR promotes the invasiveness of DU-l45 human prostate carcinoma cells both in vitro (20) and in vito (24), whereas expression of an EGFR variant that is fully mitogenic but lacks the capacity to stimulate cell motility (c’973) results in decreased invasiveness secondary to down-regulation of endogenous EGFR. The question is whether the operational signal for tumor invasiveness is that of cell motility. PLC-y activation is required for motility signaled by the EGFR (25) as well as the related receptors for PDGF (26) and IGF-l (27). Initial experiments demonstrated that inhibition of 4 The abbreviations used are: EGFR. epidermal growth factor receptor: PLC y. phospholipase C : PLCz, PLC’y from the Z-region: PDGF. platelet-derived growth factor; IGF, insulin-like growth factor: WT. wild type: MMTV, Moboney murine tumor virus; LTR, long terminal repeat: FBS. fetal bovine serum: IP. inositol phosphate: MiT. 3-(4.5dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide. Research. on October 16, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from