Protease-Activated Receptor-1 (hPar1 ), A Survival Factor Eliciting Tumor Progression Zaidoun Salah, Myriam Maoz, Elisheva Pokroy, Michal Lotem, Rachel Bar-Shavit, and Beatrice Uziely Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel Abstract Although ample evidence point to the central involvement of protease activated receptor-1 (PAR1 ) in tumor progression, little is known about the fate of the tumor when hPar1 is being silenced. We observed that hPar1 antisense clones exhibit low PAR1 levels, attenuated cell proliferation and invasion in vitro , and tumor formation in vivo . These clones showed noticeably reduced paxillin phosphorylation compared with the parental A375SM cells, whereas no change in the integrin levels was noticed. Antisense clones injected into the mice resulted in very few and only occasional small tumors, whereas advanced and vascularized tumors were observed in A375SM cells. The antisense-derived tumor sections expressed active caspase-3, increased terminal deoxynucleotidyl transferase–mediated nick-end labeling staining, and a markedly reduced proliferating cell nuclear antigen level compared with A375SM cell–derived tissue sections. Likewise, ablation of the hPar1 gene in a tetracycline- inducible hPar1 system leads to apoptosis in immature blood vessels, whereas mature vessels were unaffected. The activation of PAR1-induced pAkt/protein kinase B abrogated serum-deprived Bim EL induction and also markedly inhibited Bax levels. On the other hand, small interfering RNA silencing of the hPar1 gene induced the expression of Bim EL , a direct substrate of Akt/protein kinase B and also induced expression of active caspase-9 and caspase-3. These results altogether identify PAR1 as a survival factor that protects cells from undergoing apoptosis. We conclude that whereas PAR1 gene expression correlates with tumor progression, its neutralization effectively initiates an apoptotic pathway leading at least in part to significantly reduced tumor formation. (Mol Cancer Res 2007;5(3):229–40) Introduction Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR1-PAR4). Members of the family are uniquely activated by proteolytic cleavage of their extracellular portion exposing tethered ligands specific for each PAR (1, 2). PAR1, the prototypic receptor of the family, is activated via a spectrum of serine proteases taking part in the thrombotic/hemostasis cascade, including thrombin (3), plasmin (4), Xa (5), and activated protein C (6). Human Par1 (hPar1 ) plays a distinct role in the progression of malignant epithelia as well as in the physiologic invasion process of trophoblast implantation in the uterus decidua (7-10). The overexpression of PAR1 is a striking feature of epithelia malignancy, whereas none or very little PAR1 expression is observed in normal epithelial cells, shown in a wide array of cell lines and tissue biopsy specimens (7, 8). Accumulating evidence suggests that prolonged survival machinery is mostly responsible for enhanced tumor invasion process. Members of the phosphatidylinositol 3¶ kinase/Akt signaling axis are central mediators of the prosurvival pathway. Phosphatidylinositol 3¶ kinase, a lipid kinase, catalyzes and recruits inactive cytosolic molecules to the membrane where they undergo activation. One of these is Akt, a proto-oncogene originally discovered as the cellular homologue of the v-Akt oncogene (11). Upon stimulation, the inactive Akt undergoes subsequent phosphorylation by upstream kinases that lock it in an active conformation. Akt is then released into cellular compartments where it acts as a kinase and phosphorylates numerous substrates such as Forkhead transcription factors (FOXO), glycogen synthase kinase-3, Bcl-associated dimer, and murine double minute 2 (12). Hyperactivation of Akt tips the balance in favor of prosurvival pathways, whereas reduced activity of Akt tips the balance toward apoptosis. Apoptosis can be triggered in vivo by stress events like hypoxia and growth factor deprivation or by activation of the death receptor family (e.g., tumor necrosis factor–related apoptosis-inducing ligand, refs. 13, 14). These conditions lead to activation of caspases, a family of cysteine aspartyl proteases that are involved in a cascade of cleavage events that ultimately result in apoptosis. Thus, prosurvival modulation and the apoptotic machinery pathway are both key in forming the appropriate balance for normal cell maintenance. For example, factor VIIa/tissue factor, apart from its role in coagulation, also induces tumor progression partly via eliciting a survival pathway and inhibiting apoptosis (15, 16). We wished to elucidate the prosurvival potential of the hPar1 gene in the context of tumor progression. For this, we generated stable clones expressing hPar1 antisense and compared their properties with the parental super-metastatic melanoma cell lines. In parallel, in a tetracycline hPar1 - inducible system, ablation of the hPar1 gene led to a markedly reduced number of blood vessels, mediated primarily by Received 8/17/06; revised 12/4/06; accepted 1/22/07. Grant support: Imperial Cancer Research Fund and Israeli Science Foundation (R. Bar-Shavit). 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. Requests for reprints: Rachel Bar-Shavit, Department of Oncology, Hadassah- Hebrew University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Phone: 972-2-6777563; Fax: 972-2-6422794. E-mail: barshav@md.huji.ac.il Copyright D 2007 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-06-0261 Mol Cancer Res 2007;5(3). March 2007 229 Downloaded from http://aacrjournals.org/mcr/article-pdf/5/3/229/3140603/229.pdf by guest on 04 February 2024