Abstract 4101: Thrombin stimulates glioblastoma cell proliferation, adhesion and in vivo tumor growth through Rap1 activation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Rap1 is a Ras family GTPase that plays an important role in ERK1/2 activation, cell proliferation and adhesion. Little is known about the role of Rap1 in signaling through GPCRs. We report that thrombin coupling to the G-protein-coupled receptor PAR-1 in human glioblastoma cells induces a robust and sustained increase in Rap1 activation. This response is mediated through RhoA activation and phospholipase D (PLD) signaling. In addition to its role in cell proliferation, Rap1 mediates inside-out integrin activation thereby affecting cell adhesion. Accordingly, we tested the possibility that thrombin affects glioblastoma cell adhesion through Rap1. Thrombin treatment lead to a robust 4-5 fold increase in cell adhesion to fibronectin relative to control cells. Down-regulating either PLD1 or Rap1 with siRNA inhibited this response implicating PLD1 and Rap1 in thrombin stimulated cell adhesion. Thrombin was shown to increase cell adhesion through activation of β1 integrin, since a β1 neutralizing antibody blocked the ability of thrombin to increase cell adhesion to fibronectin. Thrombin treatment also increased phosphorylation of FAK at tyrosine 397 and blocking integrin activation with a β1 integrin neutralizing antibody significantly inhibited this response. FAK Y397 phosphorylation by thrombin was found to be Rap1 dependent, as it was abolished by down-regulating Rap1 expression via siRNA. We found that thrombin stimulation lead to a robust and sustained increase in ERK1/2 phosphorylation and glioblastoma cell proliferation. Down-regulation of Rap1 by siRNA inhibited thrombin stimulated ERK1/2 activation and cell proliferation in glioblastoma cells. Consistent with a connection between Rap1, integrin signaling and ERK1/2 activation, the β1 neutralizing antibody also significantly inhibited thrombin induced glioblastoma mitogenesis. Finally, we used a mouse xenograft model to assess the in vivo role of Rap1 in tumor cell growth. Lentiviral mediated Rap1 shRNA expression was used to inhibit Rap1 expression in glioblastoma cells. Nu/Nu mice were then injected with cells treated with lentiviral control scrambled or Rap1 lentiviral shRNA and tumor mass was assessed 22 days following injection. Rap1 knockdown in these cells had a profound effect, with tumor mass reduced by 90% relative to control. Rap1 gene expression was also decreased in these cells by 80% relative control. Taken together, our results suggest that thrombin induces glioblastoma cell proliferation through its ability to activate a Rho/Rap1/β1 integrin/ERK1/2 mediated pathway. These results also demonstrate a critical role for Rap1 in signaling via GPCRs in vitro and in glioblastoma tumor growth in vivo Citation Format: Jacqueline Sayyah, Dwayne Stupack, Joan Heller Brown. Thrombin stimulates glioblastoma cell proliferation, adhesion and in vivo tumor growth through Rap1 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2013-4101