Abstract 301A: A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells
Authors: Tyson Lager, Megan Thacker, Charissa Etrheim, Kristi A. Egland, Mark K. Larson, Jennifer A. A. Gubbels
Journal: Cancer Research (2015)

Abstract Over 30% of ovarian cancer (OvCa) patients present with thrombocytosis (elevated plasma platelet count above 450,000 per cubic millimeter) at the time of diagnosis. These patients exhibit shorter survival times and a higher likelihood of advanced stage disease. Platelets have also been shown to enhance metastasis and promote tumor cell survival by coating tumor cells which provides immune escape and protection from chemotherapeutic agents. While previous studies have shown that platelets exhibit differential OvCa cell binding and facilitate cancer cell growth, the mechanism and molecules involved have yet to be elucidated. SUSD2 (SUShi Domain containing 2), an 822 amino acid type I transmembrane protein, is highly abundant in ovarian tumors and normal endothelial cells that line the blood vessels. Endothelial cells do not normally adhere to platelets; therefore, we hypothesized that SUSD2 inhibits the binding of platelets to cancer cells, which subsequently decreases platelet activation. To investigate the role of SUSD2 in platelet binding, SUSD2 knock-down (SUSD2neg) and non-targeting (SUSD2pos) OVCAR3 cell lines were generated. SUSD2pos and SUSD2neg OVCAR3 cell lines were cultured and grown to confluency. Platelets were isolated, dyed with Calcein-AM, washed and added to the cancer cells. After a 15-minute incubation, the co-culture was washed to remove non-adherent platelets. We demonstrated that platelets were bound to the OVCAR3-SUSD2neg an average of 35% more compared to OVCAR3-SUSD2pos cells. In order to identify the receptor on the platelets mediating adhesion to the tumor cells, we used the drug eptifibatide to block GPIIb/IIIa, an integrin receptor on the platelet surface that primarily binds to fibrinogen. Using the same methods described above, platelets were incubated with the inhibitor before being co-incubated with the cancer cells. Treating the platelets with eptifibatide decreased platelet binding by an average of 35% to both cancer cell lines, indicating that platelet binding to tumor cells is partially, yet equally, mediated by GPIIb/IIIa regardless of the presence of SUSD2. The role of this receptor was further explored by performing flow cytometry on the supernatant collected from co-culturing platelets with the cancer cells. The conditioned media contains the platelets that were not bound to the cancer cells. We measured the activation of the unbound platelets using the anti-PAC-1 antibody that binds exclusively to the activated conformation of GPIIb/IIIa. Incubation with the OVCAR-3-SUSD2neg cells activated the unbound platelets three-fold more compared to the OVCAR3-SUSD2pos cells. These data indicate that SUSD2 expression may mediate the activation state of GPIIb/IIIa, and this, in turn, may affect adhesion mediated by other factors. Citation Format: Tyson Lager, Megan Thacker, Charissa Etrheim, Kristi A. Egland, Mark K. Larson, Jennifer A. A. Gubbels. A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 301A. doi:10.1158/1538-7445.AM2015-301A