Abstract LB-146: Investigation of Gas6/Axl signaling in clear cell renal cell carcinoma tumor cells and endothelial cells in presence of the first line treatment drug Sunitinib Malate

Abstract

Abstract Introductory sentence: The aim is to investigate whether Gas6 dependent Axl activation and downstream signaling can occur in clear cell Renal Cell Carcinoma tumor cells (ccRCCs) and Endothelial cells (ECs) during treatment with Sunitinib Malate. Experimental set up: The ccRCC cell line 786-O and ECs of human aortic origin (HAECs) were grown in vitro, serum-starved over night and pre-incubated with Sunitinib Malate for 2 hours prior 100 ng/mL Gas6 stimulation for 10 minutes. Activation of Axl in terms of induced tyrosine phosphorylation was determined by immunoprecipitation and immunoblotting. Gas6-induced downstream signaling of Axl was analyzed by immunoblotting for phosphorylation of Akt and Erk protein in total cell lysates. Gas6/Axl specific signaling was verified by pre-incubation with an Axl specific kinase inhibitor, R428. Functionality of Sunitinib Malate was verified by inhibition of VEGFR-activation in ECs. In addition, prolonged stimulation of ccRCCs with Gas6 only was analyzed up to 2 days. Longterm effects on induced Axl activation and downstream Akt and Erk signaling in ccRCCs were investigated as described above. Results: In ccRCCs and ECs, Gas6-induced Axl activation and downstream signaling are resistant to physiologically relevant and higher doses of Sunitinib Malate. Gas6/Axl-signaling activates Akt and Erk pathways in presence of Sunitinib Malate. Sunitinib Malate contributes to Gas6 signaling with an increased phospho-Axl, Akt and Erk activation in ccRCCs and ECs. Axl is constitutively activated in ccRCCs upon longterm Gas6 stimulation, with early peak Erk activation and sustained Akt activation. Conclusions and importance: RCC is a lethal cancer with bad prognosis due to development of chemoresistance and recurrence of a more aggressive tumor. The Gas6/Axl system influences many cellular processes, including survival, migration and invasion, contributing to both chemoresistance and metastasis of several tumor types. Axl overexpression is also associated with poor patient outcome. Our data indicates that the Gas6/Axl system can be constitutively activated in ccRCCs with sustained Axl activation and downstream signaling of pathways known to apply survival, growth and proliferative advantage to tumor cells. Sunitinib Malate, first-line anti-angiogenic targeted therapy drug used in advanced RCC, does not inhibit and moreover, increases Gas6/Axl signaling in both ccRCCs and ECs. Importantly this suggests that, Sunitinib Malate, while targeting ECs and tumor-angiogenesis, simultaneously possibly provide a pro-tumorigenic tumor microenvironment where Gas6/Axl signaling, not inhibited by Sunitinib Malate, stimulates ccRCCs and ECs more intensively than in the non-treated tumor microenvironment. Altogether, Gas6/Axl signaling during targeted therapy is possibly contributing to the acquired chemoresistance and recurrence of aggressive tumors in patients treated for advanced RCC. Citation Format: Anna Gustafsson, Björn Dahlbäck. Investigation of Gas6/Axl signaling in clear cell renal cell carcinoma tumor cells and endothelial cells in presence of the first line treatment drug Sunitinib Malate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-146. doi:10.1158/1538-7445.AM2014-LB-146