Abstract 3569: Changes in signaling pathways induced by vandetanib in a human medullary thyroid carcinoma model as detected by reverse phase protein arrays

Abstract

Abstract Introduction: Medullary thyroid carcinomas (MTC) frequently involve activating mutations in the proto-oncogene tyrosine-kinase receptor RET. Vandetanib (ZD6474, ZACTIMATM) is a potent inhibitor of RET and VEGFR-2 tyrosine-kinase activities which is currently in phase III clinical trial in MTC. The aim of this study was to investigate in vitro and in vivo molecular effects of vandetanib on MTC signaling pathways using a proteomic approach based on reverse-phase protein arrays (RPPA). Methods: The human MTC TT cell line, bearing a RETC634W activating mutation, was cultured in the absence or the presence of vandetanib (100, 500, 1000 nM) during 4, 24 and 72 hours. MTC TT xenografted athymic nu/nu mice received vandetanib (50 mg/kg/day p.o.) or placebo and xenografts were removed after 1 and 4 days of treatment. Twenty-eight protein extracts from in vitro (n=24) and in vivo (n=4) experiments were profiled by RPPA analysis. Equal amounts of lysates were printed in serial dilutions onto nitrocellulose-coated glass slides. Each slide was then probed with each of a 20-antibodies set, specific for oncogenic pathways (PI3K/AKT/mTOR, MEK/p44-42, STAT3, NFκB) and cell cycle/apoptotic proteins (CDK1, PARP, cleaved-PARP). Results: In in vitro experiments, vandetanib treatment induced an increase of cleaved-PARP (p<10-5) with a 3-fold rise after 72 hours at 100 nM. Phosphorylation of NFKB was not significantly modified when compared to actin. In contrast, phosphorylation of STAT3 significantly decreased after 72 hours of vandetanib treatment. Overall, the MAPK signaling pathway was dramatically inhibited by vandetanib, as substantiated by the decrease of MEK and p44-42 phosphorylation when compared to their total protein forms (p<10-3). Inhibition of p44/42 and STAT3 phosphorylation were also observed in MTC TT xenografted mice treated by vandetanib. Several proteins were re-analyzed by western-blot and/or immunohistochemistry analyses and the results were in agreement with those observed in RPPA. Conclusions: Together these observations demonstrated that, at the protein level, vandetanib induces apoptosis and mainly inhibits MAPK and AKT pathways in MTC. Furthermore, our experimental approach confirmed the interest of this method of protein arrays for analyzing changes in signaling pathways induced by tyrosine-kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3569. doi:10.1158/1538-7445.AM2011-3569