Introduction Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.

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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

Introduction: Medullary thyroid carcinomas (MTC) frequently involve activating mutations in the proto-oncogene tyrosine-kinase receptor RET. Vandetanib (ZD6474, ZACTIMA) is a potent inhibitor of VEGFR-2, EGFR and RET 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) for 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, NFKB) 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 analysis 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 inhibits MAPK and STAT3 pathways in MTC. Furthermore, our experimental approach confirmed the utility of protein arrays for analyzing changes in signaling pathways induced by tyrosine-kinase inhibitors. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3616.