Abstract LB-58: Angiogenic signaling is upregulated in human oral squamous carcinoma cells with acquired resistance to the EGFR inhibitor gefitinib
Authors: Box Carol , Mendiola Marta , Gowan Sharon , Box Gary M. , Nutting Christopher M. , Evans Peter H. Rhys , Harrington Kevin J. , Eccles Suzanne A. 
Published: February 2011

Abstract
Tyrosine kinase inhibitors (TKIs) have improved the therapeutic options for patients with EGFR-driven tumors; however initially responsive patients frequently relapse whilst on therapy. To investigate acquired resistance to EGFR TKIs we developed and characterized in vitro and in vivo models of gefitinib-resistance through chronic drug exposure of a squamous carcinoma cell line, CAL-27. The derived CALR subline showed 166-fold resistance to gefitinib compared with CALS cells (drug-sensitive controls). Constitutive levels of EGFR were unchanged but phospho-EGFR was reduced and phospho-AKT increased in CALR cells. No upregulation of MET or IGF-1R signalling pathways, previously reported in acquired resistance to EGFR TKIs, was seen. The growth rate and vascularisation of CALR xenografts was significantly greater than CALS, although in vitro proliferation rates were similar, observations recapitulated in a second, independently-derived gefitinib-resistant cell line. We thus hypothesised that the differential in vivo growth may be attributed to differences in angiogenic signalling between gefitinib-sensitive and -resistant tumors. To test this hypothesis we examined the expression of HIF1α (hypoxia-inducible factor 1α) a key mediator of tumor angiogenesis through transcriptional regulation of angiogenic factors, most notably VEGF-A. Here we present the novel finding that HIF1α is constitutively upregulated in gefitinib-resistant CALR cells in normoxia in vitro, and in xenograft tumors. This is accompanied by a concomitant increase in constitutive VEGF-A secretion from the resistant cells determined by ELISA, demonstrating that HIF1α is transcriptionally active. Multiplex antibody arrays (Proteome Profiler™, R&D Systems) were used to assess the expression of 55 angiogenesis-related proteins in gefitinib-sensitive and -resistant cells. Membranes spotted with capture antibodies were incubated with lysates or mouse sera and signals from a chemiluminescent substrate were quantified by densitometry using ImageQuant software (Nonlinear Dynamics). We identified 22 angiogenic proteins differentially expressed between CALS and CALR cells in vitro, several of which are transcriptionally regulated by HIF1α. These include pro-angiogenic factors upregulated in the CALR cells such as VEGF-A (confirming ELISA data) whilst a number of anti-angiogenic proteins were down-regulated, two of which were independently identified in xenograft-bearing mouse sera. In summary, using our model of gefitinib-resistant squamous carcinoma we have identified HIF1α as a potential novel mediator of gefitinib resistance and, with the aid of novel antibody array technology, have identified angiogenesis-related molecules not previously reported to associate with EGFR TKI acquired resistance. Together these data support our hypothesis that increased signalling via the EGFR-HIF-AKT axis is associated with the acquisition of resistance to gefitinib in oral squamous carcinoma.
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 LB-58.