Abstract 1030: The intriguing interplay between EGFR inhibitors and the hypoxic microenvironment: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines.
Authors: Boeckx Carolien , Van den Bossche Jolien , Baay Marc , Deschoolmeester Vanessa , Specenier Pol , Vanderveken Olivier , Van den Weyngaert Danielle , Vermorken Jan B. , Peeters Marc , Lardon Filip , Wouters An 
Published: August 2014

Abstract
Introduction: Since the epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of all head and neck squamous cell carcinomas (HNSCC) and activation of EGFR initiates important signal transduction pathways in carcinogenesis, it has emerged as a promising therapeutic target. Nevertheless, one main challenge alongside the treatment with EGFR inhibitors remains, being drug resistance. An important potential mechanism involves the hypoxic tumor microenvironment. As our knowledge about the exact impact of tumor hypoxia on the activity of EGFR inhibitors and vice versa remains limited, the present study investigated the cytotoxic effect of the EGFR-targeting monoclonal antibody cetuximab and the EGFR tyrosine kinase inhibitor erlotinib under normoxic versus hypoxic conditions.
Materials & Methods: Three cetuximab-sensitive HNSCC cell lines (SC263, LICR-HN2 and LICR-HN5) were treated either with 0 - 10 nM cetuximab for 168 hours or with 0 - 20 μM erlotinib for 72 hours. Cells were incubated under normal or reduced oxygen conditions for 24 or 72 hours immediately after addition of the drug, with hypoxia (&lt;0.1% O2) being achieved in a Bactron IV anaerobic chamber. Cell survival was assessed with the sulforhodamine B assay and IC50 values (representing the drug concentration reducing cell growth to 50%) were calculated.
Results: Cetuximab and erlotinib establish a dose-dependent growth inhibition under both normal and reduced oxygen conditions in all three HNSCC cell lines. Considering the effect of cetuximab on cell survival, no significant differences were observed in IC50 values when cells were incubated under normoxia versus hypoxia for 24 or 72 hours (p=0.790, p=0.807 and p=0.288 for SC263, LICR-HN2 and LICR-HN5 respectively). For example, in LICR-HN2 HNSCC cells, cetuximab treatment resulted in IC50 values of 0.14±0.09 nM, 0.19±0.12 nM, and 0.15±0.07 nM for incubation under normoxia versus hypoxia for 24 or 72 hours, respectively. Similarly, the efficacy of erlotinib was not influenced by the oxygen tension in LICR-HN2 and LICR-HN5 cells (p=0.171 and p=0.387, respectively). In SC263 cells, however, a significantly increased sensitivity to erlotinib (p=0.006) was shown when cells were incubated under hypoxic conditions, with IC50 values of 1.53±1.07 μM, 0.81±0.28 μM, and 0.40±0.00 μM for incubation under normoxic conditions versus hypoxic conditions for 24 hours or 72 hours, respectively.
Conclusion: Our results suggest that, in the HNSCC cell lines included in our study, both EGFR-inhibitors cetuximab and erlotinib maintain their growth inhibitory effect under hypoxia. As poor oxygenation is a pathophysiological property of the majority of human solid tumors, including HNSCC, further studies are warranted to define the precise mechanistic and therapeutic implications of these observations.
Citation Format: Carolien Boeckx, Jolien Van den Bossche, Marc Baay, Vanessa Deschoolmeester, Pol Specenier, Olivier Vanderveken, Danielle Van den Weyngaert, Jan B. Vermorken, Marc Peeters, Filip Lardon, An Wouters. The intriguing interplay between EGFR inhibitors and the hypoxic microenvironment: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2013-1030