Abstract 4102: Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines
Authors: Pauw Ines De , Wouters An , Bossche Jolien Van den , Deschoolmeester Vanessa , Pauwels Patrick , Vermorken Jan B. , Peeters Marc , Lardon Filip 
Published: July 2017

Abstract
Aberrant signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After the initial promising results of EGFR-targeted therapies, the problem of therapeutic resistance is emerging and new treatment options are necessary. In contrast to the first generation EGFR inhibitors, afatinib is an irreversible ErbB family blocker that inhibits EGFR as well as HER2 and HER4. The objective of this study was to investigate whether afatinib is able to overcome intrinsic and acquired cetuximab resistance in human papillomavirus (HPV) positive and negative HNSCC cell lines under normal and reduced oxygen conditions and to identify the molecular mechanisms underlying afatinib’s cytotoxic effect.
Sensitivity to cetuximab treatment was investigated in a panel of HPV positive and negative HNSCC cell lines using the colorimetric sulforhodamine B (SRB) assay (168h, 0-2μM). Cetuximab sensitive and intrinsically resistant cell lines were identified. Acquired cetuximab resistant cell lines were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines were established by exposing these cells to the vehicle control (PBS). Cytotoxicity of afatinib (72h, 0 - 5μM) was assessed under normoxic and hypoxic (1% O2) conditions. Cell cycle distribution and induction of apoptotic cell death were assessed flow cytometrically using the Vindelov method and Annexin V-FITC/PI assay, respectively. Data analysis was performed using WinNonlin and FlowJo software.
We identified intrinsic cetuximab resistance in 5 out of 7 HNSCC cell lines (i.e. LICR-HN1, Cal-27, SQD9, 93-VU-147T and UM-SCC104) and generated acquired cetuximab resistant cell lines (i.e. SCC22b-R and SC263-R). Afatinib showed a clear concentration-dependent cytotoxic effect in both cetuximab sensitive and resistant cell lines with IC50 values ranging between 0.019 and 4.04 μM. Furthermore, afatinib maintained its cytotoxic effect under hypoxic conditions. Treatment with afatinib led to an increase of the proportion of cells in the G0/G1 phase of the cell cycle. Afatinib also induced an increase in the percentage of AnnV+/PI- and AnnV+/PI+ cells with a corresponding decrease of the percentage viable (AnnV-/PI-) cells.
In conclusion, our results suggest that afatinib has the potential to overcome cetuximab resistance, as it was able to establish cytotoxicity in HPV positive and negative HNSCC cell lines that were intrinsically and acquired resistant to cetuximab. Furthermore, treatment with afatinib caused a G0/G1 cell cycle arrest and induced apoptotic cell death. These data support the hypothesis that afatinib might be a promising novel therapeutic strategy to treat HNSCC patients experiencing intrinsic or acquired cetuximab resistance.
Citation Format: Ines De Pauw, An Wouters, Jolien Van den Bossche, Vanessa Deschoolmeester, Patrick Pauwels, Jan B. Vermorken, Marc Peeters, Filip Lardon. Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2017-4102