Abstract 5628: Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6.
Authors: Boeckx Carolien , Deschoolmeester Vanessa , Wouters An , Op de Beeckx Ken , Pauwels Patrick , Vanderveken Olivier , van Camp Guy , Vanden Berghe Wim , Specenier Pol , Vermorken Jan B. , Peeters Marc , Baay Marc , Lardon Filip 
Published: August 2014

Abstract
Introduction: As the epidermal growth factor receptor (EGFR) is expressed in up to 90% of all HNSCC tumors and initiates important signaling pathways in carcinogenesis, cetuximab, an EGFR targeting agent, is a promising therapeutic agent. However, many tumors remain non-responsive. Therefore, unraveling the underlying mechanism of resistance is of major importance.
Previously, we performed gene expression studies of cetuximab sensitive (LICR-HN2 and SC263) and cetuximab resistant (LICR-HN1 and Cal27) cell lines. These results indicated an upregulation of Aurora kinase B (AURKB) in LICR-HN1 cells and a downregulation of dual specificity phosphatase 6 (DUSP6) in Cal27 cells.
Methods: Growth inhibitory experiments for barasertib, an AURKB targeting agent, and apigenin, a MAPK inhibitor, were performed as single-agent (barasertib: 0-25nM and apigenin: 0-40μM), and in combination with 15nM cetuximab for 168h using simultaneous and sequential treatment schedules. Induction of apoptotic cell death was investigated in LICR-HN1 cells using the Annexin V-FITC apoptosis detection kit for all treatment schedules. Immunohistochemical staining for AURKB was performed on formalin fixed, paraffin-embedded tissues from 52 newly diagnosed HNSCC patient samples, using an automated IHC staining platform and will be correlated with clinicopathological parameters. The phosphorylation status of ERK was investigated using the NanoPro technology and the methylation status of DUSP6 will be analyzed using the Pyrosequencing method.
Results: The mitosis-targeting drug barasertib induced cytotoxicity in the cetuximab resistant HNSCC cell lines, Cal27 and LICR-HN1, with IC50 values of 13.15 ± 0.60nM and 17.6 ± 1.38nM, respectively. In combination therapy with cetuximab, no additional benefit over barasertib as single agent was observed. However, preliminary results showed that pretreatment of LICR-HN1 cells with 15nM cetuximab followed by barasertib (10 or 20nM) resulted in less viable cells compared to cetuximab or barasertib treatment alone. AURKB expression was present in HNSCC patients, with intensities ranging from 0 - 100%.
Apigenin induced cytotoxicity in the two cetuximab resistant cell lines, Cal27 and LICR-HN1, with IC50 values of 22.29 ± 0.60μM and 30.66 ± 0.97μM respectively. Preliminary results showed that the combination of cetuximab with apigenin had a synergistic effect in the Cal27 cell line. Furthermore, increased phosphorylation of ERK was present in the cetuximab-resistant cell lines compared to the cetuximab-sensitive cell lines.
Conclusion: These results seem to confirm that overexpression of AURKB and/or downregulation of DUSP6 are involved in mechanisms of cetuximab resistance in HNSCC cell lines and inhibition of these pathways might, therefore, be a new strategy for anticancer therapy.
Citation Format: Carolien Boeckx, Vanessa Deschoolmeester, An Wouters, Ken Op de Beeckx, Patrick Pauwels, Olivier Vanderveken, Guy Van Camp, Wim Vanden Berghe, Pol Specenier, Jan B. Vermorken, Marc Peeters, Marc Baay, Filip Lardon. Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6. [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 5628. doi:10.1158/1538-7445.AM2013-5628