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Open Access Research article

Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck

Hanneke Stegeman1*, Johannes H Kaanders1, Deric L Wheeler2, Albert J van der Kogel1, Marieke M Verheijen1, Stijn J Waaijer1, Mari Iida2, Reidar Grénman3, Paul N Span1 and Johan Bussink1

Author Affiliations

1 Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands

2 Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, 1111 Highland Ave, Madison, WI 53705, USA

3 Department of Otorhinolaryngology-Head and Neck Surgery and Department of Medical Biochemistry, Turku University Hospital and University of Turku, PO Box 52, FI-20521, Turku, Finland

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BMC Cancer 2012, 12:463  doi:10.1186/1471-2407-12-463

Published: 10 October 2012

Abstract

Background

Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.

Methods

Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O2 and treated with MK-2206.

Results

We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs=0.77, p=0.10, rs=0.89, p=0.03) and rs=0.93, p=0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all rs<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs=0.51, p=0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.

Conclusions

These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.

Keywords:
Head and neck cancer; Tumor microenvironment; Hypoxia; pAKT; EGFR