Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct 10:12:463.
doi: 10.1186/1471-2407-12-463.

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

Hanneke Stegeman  1 , Johannes H Kaanders, Deric L Wheeler, Albert J van der Kogel, Marieke M Verheijen, Stijn J Waaijer, Mari Iida, Reidar Grénman, Paul N Span, Johan Bussink
Affiliations

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

Hanneke Stegeman et al. BMC Cancer. .

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% O(2) 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 r(s)<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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
In vitro and in vivo expression of EGFR, pEGFR, pAKT and AKT in 6 HNSCC lines. Cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo) and expression levels were determined with western blot. Expression of α-tubulin was used as loading control. A) In vitro expression of EGFR, pEGFR, pAKT and AKT. B) In vivo expression of EGFR, pEGFR, pAKT and AKT. Number of harvested tumors ranged from 2 to 4 per cell line.
Figure 2
Figure 2
Correlation between in vitro and in vivo expression of EGFR/HER2 and pAKT/pSTAT3/pERK1/2 in 6 HNSCC lines.A) Correlation between in vitro and in vivo expression of EGFR and HER2. B) Correlation between in vitro and in vivo expression of pAKT, pERK1/2 and pSTAT3. Expression was assessed by western blot analysis and depicted in relative units. The integrated optical density (IOD) was measured and all values were normalized to those of α-tubulin by dividing the IOD value for that specific marker by the IOD value of α-tubulin. In vitro expression of UT-SCC5 was set as standard. Error bars represent standard error of the mean and all axes are in log scale. Correlations between in vitro and in vivo expression were assessed using the Spearman correlation test.
Figure 3
Figure 3
Expression of EGFR, pAKT and hypoxia in vivo. Expression of EGFR and pAKT in relation to hypoxia was analyzed by immunohistochemical analysis in UT-SCC xenograft tumors of 4 different lines. Left column: EGFR (red), pAKT (green), vessels (blue). Right column: EGFR (red), hypoxia (green), vessels (blue). Non-specific staining present in necrotic regions. Scale bars represent 500 μm. Magnification: 100X.
Figure 4
Figure 4
Correlation between pAKT and hypoxia in vivo . Correlation between the pAKT and hypoxic fraction in tumors of 6 HNSCC lines was assessed with the Spearman correlation test. Symbols represent individual tumors of the different UT-SCC lines.
Figure 5
Figure 5
Effect of hypoxia and MK-2206 on pAKT levels and cell survival in vitro. A) Expression of (p)AKT after treatment with MK-2206 under normoxic and hypoxic (1h, 0.5% O2) conditions in UT-SCC5 and UT-SCC15. B) Cell survival after treatment with MK-2206 under normoxic and hypoxic (72h, 0.5% O2) conditions in UT-SCC5 and UT-SCC15. **: p<0.01 compared to control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Davies L, Welch HG. Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg. 2006;135(3):451–457. doi: 10.1016/j.otohns.2006.01.029. - DOI - PubMed
    1. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, Sur R, Raben D, Jassem J. et al.Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354(6):567–578. doi: 10.1056/NEJMoa053422. - DOI - PubMed
    1. Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nat Rev Clin Oncol. 2010;7(9):493–507. doi: 10.1038/nrclinonc.2010.97. - DOI - PMC - PubMed
    1. Erjala K, Sundvall M, Junttila TT, Zhang N, Savisalo M, Mali P, Kulmala J, Pulkkinen J, Grenman R, Elenius K. Signaling via ErbB2 and ErbB3 associates with resistance and epidermal growth factor receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells. Clin Cancer Res. 2006;12(13):4103–4111. doi: 10.1158/1078-0432.CCR-05-2404. - DOI - PubMed
    1. Yamatodani T, Ekblad L, Kjellen E, Johnsson A, Mineta H, Wennerberg J. Epidermal growth factor receptor status and persistent activation of Akt and p44/42 MAPK pathways correlate with the effect of cetuximab in head and neck and colon cancer cell lines. J Cancer Res Clin Oncol. 2009;135(3):395–402. doi: 10.1007/s00432-008-0475-2. - DOI - PubMed

Publication types

MeSH terms