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. 2013 Jun;7(3):359-68.
doi: 10.1016/j.molonc.2012.11.001. Epub 2012 Nov 14.

Molecular phenotype predicts sensitivity of squamous cell carcinoma of the head and neck to epidermal growth factor receptor inhibition

Natalie R Young  1 , Jing Liu, Carolyn Pierce, Tai-Fen Wei, Tatyana Grushko, Olufunmilayo I Olopade, Wanqing Liu, Christine Shen, Tanguy Y Seiwert, Ezra E W Cohen
Affiliations

Molecular phenotype predicts sensitivity of squamous cell carcinoma of the head and neck to epidermal growth factor receptor inhibition

Natalie R Young et al. Mol Oncol. 2013 Jun.

Abstract

Despite nearly universal expression of the wild-type epidermal growth factor receptor (EGFR) and reproducible activity of EGFR inhibitors in patients with squamous cell carcinoma of the head and neck (SCCHN), the majority of patients will not have objective responses. The mechanisms of this intrinsic resistance are not well established. We hypothesized that sensitivity to EGFR inhibitors can be predicted based on the inhibitors' effects on downstream signaling. Cell viability assays were used to assess sensitivity to the EGFR inhibitor gefitinib (ZD1839) in 8 SCCHN cell lines. Fluorescence in-situ hybridization showed the two most sensitive lines to be highly gene-amplified for EGFR. Western blotting confirmed that phosphoEGFR was inhibited at low concentrations of gefitinib in all lines tested. Phosphorylation of downstream signaling protein AKT was inhibited in sensitive lines while inhibition of phosphoERK displayed no relationship to gefitinib efficacy. Phosphatase and tensin homolog (PTEN) expression was evident in all cell lines. Activating PIK3CA mutations were found in two resistant cell lines where pAKT was not inhibited by gefitinib. In resistant cell lines harboring PIK3CA mutations, a PI3K inhibitor, LY294002, or AKT siRNA reduced cell viability with an additive effect demonstrated in combination with gefitinib. Additionally, LY294002 alone and in combination with gefitinib, was effective at treating PIK3CA mutated tumors xenografted into nude mice. Taken together this suggests that constitutively active AKT is a mechanism of intrinsic gefitinib resistance in SCCHN. This resistance can be overcome through targeting of the PI3K/AKT pathway in combination with EGFR inhibition.

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Figures

Figure 1
Figure 1
Phosphorylation of EGFR, AKT and ERK, and PTEN expression in SCCHN cell lines. A, EGFR phosphorylation at tyrosine 1068 by Western blotting was reduced in all cell lines upon treatment with increasing concentrations of gefitinib. Tubulin and EGFR serve as loading controls. B, Phosphorylation of AKT and C, phosphorylation of ERK proteins with increasing concentrations of gefitinib are shown by Western blotting. Tubulin serves as loading controls. D, Expression of PTEN in all cell lines is shown by Western blotting. Tubulin serves as loading control.
Figure 2
Figure 2
EGFR gene amplification. Images of interphase nuclei after FISH are presented. The EGFR gene is localized by red fluorescent signals, and chromosome 17 centromere (CEP7) is localized by green fluorescent signals. The cells were counterstained with DAPI (blue). Magnification ×1200.
Figure 3
Figure 3
Sequencing chromatograms of PIK3CA mutations in SCC61 and Detroit 562 cell lines.
Figure 4
Figure 4
Inhibition of EGFR and PI3K/AKT pathway has additive effects in SCCHN cell lines with constitutively active PIK3CA mutations, while removing negative regulation of PI3K/AKT pathway increases resistance in sensitive SCCHN cell line. A, results from MTT assays on cells treated with PI3K inhibitor, LY294002, and gefitinib. By repeated measures one‐way ANOVA, means are significantly different; p < 0.0001. By Tukey's multiple comparison test; *p < 0.05. B, results from MTT assays on cells treated with Akt1 and Akt2 siRNA and gefitinib. By repeated measures one‐way ANOVA, means are significantly different; p < 0.0001. By Tukey's multiple comparison test differences between siAkt 1 + 2 with and without gefitinib and as indicated; *p < 0.05. C, Western blot results showing Akt protein levels decrease upon siRNA treatment. Actin serves as loading control. D, results from MTT assays on cells treated with PTEN siRNA and gefitinib. By repeated measures one‐way ANOVA, means are significantly different; p < 0.0001. *p < 0.05. E, Western blot results showing PTEN protein levels decrease upon siRNA treatment. α‐tubulin serves as loading control.
Figure 5
Figure 5
SCC61 or Detroit 562 xenografted tumors respond to treatment by PI3K inhibitor, LY294002 alone, or in combination with gefitinib. % tumor growth is shown. By two‐way ANOVA, the effect of treatment and time is considered significant; SCC61 treatment p < 0.001, time p = 0.0006; Detroit 562 treatment p = 0.0043; time p < 0.0001. By Bonferroni multiple comparison test, the indicated time points are significantly different from control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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