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Comparative Study
. 2006 Dec 6:6:277.
doi: 10.1186/1471-2407-6-277.

Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation

Rintaro Noro  1 , Akihiko Gemma, Seiji Kosaihira, Yutaka Kokubo, Mingwei Chen, Masahiro Seike, Kiyoko Kataoka, Kuniko Matsuda, Tetsuya Okano, Yuji Minegishi, Akinobu Yoshimura, Shoji Kudoh
Affiliations
Comparative Study

Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation

Rintaro Noro et al. BMC Cancer. .

Abstract

Background: Phase III trials evaluating the efficacy of gefitinib (IRESSA) in non-small cell lung cancer (NSCLC) lend support to the need for improved patient selection in terms of gefitinib use. Mutation of the epidermal growth factor receptor (EGFR) gene is reported to be associated with clinical responsiveness to gefitinib. However, gefitinib-sensitive and prolonged stable-disease-defined tumors without EGFR gene mutation have also been reported.

Methods: To identify other key factors involved in gefitinib sensitivity, we analyzed the protein expression of molecules within the EGFR family, PI3K-Akt and Ras/MEK/Erk pathways and examined the sensitivity to gefitinib using the MTT cell proliferation assay in 23 lung cancer cell lines.

Results: We identified one highly sensitive cell line (PC9), eight cell lines displaying intermediate-sensitivity, and 14 resistant cell lines. Only PC9 and PC14 (intermediate-sensitivity) displayed an EGFR gene mutation including amplification. Eight out of the nine cell lines showing sensitivity had Akt phosphorylation without ligand stimulation, while only three out of the 14 resistant lines displayed this characteristic (P = 0.0059). Furthermore, the ratio of phosphor-Akt/total Akt in sensitive cells was higher than that observed in resistant cells (P = 0.0016). Akt phosphorylation was partially inhibited by gefitinib in all sensitive cell lines.

Conclusion: These results suggest that Akt phosphorylation without ligand stimulation may play a key signaling role in gefitinib sensitivity, especially intermediate-sensitivity. In addition, expression analyses of the EGFR family, EGFR gene mutation, and FISH (fluorescence in situ hybridization) analyses showed that the phosphorylated state of EGFR and Akt might be a useful clinical marker of Akt activation without ligand stimulation, in addition to EGFR gene mutation and amplification, particularly in adenocarcinomas.

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Figures

Figure 1
Figure 1
IC50 values for 23 lung cancer cell lines using the MTT assay. Cell lines were classified as: sensitive (IC50≤1 μM), intermediate-sensitive (1 μM < IC50≤10 μM), and resistant (10 μM <IC50) to gefitinib. The bars represent the IC50 and standard deviation (SD) obtained in three indpendent MTT assays. Point: mean of three independent experiments, bar: ± SD.
Figure 2
Figure 2
Western blot analyses: protein expression and phosphorylation of epidermal growth factor receptor (EGFR), Akt, PTEN, Her2, Her3, p44/42 mitogen activated protein kinase (MAPK), and p38 MAPK in adenocarcinoma cell lines cultured for 24 h with (+) or without (-) serum.
Figure 3
Figure 3
Summary of expression of PTEN and phosphorylation of Akt in 23 lung cancer cell lines grown under serum-containing (cross bars) and serum-free (black bars) conditions. (A) The ratio of phosphorylated Akt signal/total Akt signal (pAkt/Akt); (B) the ratio of PTEN signal/β-actin signal; The density of expression of each of the molecules was measured by NIH Image. Point: mean of three independent experiment; bars, ± standard deviation (SD). The expression ratio of the molecules in serum-free conditions was compared between gefitinib-resistant and gefitinib-sensitive cell lines using the Mann-Whitney test; point, mean, bars, ± standard error (SE). *P < 0.05 was considered to be statistically significant.
Figure 4
Figure 4
Summary of expression of Her3 and phosphorylation of p44/42 MAPK in 23 lung cancer cell lines grown under serum-containing (cross bars) and serum-free (black bars) conditions. (A) the ratio of phosphorylated p44/42 MAPK signal/total p44/42MAPK signal (MAPKp-p44/42/MAPKp44/42); (B) the ratio of Her3 signal/β-actin signal; The density of expression of each of the molecules was measured by NIH Image. Point: mean of three independent experiment; bars, ± standard deviation (SD). The expression ratio of the molecules in serum-free conditions was compared between gefitinib-resistant and gefitinib-sensitive cell lines using the Mann-Whitney test; point, mean, bars, ± standard error (SE). *P < 0.05 was considered to be statistically significant.
Figure 5
Figure 5
Summary of expression of Her2 and phosphorylation of EGFR in 23 lung cancer cell lines grown under serum-containing (cross bars) and serum-free (black bars) conditions. (A) the ratio of phosphorylated EGFR signal/EGFR signal; (B) the ratio of Her2 signal/β-actin signal. The density of expression of each of the molecules was measured by NIH Image. Point: mean of three independent experiment; bars, ± standard deviation (SD). The expression ratio of the molecules in serum-free conditions was compared between gefitinib-resistant and gefitinib-sensitive cell lines using the Mann-Whitney test; point, mean, bars, ± standard error (SE). *P < 0.05 was considered to be statistically significant.
Figure 6
Figure 6
Fluorescence in situ hybridization (FISH) analyses of lung cancer cell lines, and EGFR mutations and expression. EGFR gene copy numbers were counted and were classified into six FISH strata [9,19]. EGFR gene amplification was detected in PC9 and PC14 cells. In the ABC1 cell line, the EGFR gene copy number was five in over 40% of cells (high polysomy:FISH negative); in the PC7 and LCKJ cell lines, the EGFR gene copy number was three in over 40% of cells (high trisomy:FISH negative).
Figure 7
Figure 7
Lung cancer cell lines: Effect of gefitinib on phosphorylated EGFR(p-EGFR), phosphorylated Akt (p-Akt) and the molecules in the Ras/MEK/Erk pathway with or without EGF.
Figure 8
Figure 8
Summary of the effect of gefitinib on the molecules downstream of EGFR (Fig.7.). These images were quantified by measuring signal intensity using NIH Image (ImageJ1.32j).
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