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Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor

Kazuko Sakai^*,, Tokuzo Arao^*, Tatsu Shimoyama^*, Kimiko Murofushi, Masaru Sekijima^||, Naoko Kaji^||, Tomohide Tamura, Nagahiro Saijo and Kazuto Nishio^*,,1

^* Shien-Lab, Medical Oncology,
National Cancer Center Hospital and
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan; and
Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, Japan; and
^|| Mitsubishi Chemical Safety Institute Ltd., Ibaraki, Japan

¹ Correspondence: Shien-Lab, Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan. E-mail: knishio{at}gan2.res.ncc.go.jp

SPECIFIC AIM

A short, in-frame deletional mutant (E746-A750del) a major mutant form of EGFR in non-small cell lung cancer, and has been reported to be a major determinant of response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. However, the biological and pharmacological functions of mutational EGFR remain unclear. The aim of this study is to clarify whether it is constitutively active or not and whether alteration occurs downstream of the intracellular signaling.

PRINCIPAL FINDINGS

1. A short, in-frame deletional mutant (E746-A750del) induced dimerization and phosphorylation of EGFR without any ligand stimulation
To determine the biological functions of deletion mutant (E746-A750del) EGFR, we used the stable transfected cells of wild-type and deletion mutant of EGFR. Previously, we demonstrated that the 293(D) cells transfected with the deletional EGFR were hypersensitive to EGFR-targeted tyrosine kinase inhibitors such as gefitinib and ZD6474 as compared with the 293(W) cells transfected with wild-type EGFR. Dimerization and phosphorylation of EGFR in these cells were determined by using chemical cross-linker and by immunoblot analysis (Fig. 1 ). No expression of EGFR dimer or monomer was detected in the 293(M) cells. Increased dimerization and phosphorylation of the deletional EGFR with a molecular weight of 400 kDa were detected without EGF stimulation in the 293(D) cells. When stimulated with the EGF, increased dimerized and phosphorylated EGFR were observed in the 293(W) cells, whereas no response of EGFR to EGF was observed in the 293(D) cells. The ratio of dimerized to monomeric EGFR in 293(W) and 293(D) cells was analyzed densitometrically (Fig. 1 , right). The dimer/monomer ratio in the 293(W) cells was markedly increased (3-fold) by addition of EGF. Under unstimulated conditions, the dimer/monomer ratio of the 293(D) cells was higher than that of the 293(W) cells and the ratio was unchanged by addition of EGF. These results suggest that the cells expressing the wild-type of EGFR responded to EGF for their dimerization and phosphorylation and that the deletional mutant of EGFR was dimerized and phosphorylated constitutively without any ligand stimulation.

View larger version (28K): [in this window] [in a new window]   Figure 1. Dimerization and phosphorylation of wild-type EGFR and deletional EGFR A. The 293 cells transfected with the empty vector (293(M)), wild-type EGFR (293(W)), and deletional EGFR (293(D)) were treated with or without EGF (10 ng/mL) for 10 min after serum starvation. After two washes with ice-cold PBS(+), monolayer cells were incubated with the chemical cross-linking reagent BS3 (1.5 mM) in PBS(+). Glycine (20 mM) was added for an additional 5 min to terminate the reaction. The lysates (twenty µg protein) were subjected to 2–15% SDS-PAGE followed by immunoblot analysis using anti-EGFR and anti-phospho-EGFR. Right panel: ratio of dimerized to monomeric EGFR.

2. p44/42 MAPK and AKT pathways are activated in the cells expressing deletional EGFR without ligand stimulation
We examined the phosphorylation status of p44/42 MAPK and AKT that are major downstream targets of EGFR in the transfectants. Even under unstimulated conditions, increased phosphorylation of p44/42 MAPK and AKT was observed in the 293(D) cells. In the 293(W) cells, increased phosphorylation of p44/42 MAPK and AKT was observed with the addition of EGF but p44/42 MAPK was remarkably phosphorylated. On the other hand, no increased phosphorylation of p44/42 MAPK and AKT was observed with the addition of EGF in the 293(D) cells. This result suggests that the p44/42 MAPK and AKT pathways are activated in cells expressing the deletional EGFR without ligand stimulation.

3. Gefitinib inhibited the AKT signaling pathway more strongly than the p44/42 MAPK signaling pathway
We next determined the action of EGFR-targeted tyrosine kinase inhibitor gefitinib on downstream of deletional EGFR (Fig. 2 A). In the 293(W) cells, phosphorylation of p44/42 MAPK was not inhibited by exposure to a low dose of gefitinib (0.01 µM) but phosphorylation of AKT was inhibited by exposure to gefitinib (70%, Fig. 2C ). In contrast, exposure to gefitinib decreased phospho-EGFR in the 293(D) cells. Phosphorylation of AKT was completely inhibited by 0.01 µM gefitinib exposure (99%, Fig. 2C ), whereas inhibition of p44/42 MAPK phosphorylation was not remarkable in the 293(D) cells (20%, Fig. 2B ). These data suggest that gefitinib inhibited the AKT signaling pathway more strongly than the p44/42 MAPK signaling pathway in the cells expressing the deletion mutant EGFR.

View larger version (48K): [in this window] [in a new window]   Figure 2. Effect of gefitinib on phosphorylation of EGFR, p44/42 MAPK, and AKT in the EGFR transfected 293 cells. A) The 293(M), 293(W), and 293(D) cells were incubated with gefitinib (0.01 µM) for 3 h or 12 h under nonstarved conditions. After two washes with ice-cold PBS(+), monolayer cells were lysed. Equivalent amounts of protein were separated by 2–15% gradient SDS-PAGE for EGFR or 10–20% for p44/42 MAPK, phospho-p44/42 MAPK, AKT, and phospho-AKT, then subjected to immunoblot analysis. B) Histogram of the degree of p44/42 MAPK activation expressed as phospho-p44/42 MAPK per total p44/42 MAPK. C) Histogram of the degree of AKT activation expressed as phospho-AKT per total AKT.

4. AKT pathway was activated in the PC-9 cells expressing deletional EGFR intrinsically
To examine whether increased phosphorylation is also observed in the lung cancer cells intrinsically expressing deletional EGFR, we monitored the phosphorylation of EGFR and its related molecules in the PC-9 cells expressing deletional EGFR by using a beads-based mulitiplex assay. We found increased phosphorylation of EGFR and downstream molecules of AKT pathway including IB- in PC-9 cells. This finding is consistent with the result of the previous experiments with the 293(D) cells. It is suggested that AKT pathway is activated in the cells expressing deletional EGFR intrinsically.

CONCLUSIONS AND SIGNIFICANCE

To clarify the function of deletional EGFR, we used the cell transfectants with deletional EGFR [293(D)] that is hypersensitive to tyrosine kinase inhibitors (e.g., gefitinib). We detected significantly higher levels of dimerization and phosphorylation of deletional EGFR without any ligand stimulation in the cells deletional EGFR. Increased phosphorylation of p44/42 MAPK and AKT was observed in the 293(D) cells. These results suggest that deletional EGFR is constitutively active. When the 293(D) cells were exposed to gefitinib (0.01 µM), AKT phosphorylation was completely suppressed, suggesting that deletional EGFR signaling inclines toward the AKT pathways. A summary of characteristics of deletional EGFR is shown in Fig. 3 .

View larger version (27K): [in this window] [in a new window]   Figure 3. Function of deletional EGFR. Wild-type EGFR is dimerized and phosphorylated by EGF the wild-type EGFR and MAPK and AKT pathways are activated. The deletion mutant EGFR is dimerized and phosphorylated without EGF. Both MAPK and AKT pathways are activated; but phospho-AKT was inhibited by TKI predominantly in the cells expressing deletional EGFR. MEK, MAP kinase/extracellular regulated kinases; PI3K, phosphoinositide-3-kinase; TKI, tyrosine kinase inhibitors.

An additional experiment using a PC-9 lung cancer cell line intrinsically expressing deletional EGFR confirmed the gain of function of deletional EGFR and activated AKT signaling pathway.

Results from this study have provided the understanding for biological functions of deletional EGFR and cellular hypersensitivity to the EGFR-targeted tyrosine kinase inhibitor.

Now over 30 types of mutation have been reported in clinical lung cancer specimens. We will examine the biological function of other types of EGFR mutants differentially, with the aim of selecting clinically meaningful mutations.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4034fje;

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