Sakai Kazuko, Arao Tokuzo, Shimoyama Tatsu, Murofushi Kimiko, Sekijima Masaru, Kaji Naoko, Tamura Tomohide, Saijo Nagahiro, Nishio Kazuto
Shien-Lab, Medical Oncology, National Cancer Center Research Institute, Tokyo, Japan.
FASEB J. 2006 Feb;20(2):311-3. doi: 10.1096/fj.05-4034fje. Epub 2005 Dec 22.
A short, in-frame deletional mutant (E746-A750del) is one of the major mutant forms of epidermal growth factor receptor (EGFR) and has been reported to be a determinant of response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. However, the biological and pharmacological functions of mutational EGFR remain unclear. To clarify these biological functions of deletional EGFR, we examined the cellular response to EGF ligand stimulation. Dimerization and phosphorylation of EGFR were observed without any ligand stimulation in the 293(D) cells transfected with deletional EGFR as compared with those transfected with wild-type EGFR (293(W) cells). When the 293(D) cells were exposed to gefitinib, an immunoblotting analysis revealed remarkable inhibition of AKT phosphorylation but not phospho-p44/42 MAPK. To examine the cellular response in a lung cancer cell line intrinsically expressing deletional EGFR, phospho-EGFR, and downstream reactions were monitored under EGF stimulation with a beads-based mulitiplex assay. EGFR and its downstream proteins were constitutively phosphorylated in the PC-9 cells without any ligand stimulation as compared with A549 lung cancer cells expressing wild-type EGFR. In conclusion, deletional EGFR is constitutively active and phosphorylates p44/42 MAPK and AKT in the cells, although the fact that the EGFR phosphorylation in the PC-9 cells is still modulated by EGF stimulation cannot be ignored. Gefitinib-inhibited phospho-AKT predominantly in deletional EGFR expressing cells.
一种短的、框内缺失突变体（E746 - A750del）是表皮生长因子受体（EGFR）的主要突变形式之一，据报道它是对吉非替尼和厄洛替尼等EGFR酪氨酸激酶抑制剂反应的决定因素。然而，突变型EGFR的生物学和药理学功能仍不清楚。为了阐明缺失型EGFR的这些生物学功能，我们检测了细胞对EGF配体刺激的反应。与转染野生型EGFR的细胞（293（W）细胞）相比，在转染缺失型EGFR的293（D）细胞中，未施加任何配体刺激时就观察到了EGFR的二聚化和磷酸化。当293（D）细胞暴露于吉非替尼时，免疫印迹分析显示AKT磷酸化受到显著抑制，但磷酸化的p44/42 MAPK未受抑制。为了检测在内在表达缺失型EGFR的肺癌细胞系中的细胞反应，使用基于磁珠的多重分析监测了EGF刺激下的磷酸化EGFR及其下游反应。与表达野生型EGFR的A549肺癌细胞相比，PC - 9细胞在未施加任何配体刺激时，EGFR及其下游蛋白就组成性磷酸化。总之，缺失型EGFR在细胞中组成性激活并使p44/42 MAPK和AKT磷酸化，尽管不能忽视PC - 9细胞中EGFR磷酸化仍受EGF刺激调节这一事实。吉非替尼主要在表达缺失型EGFR的细胞中抑制磷酸化AKT。
