Abstract
The most frequently found alteration of the epidermal growth factor receptor (EGFR) in human tumors is a deletion of exons 2-7. This receptor, termed EGFRvIII, can transform NIH 3T3 cells, and the frequent expression of this variant implies that it confers a selective advantage upon tumor cells in vivo. Although EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras.GTP levels and low levels of mitogen-activated protein kinase activity in NIH 3T3 cells expressing this variant. We investigated whether phosphatidylinositol (PI) 3-kinase was an effector in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to basal levels after 12 h of continuous EGFR activation, there was a 3-fold increase in PI 3-kinase activity in cells expressing normal EGFR and an 8-fold increase in cells expressing EGFRvIII after 48 h. This increased activity may reflect enhanced binding to EGFRvIII and the presence of novel PI 3-kinase isoforms. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and also resulted in morphological reversion of EGFRvIII-transformed cells. These results support an essential role for PI 3-kinase in transformation by this EGFR variant.
Highlights
Overexpression of the EGFR1 has been implicated in the pathogenesis of many human tumors, including those derived from the brain, breast, lung, ovary, prostate, and skin [1, 2]
The smaller effect of these inhibitors on HC2 20d2/c cells may reflect the higher endogenous level of PI 3-kinase activation in these cells. These results indicate that PI 3-kinase activity is essential for anchorage-independent growth in EGFRvIIItransformed cells. Work in this and other laboratories has demonstrated that expression of the EGFRvIII results in neoplastic transformation and enhanced tumorigenicity, which is due to its constitutive kinase activity [7, 12, 15]
While activation of the normal EGF receptor results in activation of MAP kinase via Ras [15, 18], our studies on EGFRvIII showed only a low level of activation of the Ras-MAP kinase pathway, which was due to decreases in Shc and Grb2 levels and induction of a MAP kinase phosphatase [15, 20]
Summary
Overexpression of the EGFR1 has been implicated in the pathogenesis of many human tumors, including those derived from the brain, breast, lung, ovary, prostate, and skin [1, 2]. EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras1⁄7GTP levels and low levels of mitogenactivated protein kinase activity in NIH 3T3 cells expressing this variant. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and resulted in morphological reversion of EGFRvIII-transformed cells.
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