The development and neoplastic progression of human astrocytic tumors appears to result through an accumulation of genetic alterations occurring in a relatively defined order. One such alteration is amplification of the epidermal growth factor receptor (EGFR) gene. This episomal amplification occurs in 40-50% of glioblastomas, which also normally express endogenous receptors. Moreover, a significant fraction of amplified genes are rearranged to speifically eliminate a DNA fragment containing exons 2-7 of the gene, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain. Here we used retroviral transfer of such a mutant receptor (de 2-7 EGFR) into glioblastoma cells expressing normal endogenous receptors to test whether the mutant receptor was able to augment their growth and malignancy. Western blotting analysis showed that these cells expressed endogenous EGFR of 170 kDa as well as the exogenous de 2-7 EGFR of 140-155 kDa. Although holoEGFRs were phosphorylated on tyrosine residues only after exposure of the cells to liand, de 2-7 EGFRs were constitutively phosphorylated. In tissue culture neither addition ofEGF nor expression of the mutant EGFR affected the rate of cell growth. However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously or intracerebrally, tumorigenic capacity was greatly enhanced. These results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions oftumor cells with their microenvironment in ways not easily assayed in vitro.

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named ⌬EGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active ⌬EGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of ⌬EGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation. Ligand binding to wild-type epidermal growth factor receptor (wt EGFR)1 results in receptor dimerization, kinase activation, and autophosphorylation that provides both docking sites for proteins involved in signal transduction and exposure of endocytic and lysosomal targeting sequence codes required for receptor internalization and down-regulation (1). The biochemical and biological roles of each autophosphorylation site in wt EGFR have been explored by mutational analysis, and mutation of any single autophosphorylation site does not significantly abrogate binding of the activated receptor to specific SH2-containing proteins associated with distinct signaling pathways (2). Likewise, such single mutations are generally incapable of reducing the biological functions of the receptor in in vitro models (3, 4). Correspondingly, the mitogenic and transforming activities of wild-type receptor were diminished only when combinations of favorable autophosphorylation sites (i.e. Tyr-1068, Tyr-1148, and Tyr-1173) were mutated (3), suggesting that the autophosphorylation sites of wt EGFR may have less specificity for signaling proteins and can compensate for each other. Sites of tyrosine phosphorylation may be provided via heterodimerization with other members of the erb B family of receptors (5-7). Point mutations that inactivated the tyrosine kinase activity of wt EGFR eliminated occupancyinduced receptor internalization (8,9,40,41), whereas mutant receptors lacking multiple autophosphorylation sites also lacked the ability to undergo ligand-induced endocytosis, suggesting that kinase-regulated re...

Alterations of the epidermal growth factor receptor (EGFR) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2-7, resulting in truncation of the extracellular domain (⌬EGFR or EGFRvIII), which occurs in a large fraction of de novo malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-X L . Here, we demonstrate that the ⌬EGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-X L was elevated in U87MG.⌬EGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.⌬EGFR cells. These responses were highly specific to constitutively kinase-active ⌬EGFR, because overexpression of kinase-deficient ⌬EGFR (DK) or wild-type EGFR had no such effects. Correspondingly, ⌬EGFR specific tyrosine kinase inhibitors reduced Bcl-X L expression and potentiated CDDP-induced apoptosis in U87MG.⌬EGFR cells. Ectopic overexpression of Bcl-X L in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing ⌬EGFR.

In some respects, the EGFR appears to be an attractive target for tumor-targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti-tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2-7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2-7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806 The delta 2-7 epidermal growth factor receptor (de2-7 EGFR) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. [1][2][3][4] In adults, the expression of the de2-7 EGFR appears restricted to tumors exhibiting amplification of the EGFR gene. 5 Prostate cancer maybe an exception to this observation, as the expression level of de2-7 and wild type EGFR appear to be inversely related in this tumor type. 2 The rearrangement seen in the de2-7 EGFR results in an in-frame mature mRNA lacking 801 nucleotides spanning exons 2-7. 6 -9 The corresponding EGFR protein contains a 267 amino acid deletion and a novel glycine residue at the fusion junction. 9 Although this truncated receptor does not bind ligand, it does possess low constitutive activity and imparts a significant growth advantage to glioma cells grown as tumor xenografts in nude mice 10 and is able to transform NIH3T3 cells. 11 The cellular mechanisms utilized by the de2-7 EGFR in glioma cells are not fully defined but include a decrease in apoptosis 12 and a small enhancement of proliferation. 12 The aberrant splicing event that generates the de2-7 EGFR leads to the formation of an unique junctional peptide in the N-terminal of the extracellular domain. 13 As this peptide sequence is not found in the wild type EGFR, its expression is restricted to tumor cells making it an attractive target for antibody therapy. Indeed, the production of both polyclonal 14 and monoclonal 3,15,16 antibodies specific to this unique peptide have been described. One potential drawback in targeting of the de2-7 EGFR is that it is only expressed in a portion of tumors containing EGFR gene amplification. Expression of the de2-7 EGFR has been analyzed in glioma by a number of groups with most reporting that approximately 30% of gliomas express the mutant receptor 13 with expression being lowest in anaplastic astrocytomas and highest in glioblastoma multiforme. 17 The proportion of positive cells within de2-7 EGFR expressing gliomas has been reported to range from 37-86%. 1 A very recent report found that all high-grade prostate carcinomas expressed the de2-7 EGFR 2 although this observation has not been confirmed. Expression in ...

A mutant epidermal growth factor receptor (⌬EGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.⌬EGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ⌬EGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTPbound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.⌬EGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ⌬EGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

CorrectionsArai, and Glenn D. Prestwich, which appeared in number 1, January 7, 2003, of Proc. Natl. Acad. Sci. USA (100, 131-136; First Published December 26, 2002; 10.1073͞pnas.0135855100), Fig. 4 should have appeared in color. The correct figure and its legend appear below. Fig. 4.LPA stimulates lipid accumulation, CD36 expression, and oxidized LDL uptake through a PPAR-responsive element. (a) LPA stimulates monocyte uptake of oxidized LDL. Freshly elutriated human monocytes were allowed to interact with an anti-ICAM3-coated well, which leads to rapid PPAR␥ expression (13), and then stimulated, or not (negative, oxLDL), with oleoyl LPA. Some cells were then briefly exposed to oxidized LDL before intracellular lipid stores were visualized with oil red O stain. (b) LPA increases the expression of CD36 on the surface of primary human monocytes. Monocytes engaging anti-ICAM3 were treated or not with LPA, and then recovered by gentle scraping and washing by centrifugation before their surface CD36 was assessed by flow cytometry. (c) LPA and the LPA analogs XY4 and XY8 stimulate CD36 promoter function only when the PPRE is present. RAW264.7 cells were transfected with the human CD36 promoter containing the PPRE (CD36 Ϫ273 ) or a reporter that lacks only this element (CD36 Ϫ261 ) and then stimulated with oleoyl LPA, azPC, XY4, or XY8. Expression of luciferase normalized to ␤-galactosidase was determined as above. (d) Anti-CD36 blocks LPA-stimulated accumulation of cholesterol from oxidized LDL. Freshly isolated human monocytes were treated as in a, but after being preincubated with a blocking anti-CD36 antibody before exposure to oxidized LDL. G rowth factor receptors are promising targets for antibodybased cancer therapies. Because of their cell-surface location, they are readily accessible, and therapeutic antibodies can exert their inhibitory effects by either interfering with cellular signaling or targeting toxic molecules or biological effectors to the tumor site (1).The epidermal growth factor receptor (EGFR) is expressed in normal tissues and neoplastic lesions of most organs, and its expression level has been associated with biological characteristics of tumors (2). Elevated levels of EGFR have been demonstrated in many different types of cancer including glioblastoma, and EGFR overexpression seems to be associated with poor prognosis in several neoplasms (3). EGFR overexpression is often associated with gene amplification (4-8). In glioblastoma, EGFR amplification has been shown to be accompanied by gene rearrangement (9-11), frequently with deletions in the coding region. Several mutant forms have been found (12, 13), and among these the most common mutation is the ⌬2-7 deletion (⌬EGFR), which lacks exons 2-7 of the external EGFR domain, resulting in the loss of an 801-bp fragment of the wild-type (wt) gene (14). Several studies have indicated that the presence of ⌬EGFR enhances the tumorigenic behavior of cancer cells (15-17). ⌬EGFR has only been found in neoplastic lesions and not in any normal tissue. ...

EGFR amplification is a frequent genetic alteration in primary (de novo) glioblastomas, and is often associated with structural alterations. Most common is variant III (EGFRvIII), which results from a non-random 801 bp in-frame deletion of exons 2 to 7 of the EGFR gene. We assessed amplification and overexpression of EGFRvIII and wild-type EGFR in 30 glioblastoma biopsies. Immunohistochemically, EGFR overexpression was observed in 20 (67%) of 30 glioblastomas. Eight (27%) cases also showed immunoreactivity to an EGFRvIII antibody. In 6 of these cases, the pattern of EGFR and EGFRvIII overexpression was compared in serial sections: In 4 cases, areas with immunoreactivity to EGFRvIII largely coincided with wild-type EGFR expression. In the other 2 cases, the areas immunoreactive to EGFRvIII were significantly less extensive than EGFR-positive areas. To assess whether EGFRvIII is predominantly amplified in tumors with concurrent wild-type EGFR amplification, we carried our real-time quantitative PCR using 2 sets of primers located in exon 2 and intron 15 of the EGFR gene. A > 5-fold ratio of relative copy numbers between intron 15 (present both in wild-type EGFR and EGFRvIII) and exon 2 (present only in wild-type EGFR, but missing in EGFRvIII) suggested predominant amplification of EGFRvIII in only 3 (10%) of 30 glioblastomas. The observation that intratumoral wild-type EGFR overexpression is often more extensive and that predominant amplification of EGFRvIII is a rare event would limit the effectiveness of therapeutic approaches based on selective targeting of EGFRvIII.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as ⌬EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize ⌬EGFR as a cellular substrate. TC45 dephosphorylated ⌬EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of ⌬EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express ⌬EGFR. ⌬EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of ⌬EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of ⌬EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the ⌬EGFR-mediated activation of ERK2 and suppress the tumorigenicity of ⌬EGFR-expressing glioblastoma cells in vivo. Glioblastoma multiforme (GBM)1 is the most malignant form of brain cancer with 50% of patients dying within the first year of diagnosis. Common in many de novo GBMs is the overexpression of wild type or mutant epidermal growth factor (EGF) receptor. The EGF receptor is a protein tyrosine kinase (PTK) that regulates fundamental cellular processes such as proliferation, migration, differentiation, and survival. A common rearrangement of the EGF receptor gene results in the expression of a truncated protein known as ⌬EGFR (also known as EGFRvIII and de2-7EGFR) that has an in-frame deletion of 267 amino acids from the extracellular domain (reviewed in Refs.