Epidermal Growth Factor Receptor, EGFRvIII, and Human Cancer
Epidermal Growth Factor Receptor, EGFRvIII, and Human Cancer
By Careen K. Tang, PhD
In the last decade, numerous studies have indicated that growth factors and their receptors play an important role in cancer biology. In particular, the class I subfamily of growth factor receptors (EGFR, HER2/p185erbB-2/neu, HER3/p160erbB-3, and HER4/p180 erbB-4) has received increased attention.1-4 This epidermal growth factor receptor (EGFR) family is one group of tyrosine kinases, frequently overexpressed in a variety of carcinomas. The EGFR family may offer several potential targets for a variety of therapeutic interventions. This mini review will describe the current development of the mutant EGFR, EGFRvIII, and its involvement in human cancer.
Clinical Significance of EGFRvIII in Human Cancer
The EGFR provided one of the first pieces of evidence that an activated oncogene is associated with human tumor biology.1,5 Enhanced expression of EGFR frequently is detected in a variety of carcinomas, including breast, lung, head, and neck, as well as glioblastoma.1,5,6 EGFR overexpression in human malignancy has been extensively studied; evidence is accumulating that EGFR gene alterations may be as important as amplification toward oncogenic effects.7,8 Spontaneous rearrangements within the EGFR gene were first identified in primary human glioblastoma tumors, and in nearly all cases the alterations have been reported in tumors with EGFR amplification.9-13 Three different types of mutants result from these rearrangements. The most common is the Type III epidermal growth factor deletion-mutant receptor (EGFRvIII), which is characterized by the deletion of exons 2-7 in the EGFR mRNA. These deletions correspond to cDNA nucleotides 275-1075, which encode amino acids 6-276, presumably through alternative splicing or rearrangements.10-13 Deletion of 801 bp within in the extracellular domain of the EGFR gene causes an in-frame truncation of the normal EGFR protein, resulting in a 145-kDa receptor.10-13 Recent reports have demonstrated that 52-67% of primary human glioblastoma tumors detected EGFRvIII expression.13,14 EGFRvIII also is frequently detected in various human cancers, including breast, prostate, ovarian, lung, and medulloblastoma tumors.11,14-17 The expression of EGFRvIII increased with de-differentiation of prostatic epithelial cells with a concomitant decrease in wild-type EGFR expression.16 No tested normal adult human tissues, including those from the peripheral and central nervous systems, the lymphoid system, skin, breast, liver, lung, ovary, placenta, endometrium, testes, and colon, have been found to express EGFRvIII by immunohistochemical and/or genetic analysis.15,16
Biochemical and Biological Characteristics and Functions of EGFRvIII
Several functional differences between EGFRvIII and normal EGFR have been characterized. Overexpression of EGFRvIII in NIH3T3 and NR6 cells results in transformed morphology, enhanced growth, and tumorigenicity in athymic mice.17,18 The EGFRvIII molecule does not appear to be regulated by EGF or TGF-a,18-20 and is constitutively activated in various systems. Even though TGF-a can not bind to EGFRvIII when expressed in CHO cells, these cells had increased DNA synthesis levels.19 Similar observations are seen in the EGFRvIII-transfected 32D-cell system, a non-tumorigenic, IL-3-dependent murine hematopoietic cell, which does not express endogenous levels of EGF family receptors. Comparing 32D/EGFR and 32D/EGFRvIII transfectants, the 32D/EGFRvIII transfectants displayed low levels of EGF- and BTC-stimulated mitogenic activities with very little response to TGF-a.21 32D/EGFRvIII is capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, parental, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells all depended on IL-3 or EGF-like ligands for growth. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3, low-expressing clone 32D/EGFRvIII, or parental 32D cells. Such profound transforming activity has not been observed in any homo- or heterodimers of wild-type ErbB-family receptors in this system. Transformation ability of EGFRvIII is dependent upon the level of EGFRvIII expression. The level of EGFRvIII expression is a critical driving force for the IL-3-independent phenotype.21
The biological significance of EGFRvIII in human cancer has been explored by exogenous expression of EGFRvIII in gliomas and breast cancer cells. Exogenous expression of EGFRvIII in glioblastoma U87MG cells is associated with self-phosphorylation and a pronounced enhancement of tumorigenicity in vivo.22 A similar observation is demonstrated in breast cancer cells. Exogenous expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. These MCF-7/EGFRvIII transfectants exhibited an approximately three-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages. Expression of EGFRvIII in MCF-7 cells elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk.21 Moreover, EGFR-vIII expression significantly enhanced tumorigenicity of the MCF-7 cells in athymic nude mice (P < 0.001).21
Changes in ligand specificity support the notion of an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB.23 Unlike wild-type EGFR, constitutively active EGFRvIII is not down-regulated, suggesting that the altered conformation of the mutant does not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting.24 Mutational analysis showed that the enhanced tumorigenicity of EGFRvIII is dependent on the intrinsic tyrosine kinase activity and is mediated through the carboxyl terminus. Mutation of one or more of the major autophosphorylation sites (Y1173, Y1068, and Y1148) of EGFRvIII abolished the enhanced tumorigenesis characteristic of EGFRvIII. These characteristics distinguish EGFRvIII from wild-type EGFR for which it is necessary to mutate multiple tyrosine autophosphorylation sites to block ligand-dependent transformation.24 Furthermore, EGFRvIII lacks the ability to undergo ligand-induced internalization, suggesting that kinase-regulated receptor internalization is mediated by phosphotyrosine residues. The potential role for altered conformation includes the possibility to recruit different downstream effectors, contributing potent mitogenic activities and tumorigenicity in vivo.25
Potential Promising Clinical Applications of Targeting EGFRvIII Receptor
EGFRvIII is a tumor-specific cell surface molecule. Clearly, EGFRvIII plays an important role in human cancer. The unique characteristics of EGFRvIII make it an attractive therapeutic target candidate.
The rationale to inhibit the EGFR tyrosine kinase family as an approach to cancer chemotherapy has grown stronger over the last 10 years. In response, a variety of clinical approaches have been developed. Immunotherapy has long been considered a promising approach for the treatment of cancer.26 A human-mouse chimeric anti-EGFR mAb C225 is currently being investigated in phase II and III clinical trials.26 Responses are reported in lung, head, neck, and other malignancies.26 Pivotal breast cancer trials with a humanized monoclonal antibody against HER2/neu (Herceptin) showed that it has activity as a single agent in a subset of patients whose tumors express high levels of ErbB-2. The results are even more impressive when used in combination with chemotherapy.27 A similar strategy has been applied to targeting EGFRvIII. A recent report describes a specific EGFR-vIII monoclonal antibody (designated Y10) that inhibits DNA synthesis and cellular proliferation. It also induces autonomous, complement-mediated, and antibody-dependent, cell-mediated cytotoxicity. Treatment of brain tumor in mice with a single intratumoral injection of Y10 increases median survival by an average 288%, with 26% long-term survival (n = 177; P < 0.001).28 Another approach fuses a single-chain antibody specific for the EGFRvIII to a Pseudomonas exotoxin, which has significant efficacy and no toxicity at therapeutically effective doses.29
During the past four years, significant progress has been made in the area of EGFR and ErbB-2 tyrosine kinase inhibitors, and new structural classes have emerged that exhibit enormous improvements with regard to potency, specificity, and activity both in vitro and in vivo.30 A specific tyrosine kinase inhibitor targeting EGFR recently entered clinical trial.30 Since EGFRvIII is a constitutively activated receptor, tyrosine kinase inhibitors potentially could be useful as therapeutic agents for the treatment of EGFRvIII-expressing human cancers. It is highly likely that blockage of EGFRvIII-mediated signal pathways, in combination with chemo- or radiotherapy, will provide new avenues for treatment of human cancer. A better understanding of the biology of breast cancer will lead to a new therapeutic direction of genetic intervention or prevention. (Dr. Tang is an Assistant Professor of Oncology and Principal Investigator, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC.)
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