Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer

Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligan d-independent, constitutively active variant of the EGFR, Its expression ha s been detected in gliomas and various other human malignancies. To more fu lly characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopo ietic cell line (32D cells). We observed 32D cells expressing high levels o f EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EG FR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-li ke ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII e xpression levels, These results suggested that the IL-3-independent phenoty pe is mediated by EGFRvIII, The level of expression is a critical driving f orce for the IL-3-independent phenotype, Dose-response analysis revealed 32 D/ EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of E GF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml), Similar effects were also observed in beta-cellulin-med iated proliferation. Moreover, 32D cells expressing high revels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was ad ministered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D /ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C 2 cells or the parental 32D cells, The changes of the ligand specificity su pport the notion for an altered conformation of EGFRvIII to reveal an activ ated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB, These studies clearly demonstrate that EGFRvIII is capable of trans forming a nontumorigenic, IL-3-dependent murine hematopoietic cell Line (32 D cells) into an IL-3-independent and ligand-independent malignant phenotyp e in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer , we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Express ion of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosp horylation, presumably through heterodimerization and cross-talk, These MCP -7/EGFRvIII transfectants exhibited an similar to 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percen tages of serum. A similar result was also seen in anchorage-dependent assay s, Furthermore, EGFRvIII expression significantly enhanced tumorigenicity o f MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these resu lts provide the first evidence that EGFRvIII could play a pivotal role in h uman breast cancer progression.