Trans receptor inhibition of human glioblastoma cells by erbB family ectodomains

Abstract

Our aim has been to understand the features of erbB receptor homo-and heterodimer assembly to develop approaches to disrupt receptor activation. We have developed a general approach to cause erbB receptor-specific trans inhibition of human neoplasia. The clonal progression of human astrocytomas to a more malignant phenotype often involves the amplification and overexpression of the epidermal growth factor receptor (EGFr) gene. We have selectively targeted the EGFr in human glioblastoma cells with kinasedeficient mutants of the erbB family derived from the ectodomain of the Neu oncogene that are able to form heterodimers with EGFr and inhibit EGFr-dependent phenotypes. In EGFrpositive U87MG human glioblastoma cells, expression of the Neu ectodomain inhibits EGF-, but not platelet-derived growth factor-, induced DNA synthesis; inhibits cell proliferation in the presence of EGF, but not platelet-derived growth factor; inhibits the ability of U87MG to form colonies in soft agar; and inhibits transforming efficiency in athymic mice. These studies establish that EGFr-mediated signal transduction is important in the maintenance of malignant glioma, and that trans receptor inhibition is a novel way to abrogate abnormal growth of these tumors. Neu ectodomains will be useful in determining the manner in which the EGFr contributes to glial tumorigenesis and in the design of pharmaceuticals that disable erbB family oncoproteins. In addition, these studies provide a rationale for the application of the Neu ectodomain in gene therapy approaches to human malignant glioma and, potentially, to other systemic epithelial malignancies expressing erbB family receptors.

Inhibition of EGFR-mediated phosphoinositide-3-OH kinase (PI3-K) signaling and glioblastoma phenotype by Signal-Regulatory Proteins (SIRPs)

“…EGFR-dependent signaling and phenotypes in glioblastoma cells have been characterized by our laboratory (Qian et al, 1996;O'Rourke et al, 1997aO'Rourke et al, , 1998aWu et al, 1999) and others (Nishikawa et al, Figure 1 SIRPa1 inhibits EGFR-mediated transformation of U87MG human glioblastoma cells but does not a ect cell proliferation. (a) Transforming e ciency of SIRPa1-overexpressing U87MG-derived subclones.…”

“…Our previous work utilized a trans-dominant mutant receptor derived from p185 neu that inhibits EGFR kinase activity to show that EGFR signaling determines transforming and apoptotic phenotypes in U87MG glioblastoma cells (O'Rourke et al, 1997a;1998a,b). In order to investigate the relationship between SIRP function and EGFR signaling pathways, we overexpressed the SIRPa1 cDNA in U87MG cells, which contain high endogenous levels of EGFR, but no other detectable erbB family proteins (O'Rourke et al, 1997a).…”

“…In order to investigate the relationship between SIRP function and EGFR signaling pathways, we overexpressed the SIRPa1 cDNA in U87MG cells, which contain high endogenous levels of EGFR, but no other detectable erbB family proteins (O'Rourke et al, 1997a). Stable U87MG clones expressing elevated amounts of the SIRPa1 protein were inhibited 65% in the formation of morphologically transformed foci, as compared to clones expressing vector without insert (mean number of foci+s.d.…”

“…The pRK5(RS) plasmids were digested with NruI and XhoI and the fragments containing the SIRPa1 gene or its 4Y mutant gene were blunt-end ligated into the expression vector pIRES/Hyg (Clontech) previously linearized with BamHI and blunt-ended for ligation. Derivations of the carboxyl terminal-deleted p185 neu mutant receptor construct, T691stop, have been detailed previously (O'Rourke et al, 1997a). Growth inhibitory properties of T691stop have been reported (O'Rourke et al, 1997a(O'Rourke et al, , 1998a.…”

“…We have used the human glioblastoma U87MG cell line as a model system to characterize EGFR-mediated transforming and survival phenotypes (Nishikawa et al, 1994;Nagane et al, 1996;Huang et al, 1997;O'Rourke et al, 1997aO'Rourke et al, , 1998a. Independent studies of the PTEN tumor suppressor protein have shown that deregulated PI3-K pathway activation alters glioblastoma survival, transformation, motility and adhesion (Furnari et al, 1997(Furnari et al, , 1998Tamura et al, 1998;Wick et al, 1999).…”