Leah N. Klapper

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB‐1 and ErbB‐3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB‐2. Unlike ErbB‐1 and ErbB‐2, the intrinsic tyrosine kinase of ErbB‐3 is catalytically impaired. By using interleukin‐3‐dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB‐3 is devoid of any biological activity but both ErbB‐1 and ErbB‐2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB‐3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter‐receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB‐3‐containing complexes, especially the ErbB‐2/ErbB‐3 heterodimer, are more active than ErbB‐1 complexes. Nevertheless, ErbB‐1 signaling displays dominance over ErbB‐3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen‐activated protein kinases ERK and c‐Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase‐defective ErbB‐3.

Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors.

Carcinoma, cancer of epithelial cells, is a major cause of morbidity and mortality in Western societies. Clonal fixation and propagation of oncogenic genetic changes, sporadically accumulating in epithelial cells, depend on growth factors and their surface receptors. One of the large families of receptors is that of the ErbB tyrosine kinases, which bind multiple neuregulins and other epidermal growth factor-like molecules. Certain ErbB members and their ligands are involved in human cancers of various origins. However, most of the clinical data relate to ErbB-2, a protein whose overexpression in subsets of carcinomas can predict poor prognosis. Although no ligand has so far been assigned to ErbB-2, recent biochemical evidence implies that this oncoprotein operates as a shared receptor subunit of other ErbBs. Several biochemical attributes enable ErbB-2 to act as an epithelial cell amplifier of stroma-derived growth factor signals: It delays ligand dissociation, enhances coupling to the mitogen-activated protein kinase pathway, and impedes the rate of receptor downregulation. The realization that ErbB-2 is a master regulator of a signaling network that drives epithelial cell proliferation identifies this protein as a target for cancer therapy. Indeed, various ErbB-2-directed therapeutic approaches, including immunological and genetic therapies, demonstrate promising clinical potential.

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers

Both homo‐ and hetero‐dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)‐like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor α (TGFα); and their chimera, denoted E4T, which act on cells singly expressing ErbB‐1 as a weak, a strong, and a very strong agonist, respectively. Co‐expression of ErbB‐2, a developmentally important co‐receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFα, an effect that was partially mimicked by ErbB‐3. Analysis of the mechanism underlying this trans‐potentiation implied that EGF‐driven homodimers of ErbB‐1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB‐2 or with ErbB‐3, dissociate in the early endosome. As a consequence, in the presence of either co‐receptor, ErbB‐1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFα‐driven homodimers of ErbB‐1, and also by E4T‐bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo‐ and hetero‐dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB‐2 to shunt ligand‐activated receptors to recycling may explain, in part, its oncogenic potential.

Bivalence of EGF-like ligands drives the ErbB signaling network

Signaling by epidermal growth factor (EGF)‐like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand‐induced dimerization is unknown. We contrasted two existing models: a conformation‐driven activation of a receptor‐intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)‐induced heterodimer between ErbB‐3 and ErbB‐2 favors a bivalence model; the ligand simultaneously binds both ErbB‐3 and ErbB‐2, but, due to low‐affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C‐terminal low‐affinity site has broad specificity, but it prefers interaction with ErbB‐2, an oncogenic protein acting as a promiscuous low‐affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo‐ and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB‐2/HER2.

A subclass of tumor-inhibitory monoclonal antibodies to ErbB-2/HER2 blocks crosstalk with growth factor receptors

ErbB-2 is an orphan receptor that belongs to a family of tyrosine kinase receptors for either epidermal growth factor (EGF) or Neu differentiation factor (NDF/neuregulin). Because overexpression of the erbB-2 proto-oncogene is frequently associated with an aggressive clinical course of certain human adenocarcinomas, the encoded protein is an attractive target for immunotherapy. Indeed, certain monoclonal antibodies (mAbs) to ErbB-2 effectively inhibit tumor growth in animal models and in clinical trials, but the underlying mechanism is incompletely understood. To study this question, we generated a large battery of mAbs to ErbB-2, that were classified epitopically. Whereas most antibodies stimulated tyrosine phosphorylation of ErbB-2, their anti-tumor effect correlated with its accelerated endocytic degradation. One group of tumor-inhibitory mAbs (Class II mAbs) was elicited by the most antigenic site of ErbB-2, and inhibited in trans binding of NDF and EGF to their direct receptors. The inhibitory effect was due to acceleration of ligand dissociation, and it resulted in the reduction of the ability of ErbB-2 to transactivate the mitogenic signals of NDF and EGF. These results identify two potential mechanisms of antibody-induced therapy: acceleration of ErbB-2 endocytosis by homodimerization and blocking of heterodimerization between ErbB-2 and growth factor receptors.

The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin

The ErbB-1 receptor tyrosine kinase binds to six different growth factors, whose prototype is the epidermal growth factor (EGF). Two homologous epithelial receptors, ErbB-3 and ErbB-4, bind all isoforms of another family of growth factors, the Neu differentiation factors (NDFs/neuregulins). The fourth member of the ErbB family, ErbB-2, acts as the preferred heterodimeric partner of ligand-occupied complexes of the three other ErbB proteins. Here we report that at high concentrations, EGF can induce cell growth and differentiation in the absence of ErbB-1. This function is shared by betacellulin, but not by three other ligands, including the transforming growth factor α (TGFα). The functional receptor was identified as a heterodimer between ErbB-3 and ErbB-2, a previously identified oncogenic complex. When singly expressed, neither ErbB-3 nor ErbB-2 can mediate signaling by EGF. In addition, when co-expressed, blocking either receptor by using site-specific antibodies inhibited EGF and betacellulin activities, indicating strict cooperativity between ErbB-3 and ErbB-2. Through analysis of chimeras between EGF and TGFα, we identified the middle portion of EGF (loop B) as the site that enables activation of ErbB-2/ErbB-3. In conclusion, cooperative and promiscuous binding of stroma-derived growth factors by the epithelium-expressed ErbB-2/ErbB-3 heterodimer may be significant to cancer development. The mechanistic implications of our results for a model that attributes receptor dimerization to ligand bivalency, as well as to a recently proposed mechanism of secondary dimerization, are discussed.

c-Cbl Is a Suppressor of the Neu Oncogene

A rodent oncogenic mutant of the Neu receptor tyrosine kinase is a useful experimental model because overexpression of the respective receptor, namely HER2/ErbB-2, in human malignancies is associated with relatively aggressive diseases. Here we show that the oncogenic form of Neu is constitutively associated with the product of the c-cbl proto-oncogene and is part of a large complex that includes the phosphoinositide 3-kinase and Shc. Ectopic expression of c-Cbl, a ubiquitin-protein isopeptide ligase specific to activated tyrosine kinases, causes rapid removal of Neu from the cell surface and severely reduces signaling downstream of oncogenic Neu. c-Cbl-induced down-regulation of Neu involves covalent attachment of ubiquitin molecules and requires the carboxyl-terminal domain of Neu. The negative effect of c-Cbl is antagonized by v-Cbl, a virus-encoded oncogenic truncated form of c-Cbl. In an in vivo model, infection of a Neu-transformed neuroblastoma with a c-Cbl-encoding retrovirus caused enhanced down-regulation of Neu and correlated with tumor retardation. Our results implicate c-Cbl in negative regulation of Neu and offer a potential target for treatment of HER2/ErbB-2-positive human malignancies.

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies.

Abstract Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab′ fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab′-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab′ fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms.