Guo Cai Huang

Reduced ability of transforming growth factor-alpha to induce EGF receptor heterodimerization and downregulation suggests a mechanism of oncogenic synergy with ErbB2

The epidermal growth factor receptor (EGFR) is activated by a variety of ligands including EGF and transforming growth factor-alpha (TGFα), whereas no ligand for the homologous ErbB2 oncoprotein has yet been identified. Here we use both an ErbB2 phosphoantibody (aPY1222) and an activation-specific EGFR antibody to show that low concentrations of EGF induce more efficient tyrosine phosphorylation of ErbB2 in A431 cells than does equimolar TGFα, while EGFR is more potently activated by TGFα. Co-precipitation studies confirm that heterodimerization of activated EGFR and transphosphorylated ErbB2 is readily induced by EGF but not TGFα. EGFR downregulation is also more efficiently induced by EGF, suggesting that ligand-dependent modification of ErbB2 may be required to terminate EGFR signalling in cells expressing both receptor types. These findings indicate that EGF and TGFα differ in their abilities to induce tyrosine phosphorylation and heterodimerization of ErbB2, and raise the possibility that ErbB2 exerts its oncogenic effect in part by impairing TGFα-dependent EGFR downregulation.

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor.

Transforming growth factor‐alpha (TGFα) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid‐labile structure and potent transforming function. We recently reported that TGFα induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219–2223). Here we use isoform‐specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFα exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFα alone; the effects of TGFα and BFA on EGFR degradation are opposite, however, with TGFα sparing EGFR from downregulation but BFA accelerating EGF‐dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF‐dependent receptor signalling, whereas TGFα shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFα is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH‐labile TGFα may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine‐based downregulation signal and creating an irreversible autocrine growth loop. J. Cell. Physiol. 179:52–57, 1999.

Dominant negative knockout of p53 abolishes ErbB2-dependent apoptosis and permits growth acceleration in human breast cancer cells

We previously reported that the ErbB2 oncoprotein prolongs and amplifies growth factor signalling by impairing ligand-dependent downregulation of hetero-oligomerised epidermal growth factor receptors. Here we show that treatment of A431 cells with different epidermal growth factor receptor ligands can cause growth inhibition to an extent paralleling ErbB2 tyrosine phosphorylation. To determine whether such growth inhibition signifies an interaction between the cell cycle machinery and ErbB2-dependent alterations of cell signalling kinetics, we used MCF7 breast cancer cells (which express wild-type p53) to create transient and stable ErbB2 transfectants (MCF7-B2). Compared with parental cells, MCF7-B2 cells are characterised by upregulation of p53, p21WAF and Myc, downregulation of Bcl2, and apoptosis. In contrast, MCF7-B2 cells co-transfected with dominant negative p53 (MCF7-B2/Δp53) exhibit reduced apoptosis and enhanced growth relative to both parental MCF7-B2 and control cells. These data imply that wild-type p53 limits survival of ErbB2-overexpressing breast cancer cells, and suggest that signals of varying length and/or intensity may evoke different cell outcomes depending upon the integrity of cell cycle control genes. We submit that acquisition of cell cycle control defects may play a permissive role in ErbB2 upregulation, and that the ErbB2 overexpression phenotype may in turn select for the survival of cells with p53 mutations or other tumour suppressor gene defects.

Overexpression of ErbB2 impairs ligand-dependent downregulation of epidermal growth factor receptors via a post-transcriptional mechanism.

The mechanism by which ErbB2 exerts its oncogenic effect is poorly defined. In this article we show that ErbB2 co‐expression slows ligand‐dependent growth factor receptor downregulation in NIH 3T3 transfectants. Ligand dependence of cell growth and MAP kinase signaling are retained in epidermal growth factor receptor (EGFR) transfectants but are abolished in ErbB2‐expressing cells, which grow and signal constitutively. In association with this phenomenon, we have noticed that ErbB2‐expressing cells contain increased amounts of EGFR, which is hyperphosphorylated. EGFR overexpressors do not contain increased levels of ErbB2, however, tending to exclude a transfection artifact caused by saturation of receptor processing. EGF treatment of EGFR transfectants results in more rapid EGFR downregulation than occurs in ErbB2 transfectants, but Northern blot analysis demonstrates reduced basal EGFR gene expression in ErbB2 transfectants. We conclude that ErbB2 expression impairs EGFR downregulation via a post‐transcriptional mechanism and propose that ErbB2 overexpression may sensitize tumor cells to the mitogenic effects of heterologous growth factors by retarding degradation of liganded heterodimers. J. Cell. Biochem. 74:23–30, 1999.

Association of ErbB2 Ser1113 phosphorylation with epidermal growth factor receptor co-expression and poor prognosis in human breast cancer.

The carboxyterminal domain of the epidermal growth factor receptor (EGFR) – a putative binding site for the ubiquitin ligase Cbl – is the site of serine phosphorylation events which are essential for ligand-dependent EGFR desensitization and degradation. Using a monoclonal antibody (aPS1113) which selectively recognizes the homologous phosphorylated domain in the ErbB2 oncoprotein, we show here that wild-type ErbB2 becomes Ser1113-phosphorylated following treatment of 3T3 cells with growth factors or tyrosine phosphatase inhibitors. In EGFR-overexpressing A431 cells, ligand-inducible aPS1113 immunoreactivity declines more rapidly than other detectable phosphorylation events and is followed by EGFR downregulation. Analysis of 65 ErbB2-expressing primary breast cancers reveals a highly significant relationship between Ser1113 phosphorylation and EGFR overexpression (p < 0.0001) as well as an association with poor prognosis (p = 0.005). We submit that ErbB2 Ser1113 phosphorylation status represents a novel and informative biomarker of cancer cell biology and tumor behavior.