RTK cross-talk or transactivation refers to a mechanism by which a ligand indirectly activates an RTK for which it does not serve as a direct agonist. Multiple RTKs, such as platelet-derived growth factor receptor, EGFR, and c-Met, are potential targets of receptor transactivation by diverse ligand/receptor families (24
). G protein-coupled receptors for ligands, such as thombin, angiotensin, lysophosphoatidic acid, and endothelin, probably represent the best example of a receptor family that participates in EGFR family transactivation (29
). Janus tyrosine kinase pathway agonists, such as leptin, growth hormone, and prolactin, and the frizzled receptor agonist Wnt have also been found to activate EGFR-dependent cell signaling (36
). Exclusively cytoplasmic mechanisms of transactivation include the direct phosphorylation of EGFR tyrosines by Jak2 or by non-RTKs, such as Src (39
). Direct transphosphorylation of kinase domain tyrosines can also result from RTK heterodimerization (e.g., platelet-derived growth factor receptor/EGFR, c-Met/EGFR; refs. 24
). Non-EGFR ligands can also stimulate matrix metalloproteinases and heparinases that activate HB-EGF sequestered at the cell surface that in turn binds EGFR extracellular ligand binding domains via classic ligand-receptor interactions (28
). In vitro
analyses show that these posttranslational mechanisms of receptor transactivation characteristically occur relatively quickly, within minutes, after activating the initiating receptor. Transactivation by these mechanisms may involve the formation of a caveolin-dependent “signalplex” where participating receptors, nonreceptor kinases, docking proteins, scaffolding proteins, and proteolytic enzymes colocalize (42
In this report, we describe for the first time transcription-dependent EGFR transactivated by HGF, the ligand for the c-Met RTK. We further show that EGFR transactivation by HGF occurs via a novel mechanism distinct from the posttranslational mechanisms of EGFR transactivation discussed above. We show that EGFR transactivation by HGF is both transcription-dependent and translation-dependent and associated with the induction of two EGFR ligands — TGF-α and HB-EGF. Of these two ligands, HB-EGF was most strongly induced by HGF and seemed to have the dominant role in EGFR transactivation under our in vitro
experimental conditions. We found that the HB-EGF antagonist CRM197 potently inhibited HGF-induced EGFR phosphorylation, whereas inhibiting TGF-α gene expression induction with siRNA had only a modest effect. Consistent with the requirement for gene expression and protein synthesis, HGF-induced EGFR activation is shown to occur on a time frame (i.e., hours) that is substantially slower and more prolonged than the posttranslational receptor transactivation that takes place within seconds to minutes of ligand-dependent activation of the initiating receptor (28
). EGFR transactivation by c-Met is not likely to require the colocalization of c-Met and EGFR (42
), because the EGFR ligands secreted and/or sequestered at the cell surface in response to HGF have the potential to function at some distance from the c-Met receptor. Our finding that HGF induces TGF-α secretion and HB-EGF accumulation almost exclusively at the cell surface suggests that the former would be capable of both autocrine and paracrine EGFR activation whereas the latter is limited to autocrine effects.
It is of particular interest that HGF was found to stimulate EGFR phosphorylation at both Tyr845
is a classic RTK autophosphorylation site and potentiates docking of growth factor receptor binding protein 2 and Gab1 to EGFR and subsequent activation of MAPK/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt signaling cascades, respectively (26
). EGFR Tyr845
resides within the kinase domain activation loop, and its phosphorylation, while dependent upon EGFR stimulation, is mediated by Src kinase via a mechanism potentially independent of MAPK (27
). Src activation can occur downstream of either c-Met and EGFR, and it is possible that one or both of these potential paths to Src activation contribute to HGF-induced EGFR Tyr845
phosphorylation. Parsons and colleagues have shown that Tyr845
phosphorylation is required for EGFR-induced DNA synthesis (44
). HGF induces cell cycle progression, proliferation, and DNA synthesis in glioma cells arrested by contact inhibition and serum deprivation (32
). We report here that inhibiting HGF-induced EGFR transactivation partially blocks HGF-induced cell cycle progression at the S-G2
-M transition. Based on these effects from antagonizing HB-EGF, it seems that EGFR transactivation acts relatively specifically at the S-G2
-M transition, because CRM197 had no effect on G1
-S transition that is potently stimulated by HGF. We show that this cell cycle response is biologically relevant because inhibiting EGFR transactivation partially inhibited HGF-induced cell proliferation. In contrast to cell proliferation, inhibiting EGFR transactivation with either CRM197 had no effect on the cytoprotective actions of HGF. Thus, EGFR transactivation differentially contributes to HGF-induced cell growth and survival responses.
Our results are also interesting with regard to the apparent stability of EGFR phosphorylation after c-Met activation by HGF. Phosphorylated EGFR was easily detected between 6 and 24 h after cell stimulation with HGF, unlike the relatively rapid EGFR turnover that follows acute receptor activation by exogenously applied EGFR agonists. There are multiple potential explanations for this. The unique kinetics of autocrine receptor transactivation induced by HGF might inefficiently induce receptor down-regulation. The glioma cells used in this study might be defective in adaptor proteins, such as Cbl that mediate EGFR turnover (45
). Alternatively, c-Met activation might alter the expression/function of adaptor proteins that actively stabilize EGFR (e.g., Alix; ref. 46
). Experiments examining these and other possibilities are currently under way.
c-Met, EGFR, and their prospective ligands are overexpressed in multiple malignancies. Each receptor/ligand system is currently recognized as a potential target for anticancer therapy. EGFR inhibitors have recently been found to be clinically active in subsets of non–small cell lung cancer and glioblastoma multiforme that contain activating EGFR mutations and deletions, respectively (10
). Efficacy in tumors overexpressing wild-type EGFR has not been firmly established. HGF/c-Met pathway inhibitors are effective in preclinical animal cancer models, and clinical trials are just being initiated to test the safety and efficacy of pathway targeting in cancer patients (16
). Whereas c-Met and EGFR share multiple cell signaling mechanisms and activate common downstream second messenger cascades (e.g., Ras/MAPK, phosphoinositide 3-kinase/AKT, Stat) and biological responses (mitogenesis, motogenesis, cytoprotection), ample differences exist to support cooperative contributions to oncogenesis (4
). The previous report from Jo et al. describing posttranslational EGFR→Met receptor transactivation in hepatocytes and our current results showing transcription-dependent c-Met→EGFR transactivation in glioma cells establish that EGFR and c-Met can be coordinately coactivated through multiple mechanisms (24
). Engelman et al. have recently found that c-Met amplification promotes lung cancer resistance to EGFR inhibitors via a mechanism involving ERBB3/phosphoinositide 3-kinase pathway activation (22
). Coordinated receptor coactivation via multiple mechanisms may have considerable consequences on developmental biology and disease mechanisms. It also has implications in our understanding of RTK signaling networks and the design of therapeutic RTK-targeting strategies in diseases including cancer.