Chronic activation of RTK-dependent pathways is generally recognized to be associated with cancer. More recently, however, it has been appreciated that impaired negative regulation of receptor signaling may also be an important mechanism involved in cancer (23
). The ubiquitin ligase Cbl is considered a key regulator of down-regulation of RTKs, including the EGFR, by controlling receptor endocytosis and targeting to the lysosomal-degradative pathway. In fact, several oncogenic forms of RTK have been shown to escape Cbl-mediated degradation (27
). In general, these proteins have mutations that remove the binding site for Cbl on the receptor leading to alteration of receptor down-regulation that is an independent mechanism for increasing RTK signaling. However, for any cancer that is dependent on sustained RTK signaling for cell proliferation and survival, including situations in which receptors have activating mutations, some mechanisms for escaping down-regulation must operate, or the activated receptors would simply be degraded. Therefore, we investigated the down-regulation of mutant EGFRs in two gefitinib-sensitive NSCLC cell lines, in which the mutations were outside the known Cbl interaction site.
We found that two mutations in the EGFR frequently observed in human tumors, L858R and deletion E746-A750, are each sufficient to impair EGFR ubiquitylation and down-regulation. The mutations were also sufficient to significantly reduce the EGF-dependent association of Cbl with EGFR. A recent study had found that EGFR mutations L858R and L861Q exhibited similarly impaired receptor down-regulation when expressed in mouse hematopoietic cells (20
). Yang et al. (20
) observed that the EGFR mutations L858R and L861Q associated with Cbl even in unstimulated conditions and saw no increased association after adding ligand. Our results differ from this study because we observed that very little Cbl coimmunoprecipitated with EGFR L858R and EGFR deletion E746-A750 in the absence of EGF (). On binding EGF, the mutant EGFRs bound 5- to 10-fold more Cbl, although this was only half that bound by the WT EGFR. We found a similar result in a different assay measuring the EGF-dependent association of Cbl to a crude membrane fraction. These results are consistent with the degree of ubiquitylation we observed () and suggest that binding of Cbl to the mutant receptors is defective. The binding of Cbl to EGFR requires phosphorylation of Y1045. However, phosphorylation at Y1045 in nontransformed mouse mammary epithelial cells stably expressing EGFRs with L858R or deletion 747–753 mutations was similar to WT in a previous study (8
). We tested phosphorylation of Y1045 under our assay conditions and found no decrease in EGFR L858R and a very modest decrease in EGFR deletion E746-A750.7
Nevertheless, we show that cells expressing these mutant EGFRs recruit less Cbl in response to EGF, suggesting that binding to Cbl or to another protein that cooperates with Cbl is impaired. Presumably, these mutations alter the cytosolic domain decreasing the affinity for binding Cbl without removing the binding site because we also show that increasing Cbl expression can at least partially compensate for the effect of the mutation on down-regulation.
Contrary to our results and those of Yang et al. (20
), a recent study had reported that EGFR L858R or EGFR deletion E746-A750 expressed exogenously in H1299, a NSCLC cell line whose endogenous EGFR is not mutated, was degraded at a rate similar to exogenous WT EGFR in those cells (9
). However, the WT receptor in H1299 cells is degraded as slowly as the mutant receptors are when expressed in HBECs.7
Therefore, it is possible that H1299 cells contain defects in cellular machinery for receptor down-regulation that have the same effect for prolonging receptor half-life as do some receptor mutations. Our studies used a normal epithelial cellular background and Yang et al. (20
) used hematopoietic cells lacking endogenous EGFRs where the effects of receptor mutations can be isolated from other possible defects in a cancer cell line. In a recent review, Bache et al. (23
) compiled evidence that links deregulation of RTK degradation machinery with cancer, including alterations in Eps15, endophilin-2, Huntingtin interacting protein, and others required for receptor down-regulation. It will be interesting to learn if changes in such factors are also involved in lung cancer.
HSP90, a chaperone that plays a role in maturation and function of RTKs, has been reported to interact with EGFR containing tyrosine kinase domain mutations but not with WT receptors (20
). However, we observed that treatment with geldanamycin, an inhibitor of HSP90, increased degradation of both WT and mutant EGFRs. Thus, either the small amount of HSP90 binding we detect for WTEGFR has consequences for receptor down-regulation, or HSP90 has effects on other cellular components that influence EGFR degradation. HSP90 binds to the EGFR heterodimerization partner ErbB-2 and restrains its signaling by limiting heterodimer formation (29
). Therefore, we cannot rule out an indirect effect of geldanamycin through ErbB-2 on down-regulation of EGFRs in NSCLC cells or HBECs.
We did not observe defective internalization of EGFR L858R or EGFR deletion E746-A750 compared with WTEGFR and treatment with geldanamycin had no noticeable effect at this step. Our results indicate that the EGFR L858R and EGFR deletion E46-A750 are poorly ubiquitylated and degraded but efficiently internalized. A similar result was observed for an ubiquitylation-deficient mutant Met receptor (Y1003F), which was mistargeted for degradation but internalized with normal kinetics (14
). Thus, if HSP90 association to mutant EGFR explains impaired down-regulation, the effect occurs after receptors are removed from the plasma membrane, presumably in the processes that sort EGFR into the intraluminal vesicles of multivesicular endosomes. This interpretation is consistent with the prolonged colocalization of EGF and transferrin receptors we observed in HCC827 cells.
Defective down-regulation of RTK has been increasingly linked to cancer. For example, components of the endocytic machinery have been found as fusion oncoproteins (30
). In addition, regulators of receptor degradation other than Cbl have been linked to cancer, such as Tsg101, a component of the ESCRT-I complex (31
). Currently, there are no rationally designed therapies available that target the down-regulation machinery for EGFR or other RTKs. Previous evidence indicates that down-regulation of EGFRs can be increased by either elevating expression of ubiquitin ligases or reducing ubiquitin hydrolases in cells such as Chinese hamster ovary or HeLa (25
). We show that overexpression of Cbl accelerated degradation of EGFR in a NSCLC cell line, HCC827, suggesting that impaired ubiquitylation is a molecular defect responsible, at least in part, for impaired EGFR down-regulation in these cells. Future experiments examining whether factors that increase EGFR down-regulation have positive effects on NSCLC will be of special interest.