The outcome of RTK signaling involves a balance between various stimulatory and inhibitory mechanisms which in turn determine both the strength and duration of signals that are transmitted through networks of signaling cascades . In this respect, endosomal sorting plays a key role in the regulation of EGFR signaling .
NSCLC-associated kinase domain mutations in EGFR promote its constitutive activation, and a number of studies have focused on delineating the signaling pathways whose activation contributes to oncogenesis . The outcome of EGFR signaling is intimately linked to its endocytic traffic, which is normally triggered by ligand-induced dimerization  and phosphorylation-dependent as well as phosphorylation-independent recruitment of endocytic machinery components [51, 52]. The nature of endocytic trafficking of NSCLC-associated EGFR mutants and any relationship of altered traffic with oncogenic signaling remain poorly understood. Here, we have used NSCLC cell lines to demonstrate that oncogenic mutant EGFRs, but not wtEGFR, are constitutively endocytosed (Figure 1). Mutant EGFRs were found to localize in early and recycling endosomes based on colocalization with labeled transferrin (Figure 2) and GFP-tagged Rab4, Rab11, EHD1 and EHD3 proteins (Figure 4). Notably, blocking the exit of endocytosed receptors from endocytic recycling compartments with monensin led to a marked accumulation of mutant EGFR in a perinuclear endocytic compartment (Figure 3) and increased its colocalization with markers of sorting and endocytic recycling compartments (Figure 4B). Thus, these findings strongly suggest that mutant EGFRs transit through the endocytic recycling compartment.
Importantly, enhanced EGFR-Src as well as activated EGFR/phospho-Src colocalization was observed in endocytic vesicles of a mutant EGFR-expressing cell line (Figure 5). Furthermore, monensin treatment increased the colocalization of mutant EGFRs with Src in the perinuclear endosomal compartment (Figure 6) and enhanced the biochemical association between mutant EGFRs and Src (Figure 7). Given the emerging evidence for a critical role of the constitutive engagement of Src-mediated signaling pathways in mutant EGFR-dependent oncogenesis [7, 9, 10], our results suggest a potentially important role of altered endocytic trafficking in the oncogenic behavior of mutant EGFRs.
In view of the critical role of ligand-induced internalization and lysosomal targeting in limiting EGFR signaling, the constitutive activation of downstream signaling pathways by NSCLC-associated mutant EGFRs has generated interest into potential alterations of endocytic trafficking. For example, given the critical role for the Cbl-family of ubiquitin ligases in orchestrating EGFR ubiquitinylation and subsequent lysosomal sorting, it is notable that a recent analysis of NSCLC-associated mutant EGFRs showed reduced Cbl-dependent lysosomal downregulation [28, 29, 53]. However, another study in an NSCLC cell line reported that mutant EGFR traffics into lysosomes upon EGF stimulation . The present study extends beyond these observations by demonstrating that mutant EGFRs traffic through the endocytic recycling compartment. Our observations, that mutant EGFRs localize to the lysosomes (Figure 2) and block of their endocytic transit by low temperature incubation (Additional File 2) or monensin treatment led to reduced degradation (Additional File 4), are consistent with the idea of mutant EGFRs trafficking into lysosomes. However, our observations do not contradict the defective ubiquitin-dependent trafficking of mutant EGFRs reported by Shtiegman et al., and others [28, 29, 37], as our studies did not address this issue.
Whether the increased transit through the endocytic recycling compartment is an intrinsic property of mutant EGFRs or is a secondary consequence of their reportedly reduced interaction with Cbl and ubiquitin-mediated lysosomal sorting machinery are important questions that will need to be addressed through appropriate manipulations in NSCLC cells as well as the use of ectopic gene expression approaches. In this regard, it is noteworthy that conditions that prevent EGFR interaction with Cbl or its Cbl-dependent ubiquitinylation lead to a more prolonged stay of EGFR in early/recycling endosomal compartments [15, 55, 56]. Physiologically, ligands such as TGFα that promote EGFR recycling rather than lysosomal degradation appear to engage the Cbl and ubiquitin machinery more transiently [22, 57]. In addition to altered ubiquitinylation of mutant EGFRs, other defects in their signaling or protein-protein interactions could contribute to their propensity to enter the endocytic recycling compartment. For example, deubiquitinylating enzymes [58, 59] as well as other factors (e.g. secretory membrane carrier protein SCAMP3) can regulate EGFR recycling versus lysosomal degradation [13, 60]. Future studies to elucidate whether or not mutant EGFRs might aberrantly interact with such proteins will therefore be of considerable interest.
NSCLC-associated mutant EGFRs (both gefitinib-sensitive deletion mutants and gefitinib-resistant L858R/T790 M mutant) are constitutively active and constitutively endocytosed (Figure 1 and Additional File 1). Recent studies have demonstrated that NSCLC-associated kinase domain mutations promote constitutive dimerization of EGFR . As dimerization is critical to EGFR endocytosis and may promote internalization in a kinase-dependent  or kinase-independent [50, 62] manner, constitutive dimerization may play an important role in the transit of mutant EGFRs into the endocytic recycling compartment. In this context, our observations using kinase inhibitors indicate that the kinase activity of EGFR is not essential for the constitutive endocytic localization of mutant EGFR (Additional File 6A). The intracellular localization of mutant EGFR was also unaffected by Src inhibitor PP2, indicating that there may be another determinant of constitutive endosomal localization of mutant EGFRs.
Transit of the constitutively-active mutant EGFR through the endocytic recycling compartment is likely to be biologically relevant. Analyses of EGFR as well as other RTKs have demonstrated that endocytic recycling, in addition to returning the internalized receptors for additional rounds of ligand-binding and signaling, can directly participate in signaling events . For example, inhibition of EGFR internalization reduced the level of activation of Akt and MAPK downstream of the receptor [30, 63]. Notably, initiation of EGFR activation directly at the level of endosomes has been shown to be sufficient to activate Erk and Akt, as well as promote cell survival and proliferation [34, 64]. However, monensin treatment did not enhance Erk, Akt and STAT3 phosphorylation levels (Additional File 5B). The lack of monensin effect on downstream signaling is likely to reflect its ability to affect multiple endocytic compartments and/or its effects on other cellular processes [38, 42, 65]. Nevertheless, our observations of EGFR and Src colocalization and association are consistent with a role of signaling at the level of the endocytic recycling compartment in the biology of mutant EGFR.
Our analyses of mutant EGFR recycling in the context of Src were based on prior evidence that Src-dependent signaling is critical for EGFR-mediated oncogenesis; this has been established in vitro using Src inhibitors as well as mutational approaches , and Src is overexpressed or hyperactive in NSCLC as well as other cancers where EGFR mutations or overexpression have been implicated in oncogenesis [9, 10]. Importantly, Src has been shown to localize to endosomes , and recent studies have shown that Src specifically localizes on recycling endosomes [45, 66]. Thus, it appears plausible that mutant EGFRs, by virtue of their transit through the endocytic recycling compartment, may gain enhanced access to Src, providing a potential explanation for the higher level of constitutive Src-mutant EGFR association [7, 48]. Confocal image analyses indeed support this possibility, as Src and mutant EGFRs show a detectable colocalization (versus essentially little detectable colocalization of Src with wtEGFR) (Figure 5); moreover, this colocalization was further increased by inhibiting the exit of EGFR from the endocytic recycling compartment using monensin (Figure 6). Also, a predominant pool of activated EGFR colocalized with activated Src (Figure 5), and Src inhibitor slightly decreased the mutant EGFR-Src association (additional File 6), which suggest that Src activity might be important for colocalization and association with mutant EGFR. In a different study, a Src inhibitor did not inhibit mutant EGFR-Src association . The difference between the two studies may be due to different types of inhibitor and/or cell lines tested.
Rather interestingly, monensin treatment led to a higher level of biochemically detectable EGFR-Src complexes (Figure 7). This, together with higher constitutive Src-mutant EGFR association, suggests the likelihood that Src-mutant EGFR complexes are either formed or more stable in the endocytic recycling compartment. As Src-dependent signaling is critical for mutant EGFR-mediated oncogenic transformation , these findings suggest that altered trafficking of mutant EGFRs into the endocytic recycling compartment may contribute to their oncogenic behavior. Further studies to perturb the endocytic recycling of oncogenic EGFR mutants should help address the biological role of the altered endocytic trafficking identified here.
It has been reported that a gefitinib-resistant version of H1650 NSCLC cell line showed increased internalization of EGFR upon ligand stimulation when compared to the parental gefitinib-sensitive cell line . Notably, the wtEGFR in the gefitinib-resistant cell line did not undergo ligand-induced lysosomal sorting, even though the receptor was found in endocytic vesicles . In our analyses, we observed a comparable pattern of subcellular localization and endocytic trafficking of gefitinib-sensitive (deletion) and gefitinib-resistant (L858R/T790 M) EGFR mutants (Figures 1, 2, 3 and Additional Files 2 and 3). Similarly, both gefitinib-resistant H1975 and gefitinib-sensitive H1650 cell lines showed delayed internalization of labeled EGF in comparison to the wtEGFR-expressing cell line H358 . However, there were subtle differences among different cell lines harboring mutant EGFRs in the perinuclear accumulation of the mutant EGFR induced by monensin in the regular growth condition (Additional File 3B); the perinuclear accumulation of EGFR was dramatic in HCC827 and HCC4006, intermediate in H1650, and not readily apparent in H1975. Similarly, quantitative assessments of EGFR localization under steady-state conditions (Figure 2E) suggested differences between different NSCLC lines: the mutant EGFR is evenly divided between Tf-positive and LAMP1-positive vesicles in H1650, HCC827 and HCC4006 showed much more mutant EGFR in LAMP1-positive than in Tf-positive vesicles; and gefitinib-resistant mutant EGFR in H1975 colocalized more with Tf than with LAMP1. In addition, H1650 cell line displayed more sensitivity to EGF than other mutant EGFR-expressing cell lines (Figure 1 and Additional File 5A). Whether EGFR expression levels, the nature of EGFR mutations, and/or activities of EGFR regulatory factors such as Src, Cbl or PTEN, which has been shown to be absent in the H1650 cell line , might contribute to the differences in the localization of mutant EGFR and their endocytic trafficking remain open questions. While it is possible that altered endocytic trafficking of EGFR relates to gefitinib resistance, extensive future studies are needed to determine if this is the case.