Background
Many human tumor cells are characterized by over-expression of epidermal growth factor receptor (EGFR), a protein that promotes growth and aggressiveness and resistance of cancer cells to chemo- and radiotherapy [
1‐
5]. EGFR can be phosphorylated in response to binding of its specific ligands (EGF, TGF alpha and Amphiregulin) [
6,
7] and after exposure to unspecific stimuli like ionizing radiation [
8], UV-radiation [
9], hypoxia [
10], hyperthermia [
11], oxidative stress [
12] and trans-activation by G-protein coupled receptors [
13,
14]. Ligand-dependent as well as ligand-independent phosphorylation of EGFR results in receptor internalization [
15] and intracellular signaling [
4,
5,
16‐
18]. Up to date internalization is assumed to be essential for receptor silencing and inactivation. Indeed, EGF treatment results in internalization of EGFR into coated pits followed by receptor degradation [
19]. As reported by Khan [
12], exposure to oxidative stress can lead to internalization of EGFR by caveolae and this process is associated with peri-nuclear accumulation of EGFR.
A characteristic constituent of caveolae is caveolin. In vertebrates the caveolin gene family has three members: CAV1, CAV2, and CAV3, coding for the proteins caveolin-1, caveolin-2 and caveolin-3, respectively. Caveolins form oligomers and associate with cholesterol and sphingolipids in certain areas of the cell membrane, leading to the formation of caveolae. Caveolae are involved in receptor independent endocytosis [
20]. Furthermore Caveolin-1 is an integral transmembrane protein and an essential component in interactions of integrin receptors with cytoskeleton-associated and signaling molecules [
21]. Compartmentation into caveolae prevents EGFR degradation and simultaneously enables intracellular EGFR signaling [
12]. These findings suggest a new function of EGFR – depending on its intracellular localization -, which supplements its functions described so far. The idea of additional EGFR functions is further supported by the observation, that peri-nuclear EGFR can be transported into cell nucleus in response to irradiation [
5]. As we and others have reported earlier [
4,
22‐
24], nuclear EGFR is linked with activation of DNA-PK and regulation of non-homologous end-joining DNA-repair resulting in increased radioresistance [
5]. As reported recently [
1], nuclear EGFR detection in tumors biopsies correlated strongly with treatment resistance and bad prognosis.
In the present study, we focused on the radiation-induced nuclear translocation process of EGFR via caveolae. Evidence is provided that inhibition of src activity blocks the caveolin-dependent EGFR internalization and nuclear EGFR transport, which results in impaired DNA-repair.
Discussion
It is generally accepted, that the epidermal growth factor receptor is localized within the cell membrane and will be internalized following activation and dimerization [
30]. Indeed, such a scenario can be observed following EGF stimulation [
30], which initiates proliferation associated signaling. However, the EGFR can be also activated by oxidative stress [
12], radiation, [
5,
8] and G-coupled receptors [
13]. The molecular mechanisms of this ligand independent activation of EGFR are not fully understood. However, ligand independent stimulation of EGFR, e.g. by ionizing radiation [
8], is clearly characterized by receptor internalization also. The data presented herein, give new insights into the mechanism of EGFR internalization process and the intra-nuclear function of EGFR following exposure to ionizing radiation.
Several pathways enable endocytic transport of cargo molecules from the surface of eukaryotic cells into cytoplasm [
31]. The two best understood pathways, relevant for EGFR internalization, are the clathrin-coated pit [
31] and the caveolin [
32] driven internalization mechanisms. As shown by Khan et al. [
12], the clathrin-coated pit associated EGFR internalization can be observed following treatment with EGF and results in a fast degradation and silencing of receptor function. In contrast, treatment with H
2O
2 leads to EGFR internalization into caveolae, which sort internalized EGFR into a per-nuclear localization associated with an ongoing receptor signaling [
12]. In agreement with these data, we could show, that exposure to ionizing radiation induced a caveolin-1 associated EGFR internalization, whereas EGF treatment failed to trigger complex formation between
src, EGFR and caveolin-1. Sorting into different compartments in response to different stimuli may explain signal discrimination at the level of activated EGFR. Like for H
2O
2 treatment [
12], exposure to ionizing radiation also mediates the src driven phosphorylations of EGFR at Y845 and of caveolin-1 at Y14, which is needed for internalization of EGFR into caveolae [
12]. In response to radiation not only EGFR phosphorylation at Y845 – which is Src dependent – was observed, but also phosphorylation at Y992 and Y1173 could be observed. Both are described as autophosphorylation sites [
33]. This implicates that ionizing radiation activates not only src kinase, but also EGFR kinase and both kinases contribute to altered phosphorylation pattern of EGFR following radiation exposure. Caveolin-1 phosphorylation seems to be critical for caveolae formation [
20]. On the contrary, Y845 phosphorylation of EGFR probably is rather essential in regulation of EGFR-kinase activity than in formation of coated pits or caveolae [
15]. However, as shown by us and by Khan [
12] src activity is crucial for radiation- and H
2O
2-induced formation of caveolae. Nevertheless, the molecular mechanism responsible for activation of src has to be resolved. From our data it appears, that radiation leads to a fast activation of src, which is documented by phosphorylation of src at residue Y416. This phosphorylation is described as an autophosphorylation [
27]. As activating molecular switch several mechanisms are discussed: (i) oxidation associated structural modifications result in activation of src kinase [
34], (ii) inhibition of a phosphatase leads to auto-activation of kinase [
35], (iii) G-coupled receptor signaling mediates src activation [
14]. Which of these potential mechanisms is relevant for radiation-induced src kinase activity is currently unclear and is subject of ongoing investigations.
As shown herein, treatment with Erbitux, which binds to the extracellular domain of EGFR, results in receptor internalization and formation of an intracellular complex of EGFR, caveolin-1 and Erbitux. Internalized EGFR however can not be activated by EGF and this observation may explain growth inhibitory effects of Erbitux.
Khan et al. observed a peri-nuclear EGFR accumulation due to caveolin-1 driven internalization after exposing cells to H
2O
2 [
12]. We could also detect a peri-nuclear localization of the EGFR [
5] in irradiated cells, which is accompanied by a nuclear EGFR shuttling [
5]. Based on these results we hypothesized, that peri-nuclear EGFR serves as a pool for nuclear EGFR transport following irradiation. This hypothesis is supported by the observation that inhibition of src either by its specific inhibitor PP2 or by specific siRNA, prevents nuclear translocation of EGFR by blocking caveolin-1 driven EGFR internalization. It is noteworthy, that caveolin-1 driven EGFR internalization occurs predominantly following treatment of cells with genotoxic agents. This observation is in favor with the idea, that EGFR internalization and nuclear transport of EGFR are linked with DNA-repair processes [
23,
36]. This assumption is supported by the observation, that caveolin-1 driven EGFR internalization is not observed after EGF treatment. As shown for irradiated cells nuclear EGFR is found in complex with DNA-PK, which is an essential compound of non-homologous end-joining DNA-repair [
5]. As reported earlier [
37], inhibition of EGFR nuclear transport by Erbitux, markedly impaired radiation associated activation of DNA-PK and increased cellular radiosensitivity [
37]. In agreement with that, inhibition of src, which blocks EGFR internalization and subsequently nuclear transport after irradiation, abolished activation of DNA-PK, inhibited DNA-repair and increased radiosensitvity. Based on the data presented, it can be concluded, that the radiation-induced activation and nuclear translocation of EGFR is mediated through src kinase activity in a caveolin-1 dependent process. As blocking of these processes markedly effects repair of DNA-double strand breaks, this EGFR-coupled radiation response mechanism offers new interventional molecular targets for cancer therapy, especially by radiation therapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KD, CM and RK performed experiments and interpreted data; the authors contribution to this research are reflected in the order shown. HPR supervised all aspects of this research. KD and HPR prepared the manuscript. All authors read and approved the final manuscript.