The exon 19-deleted EGFR undergoes ubiquitylation-mediated endocytic degradation via dynamin activity-dependent and -independent mechanisms
The epidermal growth factor receptor (EGFR) is closely implicated in cancer, and sequencing analyses have revealed a high mutation rate of EGFR in lung cancer. Recent advances have provided novel insights into the endocytic regulation of wild-type EGFR, but that of mutated EGFR remains elusive. In the present study, we aim to investigate the endocytic degradation of a frequently occurred exon 19-deleted mutant in lung cancer.
The EGF-induced endocytic degradation of EGFR was examined in a panel of lung cancer cells using immunoblotting. The subcellular distribution of internalized EGFR was investigated using immunofluorescence and confocal microscopy. The effects of dynamin were assessed using its small molecule inhibitors, while the influence of RTN3 was tested using shRNA-mediated knockdown. Finally the ubiquitylation status of EGFR mutant was studied using immunoprecipitation under steady state and tyrosine kinase inhibitor-treated conditions.
EGF induced various rates of EGFR endocytic degradation in lung cancer cells. Interestingly, the exon 19 deletion mutant is constantly internalized and sorted to lysosome for degradation, and this process is independent of dynamin activity. EGF stimulation and HSP90 inhibition further enhance the endocytic degradation of the exon 19 deletion mutant, in a dynamin activity-dependent and -independent manner, respectively. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is mediated through receptor ubiquitylation.
The internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. The endocytosis of EGFR mutant occurs through both dynamin activity-dependent and -independent mechanisms. Our findings gain novel insights into the endocytic regulation of mutated EGFR and may have potential clinical implications.
KeywordsNSCLC EGFR Exon 19 deletion Ubiquitylation Endocytosis
Dulbecco’s Modified Eagle’s Medium
Epidermal growth factor receptor
Non-small cell lung cancer
Receptor tyrosine kinase
Standard error of the mean
The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases (RTK), plays fundamental roles during tissue development and adult homeostasis [1, 2]. The expression and activation of this RTK are tightly regulated both spatially and temporally to ensure proper propagation as well as timely termination of downstream signaling . Among the multiple feedback mechanisms acquired by cells to fine tune EGFR signaling, receptor endocytosis represents a multifaceted pathway that orchestrates signal transduction and receptor downregulation . Recent advances have provided compelling evidence that cells tightly control the output of EGFR activation by deploying different modes of receptor endocytosis [5, 6]. In response to various EGF concentrations, activated EGFR is internalized through clathrin-mediated endocytosis (CME) or non-clathrin endocytosis (NCE), with the former mode favors signal propagation while the latter one promotes receptor degradation to attenuate signaling. Such balance between receptor signaling and degradation is prudently maintained in normal cells but frequently decoupled in cancer cells overexpressing EGFR .
Accumulating evidence from cancer genomics has revealed that EGFR is recurrently mutated in multiple cancer types, including lung cancer, glioblastoma, head and neck squamous cell carcinoma [8, 9]. Activating mutations in EGFR renders this RTK constantly active, which in many cases behaves as a cancer driver that governs cancer growth [10, 11]. With regard to lung cancer, mutations in EGFR are more often detected from female, Asian, or non-smoker patients. In particular, the exon 19-deletion mutation of EGFR is recurrently observed in non-small cell lung cancer (NSCLC) patients, which accounts for nearly 50% of all EGFR abnormalities [10, 12, 13]. The exon 19 of EGFR encodes only 5 amino acids (from E746 to A750) that lie within the kinase domain of the receptor. The in-frame deletion of exon 19 confers enhanced kinase activity on mutated EGFR and thus leads to the overstimulation of downstream signaling cascades that promotes tumorigenesis.
Although the regulation of wild-type EGFR by endocytic pathways is becoming well established with recent advances and EGFR is deemed as a classic model substrate to study endocytosis, our understanding of the endocytic control of mutated EGFR remains controversial [14, 15, 16, 17, 18, 19]. Impaired ubiquitylation and degradation of kinase domain mutants of EGFR were observed in lung cancer cells expressing endogenous EGFR mutants and in other cell systems with exogenous overexpression [20, 21, 22, 23]. However, another study by Chen et al. compared a number of constitutively active EGFR mutants, and reported distinctive activation patterns, with the exon 19 deletion and L858R mutants showing increased ubiquitylation relative to wild-type EGFR upon EGF stimulation .
As exon 19 deletion is the most prevalent EGFR mutation (close to 50%) detected from non-small cell lung cancer (NSCLC) patients, the current study focused on this EGFR mutant and investigated its endocytosis . Interestingly, we observed that the exon 19-deleted EGFR was constantly endocytosed and sorted to lysosome for degradation in NSCLC cells. The internalization of this deletion mutant does not require dynamin activity but relies on the ubiquitylation of RTK under steady state conditions. However, upon EGF stimulation, the exon 19-deleted EGFR was internalized through a dynamin activity-dependent mechanism. The present study thus reveals the different modes of the endocytosis of the exon 19-deleted EGFR, providing unexpected evidence towards a better understanding of the endocytic regulation of mutant EGFR. Our findings will shed light on the development of novel therapeutic strategies against NSCLC containing activating EGFR mutations.
Antibodies and reagents
Mouse anti-EGFR (R1), mouse anti-RTN3, mouse anti-LAMP2, rabbit anti-EEA1, and rabbit anti-EGFR (1005) antibodies were purchased from Santa Cruz. Mouse anti-Ubiquitin (P4G7) antibody was obtained from Covance. Rabbit anti-phospho-MEK1/2 (Ser217/221) and anti-phospho-AKT (Ser473) antibodies were obtained from Cell Signaling Technology. Mouse anti-GAPDH and mouse anti-β-Actin antibodies were purchased from Proteintech (Wuhan, China). Mouse anti-α-Tubulin antibody was obtained from Sigma. Goat anti-rabbit and anti-mouse IRDye secondary antibodies (infrared-labeled) were purchased from LICOR. Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies were obtained from Invitrogen. Gefitinib, lapatinib, filipin, and dyngo-4a were purchased from Selleck. EGF was purchased from PeproTech (USA). Cycloheximide was obtained from MP Biologicals. 17-AAG was purchased from Cell Signaling Technology. Chloroquine, puromycin, and dynasore were obtained from Sigma.
HEK293T and lung cancer SK-MES-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA), while lung cancer cell lines A549, HCC827, H1975, H1650, H1299, and H226 were maintained in RPMI-1640 media (Gibco, USA). All cells were purchased from the American Type Culture Collection, and all media were supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (Thermo-Fisher Scientific). All cells were grown in a humidified cell culture incubator (Thermo) at 37 °C with 5% CO2.
Cultured cells were harvested, washed with ice-cold phosphate-buffered saline (PBS) for three times, and lysed with the RIPA buffer (10 mM Tris-HCl pH 7.5, 1% (w/v) Nonidet P-40, 150 mM NaCl, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate) as described previously . Phosphatase inhibitor cocktails (Roche) and protease inhibitor cocktails (Sigma) were added into lysis buffer immediately before use. Cell lysates were centrifuged at top speed to remove cell debris. The protein concentrations were determined using a BCA assay kit (Pierce). Each sample was mixed with the SDS-PAGE loading buffer and boiled for 5 min. Denatured protein samples were separated by SDS-PAGE, and transferred to nitrocellulose membranes. After blocking in 4% nonfat milk in PBS for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C. After incubation with secondary infrared-labeled antibodies (680 nm or 800 nm from LICOR) for 1 h at room temperature. Blots were washed 3 times with PBS and detected using a LICOR Odyssey system. Band intensity was analyzed with the Image Studio software (version 4.0) according to manufacturer’s instructions.
Immunofluorescence and confocal microscopy
Cells were seeded onto coverslips loaded in 6-well plate and incubated overnight. After treatment, cells were washed with PBS and fixed with 4% (w/v) paraformaldehyde (Sigma) for 15 min at room temperature. Then cells were permeabilized with 0.2% Triton X100, and blocked with 10% goat serum for 30 min. Cells were stained with primary antibodies and visualized with fluorescent secondary antibodies. Fluorescence images were captured using a fluorescence microscope (Olympus BX63, Japan). Confocal images were acquired using a Leica laser scanning confocal microscope (TCS SP5).
Immunoprecipitation assays were carried out as previously described . In brief, treated cells were lysed with the RIPA buffer and protein concentrations were measured. One mg of protein per condition was incubated at 4 °C with anti-EGFR (R1, Santa Cruz) antibody and protein G-agarose (Roche) for 4 h. Beads were then washed 3 times with the YP-IP buffer (10 mM Tris-HCl pH 7.5, 0.1% Nonidet P-40, 150 mM NaCl), before immunoprecipitated proteins were eluted with 1.5 X SDS-PAGE loading buffer. Samples were further analyzed by immunoblotting with anti-EGFR (1005, Santa Cruz) and anti-Ubiquitin (P4G7, Covance) antibodies.
Four RTN3 shRNA pGIPZ vectors (sh1 target sequence TGGATCTTCTAGATATGAA, sh2 target sequence AGGAGATGATGTTATTGAA, sh3 target sequence CCGAGATCAGACCAAGTCA, and sh4 target sequence CTCAGAAGCTTTCCATAAT) were purchased from Thermo Scientific. RTN3 shRNA lentiviruses were generated using the Thermo Scientific Open Biosystems TransLenti viral packaging system according to manufacturer’s instructions. HCC827 and H1650 cells were treated with the RTN3 shRNA lentiviruses for 24 h before treatment with 2 μg/ml of puromycin to remove uninfected cells. The efficiency of knockdown was validated by Western blottings.
All experiments were carried out for at least 3 times with biological repeats. Data were presented as the mean ± standard error of the mean (SEM). Statistical differences between groups were tested with two-tailed Student’s t-test using GraphPad Prism (version 5.01), with a p-value smaller than 0.05 considered as statistically significant.
EGF-induced endocytic degradation of EGFR
The exon 19-deleted EGFR is constantly internalized
Dynamin activity is dispensable for the constitutive endocytosis of the exon 19 deletion mutant
Upon EGF exposure, wild-type EGFR is stimulated for clathrin-mediated endocytosis and non-clathrin-mediated endocytosis, both of which require the activity of dynamin [5, 27]. In support of this notion, in A549 cells carrying the wild-type EGFR, 1.5 and 20 ng/ml of EGF induced differential endocytic degradation of EGFR, while the dynamin inhibitor dynasore efficiently suppressed EGFR downregulation (Fig. 3b). Therefore, we speculated that the internalization of the exon 19 deletion mutant also required dynamin activity. Unexpectedly, the levels of the exon 19 mutant remained stable in HCC827 and H1650 cells treated with dynasore or its more potent analogue, dyngo-4a (Fig. 3c and d). In accordance with these results from immunoblottings, the subcellular distribution of mutated EGFR was also unaffected in HCC827 and H1650 cells by both dynamin inhibitors as revealed by immunofluorescence studies (Fig. 3e). These observations indicate that, although the exon 19-deleted EGFR is endocytosed through the classic endosome-lysosome pathway, its constitutive internalization occurs through a dynamin activity-independent manner under steady state condition.
Stress-induced degradation of the exon 19-deleted EGFR
Additionally, HSP90 inhibition has been shown to effectively trigger the degradation of mutated EGFR . In line with this observation, the HSP90 inhibitor 17-AAG led to the effective degradation of the exon 19 deletion mutant in HCC827 cells (Fig. 4c). Immunofluorescence analyses revealed that EGFR was routed to lysosomes following 17-AAG treatment, suggesting that HSP90 inhibition promoted the endocytic degradation of EGFR (Fig. 4d). Nevertheless, dynamin inhibition by dynasore or dyngo-4a failed to preclude the 17-AAG-induced degradation of EGFR; and the cholesterol-interfering drug filipin that obstructs non-clathrin-mediated endocytosis did not mitigate the influence of HSP90 inhibition on EGFR levels neither (Fig. 4e). Therefore, these findings revealed the existence of multiple modes of internalization of EGFR under stress conditions.
Reticulon 3-independent degradation of EGFR
EGFR internalization is mediated by ubiquitylation
We wondered whether the kinase activity of the mutated EGFR was implicated in its endocytic degradation. Therefore, we treated HCC827 cells with two small molecule inhibitors of EGFR, lapatinib and gefitinib. Interestingly, when lysosomal function was blocked with chloroquine, the accumulated EGFR signals in cells treated with gefitinib and control reagents were significantly stronger than that in lapatinib-treated cells after 6 h of incubation (Fig. 6b). Furthermore, with prolonged treatment (12 and 24 h), lapatinib resulted in the apparent accumulation of EGFR on the plasma membrane of HCC827 cells (Fig. 6c). These immunofluorescence data indicated that lapatinib but not gefitinib reduced the endocytosis of the exon 19 deletion mutant. To investigate the different effects of lapatinib and gefitinib on EGFR endocytosis, we examined their influence on the ubiquitylation of EGFR. As described in Fig. 6d, lapatinib treatment caused a remarkable reduction in the ubiquitin signal on immunoprecipitated EGFR compared to those from control and gefitinib-treated cells. It thus seems that lapatinib disrupts the endocytosis of EGFR by decreasing its ubiquitylation.
Since the endogenous levels of wild-type and exon 19-deleted EGFR from A549 and HCC827 differed significantly, we ectopically expressed wild-type and the exon 19-deleted EGFR proteins to examine their ubiquitylation. As illustrated in Fig. 6e, HEK293T cells transiently transfected with wild-type and mutant EGFR-expressing constructs contained similar amounts of EGFR proteins, while the ubiquitin signal detected from immunoprecipitated mutant EGFR was significantly enhanced compared to that from wild-type protein. Furthermore, given that NSCLC H1299 cells expressed relatively low levels of EGFR, we established stable EGFR-expressing H1299 cell lines. In accordance with data from transient overexpression, stronger ubiquitin signal was detected on EGFR mutant immunoprecipitated from H1299 stable cells (Fig. 6f).
Recent years have witnessed the continuously increased mortality from lung cancer worldwide, which causes over a million of deaths annually . In the United States, mortality from lung cancer accounts for more than 25% of cancer-related deaths [30, 31]. Therefore, the demand for effective therapies against lung cancer is huge, and many efforts have been made to develop novel therapeutic strategies. Accumulating evidence from sequencing analyses has revealed the high frequency of EGFR mutations occurring in lung cancer, among which the exon 19 deletion appears to be the most prevalent one. Compared to wild-type EGFR, the exon 19 deletion renders mutated EGFR constantly active, and therefore overactivates a wide array of signaling pathways. Nonetheless, this EGFR mutant exhibited sensitivity to multiple tyrosine kinase inhibitors. EGFR-targeted therapy represents one of the most successful approaches in the treatment of lung cancer, which has been approved by the FDA as first-line therapy that effectively prolonged patient survival [32, 33]. However, cure is seldom achieved, and relapsed disease acquires drug resistance to threaten patient lives. The most prevalent mechanism of developed resistance to EGFR inhibitors, such as gefitinib and erlotinib, is attributed to secondary mutations occurring in EGFR [34, 35]. Considering the critical roles of EGFR in lung cancer tumorigenesis and drug resistance, the thorough investigation of its features in lung cancer is prerequisite for the development of novel therapeutic strategies in the treatment of lung cancer.
In the present study, we carefully examined the EGF-induced degradation of endogenous EGFR proteins in a panel of non-small cell lung cancer cell lines. Through comparing the degradation rate of wild-type and mutated EGFR in cells belong to different histologic types, we drew the conclusion that being wild-type or mutated form of EGFR alone could not determine the degradation rate upon EGF treatment; rather, the EGF-stimulated downregulation of EGFR is controlled by multiple factors that differ within various cellular contexts [3, 15, 16]. With a closer examination of the exon 19 deletion mutant of EGFR in HCC827 and H1650 cells, our observations reveal that this mutant undergoes endocytic degradation constantly. Under steady state conditions, the exon 19-deleted EGFR forms a number of intracellular punctae that colocalize with endocytic compartments; while the amounts of intracellular EGFR punctae reversely correlate with the efficiency of EGF-induced EGFR downregulation. In HCC827 where a large amount of EGFR punctae exist, the exon 19 deletion mutant appeared to be the least sensitive to EGF-induced endocytic degradation. A possible explanation to this phenomenon is that the mutated EGFR already occupies the endocytic routes to lysosome, thus causing a ‘traffic jam’ that limits the capacity of further transport; while the situation is even more severe in HCC827 cells due to a large surplus of EGFR. These features collectively led to the inefficient EGF-induced endocytic degradation of EGFR in HCC827 cells.
In our attempt to investigate the molecular determinants of mutant EGFR endocytosis, we observed dynamin activity-dependent and -independent mechanisms. Since the EGF-induced endocytosis of wild-type EGFR has been well-documented, a simple assumption is that mutated EGFR follows similar mechanisms. Initially, we speculated that the endocytosis of the exon 19 deletion mutant might primarily adopt the non-clathrin mediated pathway, resembling that of wild-type EGFR under high EGF conditions, considering that the mutation rendered EGFR constantly active. Nevertheless, under steady state conditions, the internalization of the exon 19 mutant is independent of RTN3 and dynamin activity that are involved in the non-clathrin mediated endocytosis . The dynamin activity is also dispensable for the enhanced endocytic degradation of EGFR elicited by HSP90 inhibition. Dynamin is nonetheless implicated in the EGF-stimulated endocytosis of the mutated EGFR. These findings reveal that both dynamin activity-dependent and -independent mechanisms function in the endocytic regulation of the exon 19 deletion mutant.
Further investigation confirmed the evident ubiquitylation of the exon 19 deletion mutant under normal conditions, which was less sensitive to EGF stimulation but could account for the constant endocytosis of the receptor, considering the dominant functions of ubiquitylation in receptor endocytosis. Through manipulating the ubiquitylation levels of the mutated EGFR with small molecule tyrosine kinase inhibitors (lapatinib and gefitinib), we observed accumulated cell surface distribution of the receptor in lapatinib-treated cells, indicative of impaired endocytosis with correlated reduced ubiquitylation of EGFR. Although lapatinib and gefitinib are both quinazoline derivatives, they show different binding preference towards EGFR . Lapatinib binds to the inactive conformation of EGFR and prevents its dimerization; while gefitinib binds to its active kinase domain and promoted the formation of dimers, which explain their different effects on receptor ubiquitylation . These findings emphasize the pivotal roles of ubiquitylation in receptor endocytosis, which governs receptor endocytosis with different pathways of choice.
To summarize, our observations revealed that the exon 19 deletion mutant of EGFR is constantly internalized and routed through endosome to lysosome for degradation in lung cancer cells. Interestingly, the endocytosis of EGFR mutant involved both dynamin activity-dependent and -independent mechanisms, and differed from that of wild-type receptor. Importantly, receptor ubiquitylation appeared to play a pivotal role in the endocytic degradation of EGFR. Therefore, further investigation is warranted to tease out the precise mechanisms of the internalization of mutated EGFR, which will assist the development of novel EGFR-targeted therapeutic strategy in the treatment of lung cancer.
The authors are grateful to the National Natural Science Foundation of China for financial supports.
This work was supported by the National Natural Science Foundation of China (No. 81473452 to LZ and No. 81301901 to HL).
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Study design: LZ and HL; Data Collection: TW, JZ, SW, DW, YG, YZ, LX, YW, YW, FL, and XL; Data analysis: TW, JZ, SW, XS, SL, YZ, and YW; Manuscript preparation: HL. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.