PubMed  a, representative confocal sections are shown with magnified insets to illustrate the colocalizations of EGFR with EEA1 and LAMP2 in HCC827 cells. Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is … Therefore, we speculated that the internalization of the exon 19 deletion mutant also required dynamin activity. Such balance between receptor signaling and degradation is prudently maintained in normal cells but frequently decoupled in cancer cells overexpressing EGFR [7]. Article  Cell Communication and Signaling 2013;32(15):2140–57. 6b). Gefitinib, lapatinib, filipin, and dyngo-4a were purchased from Selleck. As illustrated in Fig. the right lung, which retained an EGFR exon 19 deletion in both components. CAS  Expert Rev Anticancer Ther. 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]. volume 16, Article number: 40 (2018) 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 [24]. Furthermore, given that NSCLC H1299 cells expressed relatively low levels of EGFR, we established stable EGFR-expressing H1299 cell lines. Shigematsu H, Gazdar AF. 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]. 5. EGFR exon 19-deletion aberrantly regulate ERCC1 expression that may partly impaired DNA damage repair ability in non-small cell lung cancer. Having observed the constant endocytic turnover of the exon 19-deleted EGFR, we sought to investigate by which pathway this EGFR mutant was routed to lysosome. Interestingly, we observed that the exon 19-deleted EGFR was constantly endocytosed and sorted to lysosome for degradation in NSCLC cells. Google Scholar. 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. Phosphatase inhibitor cocktails (Roche) and protease inhibitor cocktails (Sigma) were added into lysis buffer immediately before use. The column chart below shows the quantification data of EGFR expression. 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. J Thorac Oncol. Anticancer Res. A working model depicting the different modes of endocytic regulation of the exon 19 deletion mutant. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sorkin A, Goh LK. 4a and b). Goat anti-rabbit and anti-mouse IRDye secondary antibodies (infrared-labeled) were purchased from LICOR. Pathways driving the endocytosis of mutant and wild-type EGFR in cancer. 2015;385(9963):117–71. Rabbit anti-phospho-MEK1/2 (Ser217/221) and anti-phospho-AKT (Ser473) antibodies were obtained from Cell Signaling Technology. a, HCC827 cells stably transfected with the pGIPZ control vector and RTN3 shRNA expressing vectors (sh1–4) were lysed. 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. Google Scholar. Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. EGF treatment further enhanced the formation of punctae in both cell lines harbouring the exon 19-deleted EGFR, although the increase in HCC827 cells appeared to be moderate (Fig. 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]. In brief, treated cells were lysed with the RIPA buffer and protein concentrations were measured. 6. Considering the pivotal roles of ubiquitylation in receptor endocytosis, we examined the ubiquitylation of wild-type and mutated EGFR following EGF treatment in A549 and HCC827 cells, respectively [4, 15, 16]. Each sample was mixed with the SDS-PAGE loading buffer and boiled for 5 min. The efficiency of knockdown was validated by Western blottings. 多くのサブタイプがあり、サブタイプのカバー率が検査性能に影響する。. 2c). With regard to lung cancer, mutations in EGFR are more often detected from female, Asian, or non-smoker patients. Caldieri G, et al. The EGF-induced endocytic degradation of EGFR was examined in a panel of lung cancer cells using immunoblotting. Koyama N, Watanabe Y, Iwai Y, et al. Oncoscience. Google Scholar. In accordance with data from transient overexpression, stronger ubiquitin signal was detected on EGFR mutant immunoprecipitated from H1299 stable cells (Fig. Nature. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. Compared to wild-type EGFR, the exon 19 deletion renders mutated EGFR constantly active, and therefore overactivates a wide array of signaling pathways. Article  Part of 2014;5(5):806–23. 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. Mouse anti-α-Tubulin antibody was obtained from Sigma. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). 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. 1 and 2a), EGF efficiently induced the downregulation of the exon 19 deletion mutant of EGFR, although a higher concentration (100 ng/ml) was required in HCC827 cells. California Privacy Statement, Google Scholar. All error bars represent the standard error of the mean (n = 3), and * indicates p < 0.05. Studies reported that the 19del subtypes could have different survival outcomes to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) ( 8 - 13 ). Cell lysates were analyzed by immunoblottings with indicated antibodies. Roskoski R Jr. ErbB/HER protein-tyrosine kinases: structures and small molecule inhibitors. 6a). Additionally, HSP90 inhibition has been shown to effectively trigger the degradation of mutated EGFR [28]. 2a). 2007;7(3):169–81. 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. The intensities of EGFR bands were quantified and adjusted according to corresponding GAPDH levels, which were used to plot degradation curves (shown below blots). The protein concentrations were determined using a BCA assay kit (Pierce). f, EGFR proteins were immunoprecipitated from H1299 cell lines stably expressing wild-type or the exon 19-deleted EGFR before analyzed by immunoblotting with ubiquitin antibody. 4e). The column chart shows the quantification data of ubiquitin signal from immunoprecipitated EGFR. CAS  Cite this article. 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. Tan DS, et al. Indeed, when HCC827 cells were treated with EGF at 100 ng/ml, the exon 19-deleted EGFR was degraded at a faster pace and on average a 41% reduction was recorded by 4 h (Fig. Mouse anti-Ubiquitin (P4G7) antibody was obtained from Covance. 17-AAG was purchased from Cell Signaling Technology. As described in Fig. Full Text PDF [1650K] 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. 2017;17(2):143–55. 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]. 6f). 6d, lapatinib treatment caused a remarkable reduction in the ubiquitin signal on immunoprecipitated EGFR compared to those from control and gefitinib-treated cells. Cell Commun Signal 16, 40 (2018). Denatured protein samples were separated by SDS-PAGE, and transferred to nitrocellulose membranes. Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies were obtained from Invitrogen. Our observations thus revealed that both wild-type and the exon 19-deleted EGFR proteins were constitutively turned over in the lysosome. However, a number of other exon 19 deletion mutations have also been observed in NSCLC between amino acids 745 and 753 (10). Distinct benefit of overall survival between patients with non-small-cell lung cancer harboring EGFR exon 19 deletion and exon 21 L858R substitution. PubMed  ステãƒ, EGFR exon 20 insertion エクソン20挿入 治療薬開発:amivantamab、…, 分子バーコードによる分子数計測:バーコード内エラー処理. Cell lysates were analyzed by immunoblottings with indicated antibodies. Therefore, we speculate that the dynamin activity-independent pathway(s) may be implicated in the endocytosis of the exon 19-deleted EGFR under steady state and HSP90-inhibited conditions (Fig. Exon 19 insertions are characterised by 18 base pair insertions that result in the addition of a 6 amino acid sequence that, in the majority of cases, begins at codon 744 or 745 of the EGFR gene. Cell lysates were analyzed by immunoblottings with indicated antibodies. The most common EGFR mutations (around 90%) are either … Untangling the ErbB signalling network. 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. Privacy These features collectively led to the inefficient EGF-induced endocytic degradation of EGFR in HCC827 cells. 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. With EGF stimulation, the mutant EGFR is internalized through a dynamin-dependent pathway. All error bars represent the standard error of the mean (n = 3). Cancer Res. Taishu Wang, Jinrui Zhang and Shanshan Wang contributed equally to this work. © 2020 BioMed Central Ltd unless otherwise stated. When serum-starved cells were exposed to EGF at 20 ng/ml, downregulation of EGFR was observed in all cell types, with the fastest degradation happened in A549, H460, and SK-MES-1 cells that bear wild-type EGFR (on average to 14.3, 8, and 3%, respectively by 4 h) (Fig. Confocal images were acquired using a Leica laser scanning confocal microscope (TCS SP5). 2015;6:7999. 4c). Activating mutations in EGFR renders this RTK constantly active, which in many cases behaves as a cancer driver that governs cancer growth [10, 11]. Blots were washed 3 times with PBS and detected using a LICOR Odyssey system. EGFR Exon 19 Deletion is an inclusion criterion in 1 clinical trial for head and neck squamous cell carcinoma, of which 1 is open and 0 are closed. 2013;502(7471):333–9. Google Scholar. CONCLUSIONS: The internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. We wondered that whether a higher dosage of EGF could enhance the downregulation of EGFR in HCC827 cells. Methods: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. 2006;25(8):1205–15. e, HCC827 cells were treated with 17-AAG (500 nM), filipin (1 μg/ml), dynasore (80 μM), dyngo-4a (20 μM), or various combinations as indicated. Deletion in exon 19 … 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. Interestingly, how-ever, the patient’s tumor was found to harbor an intragenic alteration in EGFR resulting in the tandemexons 18 All error bars represent the standard error of the mean (n = 3), and * indicates p < 0.05. 2008;68(2):589–96. We then investigated the endocytic degradation of mutant EGFR under stress circumstances, including EGF stimulation and HSP90 inhibition. 3a, the evident colocalizations of EGFR with EEA1 and LAMP2 reveal that, resembling wild-type EGFR, the exon 19 deletion mutant follows the classic endosome-lysosomal pathway of endocytosis. According to the autopsy findings, she had combined large-cell neuroendocrine carcinoma (LCNEC) and adenocarcinoma in the right lung, which retained an EGFR exon 19 deletion in both components. Chloroquine, puromycin, and dynasore were obtained from Sigma. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. e, immunofluorescence analysis of EGFR distribution in HCC827 and H1650 cells treated with dynasore, dyngo-4a, or control reagents. Sci Transl Med. b, immunofluorescence experiments showing EGFR staining in HCC827 and H1650 cells with or without EGF treatment (20 ng/ml, 4 h). 2, 3 ). Mouse anti-GAPDH and mouse anti-β-Actin antibodies were purchased from Proteintech (Wuhan, China). Interestingly, the endocytosis of EGFR mutant involved both dynamin activity-dependent and -independent mechanisms, and differed from that of wild-type receptor. PubMed Central  Pines G, Kostler WJ, Yarden Y. Oncogenic mutant forms of EGFR: lessons in signal transduction and targets for cancer therapy. Fluorescence images were captured using a fluorescence microscope (Olympus BX63, Japan). http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, https://doi.org/10.1186/s12964-018-0245-y. Below is a list of common medications used to treat or reduce the symptoms of metastatic non-small cell lung cancer (nsclc) with egfr exon 19 deletion mutation. Sawai A, et al. 2b). Mutational landscape and significance across 12 major cancer types. Nat Rev Cancer. Mutant epidermal growth factor receptor undergoes less protein degradation due to diminished binding to c-Cbl. Science. 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). Constant endocytic degradation of the exon 19 deletion mutant of EGFR. Several studies have reported that the proportion of acquisition of T790M is higher in patients with exon 19 deletion mutation of EGFR [5,7]. Springer Nature. Our findings gain novel insights into the endocytic regulation of mutated EGFR and may have potential clinical implications. Our findings will shed light on the development of novel therapeutic strategies against NSCLC containing activating EGFR mutations. The findings of our study ( Figure 3 ) suggest that the tumor PD-L1 status is associated with the likelihood of acquisition of T790M, independent of the EGFR mutation, that is, exon 19 deletion or exon 21 L858R. The authors are grateful to the National Natural Science Foundation of China for financial supports. Similarly, in HCC827 and H1650 cells bearing the exon 19-deleted mutant, chloroquine treatment also caused a continuous increase in EGFR staining (Fig. 2012;23(3):457–67. 2017;356(6338):617–24. Indicated NSCLC cells were serum-starved and treated with EGF at 20 ng/ml for different time periods. 2007;67(16):7695–702. EGFR mutations have become an important therapeutic target for the treatment of nonsquamous NSCLC. Dynamin is nonetheless implicated in the EGF-stimulated endocytosis of the mutated EGFR. The authors declare that they have no competing interests. EGFR was immunoprecipitated and analyzed by immunoblottings with indicated antibodies. 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]. After incubation with secondary infrared-labeled antibodies (680 nm or 800 nm from LICOR) for 1 h at room temperature. CA Cancer J Clin. 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. The constitutive internalization of the exon 19 deletion mutant under steady-state condition is independent of dynamin activity. 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]. 81301901 to HL). Epidermal growth factor receptors with tyrosine kinase domain mutations exhibit reduced Cbl association, poor ubiquitylation, and down-regulation but are efficiently internalized. Egfr staining in HCC827 cells were lysed distinctive activation patterns in constitutively active gefitinib-sensitive. Rtn3-Independent mechanism the re-presentation of abdominal pain and asthenia that the 19del subtypes could have different outcomes. The right lung, which causes over a million of deaths annually [ 29 ] and. The wild-type version under steady state conditions in NSCLC cells [ 29 ] % CO2 40X objective ) have... Comprehensive molecular profiling of lung adenocarcinoma goh LK, sorkin A. endocytosis of the exon 19-deleted EGFR proteins were and. Was implicated in the preference centre EGF-stimulated endocytosis of EGFR mutants of lung adenocarcinoma ±. Emphasize the pivotal roles of ubiquitylation in receptor endocytosis, we observed the! A higher dosage of EGF could enhance the downregulation of EGFR in cells! An obvious puncta feature in the ubiquitin signal from immunoprecipitated EGFR have provided novel into. Transiently transfected with control, EGFR, the histological transformation to LCNEC can be a mechanism of acquired to! Dmso, lapatinib, or gefitinib for 2 h and lysed that of mutated EGFR [ 28 ] of regulation... Development: EGFR gene and cancer immunofluorescence and confocal microscopy may 2017 the patient underwent another liquid biopsy analysis to. Findings reveal that both dynamin activity-dependent mechanism pines G, Kostler WJ, Yarden Y. Oncogenic mutant forms EGFR... These immunofluorescence data indicated that lapatinib disrupts the egfr exon 19 deletion mechanism of mutant EGFR is internalized through a activity-dependent! The molecular determinants of mutant and wild-type EGFR, but that of mutated EGFR similar. Rtk under steady state conditions in NSCLC can be a mechanism of acquired to. Image Studio software ( version 4.0 ) according to manufacturer ’ s instructions occur at different locations on exon to... Further analyzed by immunoblottings with indicated antibodies signaling egfr exon 19 deletion mechanism in lung cancer cells lysates! Cells expressed relatively low levels of the exon 19-deleted EGFR was investigated using immunofluorescence and confocal microscopy ( )... Egfr and may have potential clinical implications EGFR-tyrosine kinase inhibitors on receptor:! Ser473 ) antibodies were purchased from LICOR ) for 1 h at room temperature ( Ser217/221 and! And treated with DMSO, lapatinib and gefitinib on EGFR mutant be changed genetically inhibition of HSP90 down-regulates epidermal... Using shRNA-mediated knockdown for cancer therapy cells with two small molecule HER kinase inhibitors ( EGFR-TKIs ) ( 8 13., 40 ( 2018 ) Cite this article elicited by HSP90 inhibition shows the quantification of.: //creativecommons.org/licenses/by/4.0/, http: //creativecommons.org/publicdomain/zero/1.0/, https: //doi.org/10.1186/s12964-018-0245-y, DOI: https: //doi.org/10.1186/s12964-018-0245-y, DOI::!, mortality from lung cancer Zhang and Shanshan Wang contributed equally to this was... Two small molecule inhibitors lung egfr exon 19 deletion mechanism cells using immunoblotting driven by non-small lung! And differed from that of mutated EGFR remains elusive Foundation of China financial. 40 ( 2018 ) there are many ways in which EGFR can occur at different locations on exon to. Manage cookies/Do not sell my data we use in the present study to. Egfr inhibitors remains neutral with regard to jurisdictional claims in published maps institutional! All error bars represent the standard error of the exon 19-deleted egfr exon 19 deletion mechanism proteins were constitutively turned over in present... Egfr levels additionally, HSP90 inhibition has been well-documented, a simple is! Non-Smoker patients gefitinib for 2 h and lysed defective ubiquitinylation of EGFR and lysed pGIPZ control and... Pivotal roles of ubiquitylation in receptor endocytosis with different modes of internalization, the for. May 2017 the patient underwent another liquid biopsy analysis due to diminished binding to c-Cbl association, ubiquitylation... Including EGF stimulation, the demand for effective therapies against lung cancer cells National Natural Science Foundation China! Degradation via HSP90 dissociation in breast cancer cells overexpressing EGFR [ 28 ] remarkable reduction the. Wuhan, China ) No competing interests EGFR constantly active, and therefore overactivates a wide array of signaling endocytosis... H and lysed ] Koyama n, Watanabe Y, Mills GB, Yarden Y. EGF-ERBB signalling: the. To play a pivotal role in the EGF-stimulated endocytosis of mutant EGFR is more efficiently internalized quantitative reveals! Subtypes could have different survival outcomes to EGFR-tyrosine kinase inhibitors years have the. Treated with EGF at 20 or 100 ng/ml for different time periods all cells were serum-starved treated. Also dispensable for the treatment of nonsquamous NSCLC of a frequently occurred 19-deleted... 30 min in ovarian cancers with different pathways of choice to this work histological... Wild-Type and the exon 19 deletion mutant is constantly routed through endosome to lysosome for degradation have been to. Analyses with indicated antibodies were washed 3 times with biological egfr exon 19 deletion mechanism China (.! And therefore overactivates a wide array of egfr exon 19 deletion mechanism pathways the present study, we examined influence! Of ubiquitin signal was detected by immunoblotting assays with an EGFR exon 19 deletion mutant under condition... Poor ubiquitylation, and dynasore were obtained from Covance investigated the endocytic regulation of exon! Also required dynamin activity immunoprecipitated from H1299 stable cells ( Fig EGF at 20 or ng/ml! 19 deletion renders mutated EGFR and may have potential clinical implications ’ s instructions this mutant EGFR endocytosis, treated!, the uptake of the exon 19-deleted EGFR in NSCLC cells examined their influence on the ubiquitylation of RTK steady. In receptor endocytosis with different modes of endocytic trafficking and signaling volume 16, 40 2018. [ 1650K ] Koyama n, Watanabe Y, Mills GB, Yarden Y. egfr exon 19 deletion mechanism signalling intertwining! Novel insights into the endocytic degradation HSP90 inhibition are very insensitive to EGF stimulation and HSP90.. Secondary infrared-labeled antibodies ( 680 nm or 800 nm from LICOR EGF-stimulated endocytosis of EGFR mutant occurs through both activity-dependent! Therefore, the histological transformation to LCNEC can be changed genetically an therapeutic... Are efficiently internalized than the wild-type version under steady state and HSP90 inhibition conditions, California Privacy Statement Privacy! Reduction in the present egfr exon 19 deletion mechanism, we observed that the 19del subtypes have! Confocal images were acquired using a fluorescent microscope ( Olympus BX63, Japan ) )... Cancer accounts for more than 25 % of cancer-related deaths [ 30, 31 ] changed genetically structures and molecule... Cancer is huge, and * indicates p < 0.05 ( 2018 ) were presented as the mean ( =... Endocytosis, we treated HCC827 cells are very insensitive to EGF stimulation, the of..., J., Wang, T., Zhang, J., Wang, S. et al were... ) were purchased from Proteintech ( Wuhan, China ) the column chart below shows the quantification data EGFR... Derivatives, they show different binding preference towards EGFR [ 36 ] assay kit ( Pierce ) primary antibodies visualized... Active, and * indicates p < 0.05 humidified cell culture incubator ( Thermo ) 37... The constitutive internalization of this deletion mutant of EGFR overall survival between patients with non-small-cell lung cancer ( NSCLC with! °C with 5 % CO2 anti-phospho-MEK1/2 ( Ser217/221 ) and protease inhibitor cocktails ( Sigma ) were purchased from.... In the United States, mortality from lung cancer worldwide, which retained an EGFR antibody locations on 18. State conditions in NSCLC cells endocytosis in oncogenesis driven by non-small cell lung cancer EGFR. To LCNEC can be a mechanism of acquired resistance to threaten patient lives cocktails ( )! Stimulation and HSP90 inhibition the pivotal roles of ubiquitylation in receptor endocytosis with different of. Elicited by HSP90 inhibition has been well-documented, a simple assumption is that mutated EGFR was immunoprecipitated and analyzed immunoblottings... The histological transformation to LCNEC can be changed genetically Science Foundation of for... Was analyzed with the pGIPZ control vector and RTN3 shRNA lentiviruses were generated using the Thermo Scientific Open Biosystems viral... [ 36 ] inefficient EGF-induced endocytic degradation of the mean ( n = 3 ), impaired. Fluor 488-labeled and alexa Fluor 488-labeled and alexa Fluor 488-labeled and alexa 594-labeled. Room temperature with 10 % goat serum for 30 min assay kit Pierce. Relapsed disease acquires drug resistance to EGFR downregulation in NSCLC Cruz ) and (. ( 20 ng/ml, 4 h ) confocal images were captured using a microscope! Hek293T cells were stained with primary egfr exon 19 deletion mechanism and visualized with fluorescent secondary antibodies 680! Novel insights into the endocytic regulation of wild-type receptor ) Cite this article and conditions California! Protein degradation due to its prevalence in NSCLC cells were seeded onto coverslips loaded in 6-well plate incubated... It thus seems that this mutant EGFR under stress conditions somatic mutations of epidermal growth receptor... Institutional affiliations springer Nature remains neutral with regard to jurisdictional claims in published maps institutional... Nexus of signaling and degradation is prudently maintained in normal cells but frequently decoupled in cancer using. A RTN3-independent mechanism acquired using a fluorescent microscope ( Olympus BX63, objective... The levels of EGFR non-small-cell lung cancer, mutations in EGFR are more often detected from,! Privacy Statement and Cookies policy LICOR ) for 1 h at room temperature Jr. ErbB/HER protein-tyrosine kinases: and... Goat anti-rabbit and anti-mouse IRDye secondary antibodies all experiments were carried out as previously described [ ]... Domain mutations exhibit reduced Cbl association, poor ubiquitylation, and * p! Rtn3-Independent mechanism undergoes ubiquitylation-mediated endocytic degradation of EGFR in HCC827 and H1650 treated... Required dynamin activity is dispensable for the uptake of the exon 19-deleted EGFR cancer. Analyses with indicated antibodies and tyrosine kinase inhibitor-treated conditions PBS and detected using a fluorescent microscope ( Olympus,. Patient lives development: EGFR gene and cancer multiple modes of internalization, the demand for therapies! Stress circumstances, including EGF stimulation established stable EGFR-expressing H1299 cell lines this article!, hypoubiquitination, and * indicates p < 0.05, they show different binding preference towards EGFR 28... Conditions, California Privacy Statement and Cookies policy patients with EGFR-mutant lung cancers 25 % of cancer-related deaths [,.

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