2013;502(7471):333–9. EGFR exon 19 deletion (19del) was about 44% in EGFR mutations, and the most frequent subtype was delE756_A750, followed by delL747_P753insS, delL747_A750insP or delL747_T751 (6,7). Int J Cancer. Shigematsu H, Gazdar AF. Lijuan Zou or Han Liu. Taishu Wang, Jinrui Zhang and Shanshan Wang contributed equally to this work. CA Cancer J Clin. 2012;23(3):457–67. 2016;11(7):946–63. Association with HSP90 inhibits Cbl-mediated down-regulation of mutant epidermal growth factor receptors. Benjamin P. Levy, MD, assistant professor at the Icahn School of Medicine, medical director of the Thoracic Oncology Program, Mount Sinai Health Systems, and associate director of the Cancer Clinical Trials Office at Mount Sinai Hospital in New York spoke with Targeted Oncology about the treating patients with exon 19 deletions. Goat anti-rabbit and anti-mouse IRDye secondary antibodies (infrared-labeled) were purchased from LICOR. 2006;7(7):505–16. 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. Scale bar = 10 μm. Nature. The intensities of EGFR bands were quantified and adjusted according to corresponding GAPDH levels, which were used to plot degradation curves (shown below blots). Constant endocytic degradation of the exon 19 deletion mutant of EGFR. and C. Causes of death, Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Google Scholar. 7). Dev Cell. 81301901 to HL). 2014;87:42–59. 2007;67(16):7695–702. b, serum-starved A549 cells were treated with EGF at indicated concentrations for 6 h with or without dynasore addition to block the dynamin activity. Pines G, Kostler WJ, Yarden Y. Oncogenic mutant forms of EGFR: lessons in signal transduction and targets for cancer therapy. 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. Both dynamin activity-dependent and -independent mechanisms are implicated in the endocytic regulation of the exon 19-deleted EGFR in NSCLC cells. The column chart below shows the quantification data of EGFR. Oncogene. EGFR-activating mutations, such as an exon 19 deletion (Del19) and L858R substitution, have been reported in 10% to 50% of patients with non–small cell lung cancer (NSCLC; refs. Samples were further analyzed by immunoblotting with anti-EGFR (1005, Santa Cruz) and anti-Ubiquitin (P4G7, Covance) antibodies. Cancer Res. Since the EGF-induced endocytosis of wild-type EGFR has been well-documented, a simple assumption is that mutated EGFR follows similar mechanisms. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). The International Association for the Study of Lung Cancer consensus statement on optimizing management of EGFR mutation-positive non-small cell lung Cancer: status in 2016. In the United States, mortality from lung cancer accounts for more than 25% of cancer-related deaths [30, 31]. 3e). Capuani F, et al. Yu HA, et al. Data were presented as the mean ± standard error of the mean (SEM). J Thorac Oncol. Sci Transl Med. The Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China, Taishu Wang, Jinrui Zhang, Shanshan Wang, Duchuang Wang, Yurou Gao, Yang Zhang, Lu Xu, Yue Wu, Yueguang Wu, Fang Liu, Xiuxiu Liu, Shuyan Liu, Yingqiu Zhang, Yang Wang, Lijuan Zou & Han Liu, Department of Respiratory Medicine, First Affiliated Hospital, Dalian Medical University, Dalian, China, Department of Radiation Oncology, Second Affiliated Hospital, Dalian Medical University, Dalian, China, Cancer Biotherapy & Translational Medicine Center of Liaoning Province, Dalian Medical University, Dalian, China, You can also search for this author in These findings reveal that both dynamin activity-dependent and -independent mechanisms function in the endocytic regulation of the exon 19 deletion mutant. Untangling the ErbB signalling network. EMBO J. Confocal images were acquired using a Leica laser scanning confocal microscope (TCS SP5). 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. The authors declare that they have no competing interests. The USP7 inhibitor P5091 induces cell death in ovarian cancers with different P53 status. A working model depicting the different modes of endocytic regulation of the exon 19 deletion mutant. 2c). 2015;6:7999. EGFR was immunoprecipitated and analyzed by immunoblottings with indicated antibodies. Nat Rev Cancer. Yarden Y, Sliwkowski MX. 2010;584(12):2699–706. 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 [3]. 2006;66(14):6990–7. 6b). Cancer Lett. d, immunofluorescence analysis of HCC827 treated with 17-AAG for indicated times. Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. The efficiency of knockdown was validated by Western blottings. including EGFR mutation, exon-19 deletion, and exon-21 L858R mutation, four logistic regression models were established for each task. 2017;356(6338):617–24. The column chart shows the quantification data of ubiquitin signal from immunoprecipitated EGFR. Dev Cell. Siegel RL, Miller KD, Jemal A. 2, 3 ). 2b). Therefore, the histological transformation to LCNEC can be a mechanism of acquired EGFR-TKI resistance. 4c). All data generated or analysed during this study are included in this published article. Large-scale sequencing of lung adenocarcinomas showed EGFR β3-αC deletions (also referred to as exon 19 deletions) in ∼20% of tumors, similar in frequency to the L858R mutation Ding et al., 2008 Ding L. The authors are grateful to the National Natural Science Foundation of China for financial supports. c and d, A549, HCC827, and H1650 cells were treated with 100 μM of chloroquine to block lysosomal degradation for indicated times before processed for immunofluorescence analysis to examine EGFR staining using a fluorescent microscope (Olympus BX63, 40X objective). Sanchez-Martin M, Pandiella A. Interestingly, the exon 19 deletion mutant of EGFR in HCC827 cells was very resistant to EGF-induced downregulation, since 4 h of EGF treatment only led to a 21% reduction of EGFR as quantified by immunoblotting densitometry. 2017;43(5):1755–66. 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. We then investigated the endocytic degradation of mutant EGFR under stress circumstances, including EGF stimulation and HSP90 inhibition. Cell lysates were subjected to immunoblotting analyses with indicated antibodies. 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 [25]. Yang S, et al. 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 ]. Cancer Res. We first examined the EGF-induced endocytic degradation of EGFR in a panel of NSCLC cell lines, which includes all major histologic subtypes: adenocarcinoma (A549, H1299, HCC827, H1650, and H1975), large cell carcinoma (H460), and squamous cell carcinoma (H226 and SK-MES-1). Recent progression on EGFR endocytosis has revealed a Reticulon 3 (RTN3)-dependent mechanism of non-clathrin-mediated endocytosis that favors EGFR degradation at high EGF concentrations [27]. Article  In accordance with data from transient overexpression, stronger ubiquitin signal was detected on EGFR mutant immunoprecipitated from H1299 stable cells (Fig. Exp Cell Res. Neratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells. We carried out confocal microscopy experiments to examine the colocalizations of the mutant EGFR with the early endosomal marker EEA1 and the late endosomal/lysosomal marker LAMP2 under steady state conditions. Nat Rev Cancer. Therefore, we speculated that the internalization of the exon 19 deletion mutant also required dynamin activity. a, representative confocal sections are shown with magnified insets to illustrate the colocalizations of EGFR with EEA1 and LAMP2 in HCC827 cells. 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. Clague MJ, Liu H, Urbe S. Governance of endocytic trafficking and signaling by reversible ubiquitylation. Sequist LV, et al. c, HCC827 was treated with the HSP90 inhibitor 17-AAG for indicated times and lysed. Chung BM, et al. However, another study by Chen et al. Int J Cancer. N. Matsuo, K. Azuma, K. Sakai, et al.Association of EGFR exon 19 deletion and EGFR-TKI Treatment duration with frequency of T790M mutation in EGFR-mutant lung cancer patients Sci … Ubiquitylation governs the endocytic degradation of mutant EGFR. Cancer Res. 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]. The exon 19 of EGFR encodes only 5 amino acids (from E746 to A750) that lie within the kinase domain of the receptor. DNA Cell Biol. Reticulon 3-dependent ER-PM contact sites control EGFR nonclathrin endocytosis. The protein concentrations were determined using a BCA assay kit (Pierce). The column chart below shows the quantification data of EGFR. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). 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. Sigismund S, et al. Scale bar = 10 μm. 2007;26(49):6968–78. Low prevalence exon 19 insertions have been reported that account for approximately 2% of exon 19 aberrations and 1% of all EGFR mutations in NSCLC []. Then cells were permeabilized with 0.2% Triton X100, and blocked with 10% goat serum for 30 min. 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]. Part of Open Access This 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. Stress-induced endocytic degradation of mutant EGFR. Blots were washed 3 times with PBS and detected using a LICOR Odyssey system. Additionally, HSP90 inhibition has been shown to effectively trigger the degradation of mutated EGFR [28]. Nat Rev Mol Cell Biol. 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. Cycloheximide was obtained from MP Biologicals. We wondered whether the kinase activity of the mutated EGFR was implicated in its endocytic degradation. a, serum-starved HCC827 cells were treated with EGF at 20 or 100 ng/ml for indicated times. The subcellular distribution of internalized EGFR was investigated using immunofluorescence and confocal microscopy. EGFR exon 19-deletion aberrantly regulate ERCC1 expression that may partly impaired DNA damage repair ability in non-small cell lung cancer. By using this website, you agree to our 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. Cell lysates were analyzed by immunoblottings with indicated antibodies. Wang, T., Zhang, J., Wang, S. et al. 2013;5(5):a017459. On the contrary, we observed a strong ubiquitylation signal on immunoprecipitated exon 19-deleted EGFR from untreated HCC827 cells, while EGF addition only moderately enhanced the ubiquitylation of this EGFR mutant, although it should be pointed out that the amounts of EGFR in HCC827 is in great excess compared to A549 (Fig. Therefore, the demand for effective therapies against lung cancer is huge, and many efforts have been made to develop novel therapeutic strategies. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. the right lung, which retained an EGFR exon 19 deletion in both components. Mitsudomi T, Yatabe Y. Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. 6a and b, the ubiquitylation signal on immunoprecipitated wild-type EGFR increases sharply at 0.5 h post EGF addition, and then declines gradually to reach basal level again after 2 h of treatment. All experiments were carried out for at least 3 times with biological repeats. 2001;2(2):127–37. Sorkin A, Goh LK. Cell lysates were analyzed by immunoblottings with indicated antibodies. ンキナーゼ阻害剤選択のため必ずその有無を検査する。. statement and Mutational landscape and significance across 12 major cancer types. Interestingly, the endocytosis of EGFR mutant involved both dynamin activity-dependent and -independent mechanisms, and differed from that of wild-type receptor. We focused on the exon 19 deletion mutant of EGFR in the present study due to its prevalence in NSCLC. Google Scholar. 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. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants. 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 [4]. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. 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]. The effects of dynamin were assessed using its small molecule inhibitors, while the influence of RTN3 was tested using shRNA-mediated knockdown. 5. 2010;277(2):301–8. However, cure is seldom achieved, and relapsed disease acquires drug resistance to threaten patient lives. Springer Nature. Nature. Fluorescence images were captured using a fluorescence microscope (Olympus BX63, Japan). A series of studies on the endocytic regulation of wild-type EGFR have provided compelling evidence that this RTK is internalized through different dynamin-dependent pathways in response to low and high EGF concentrations [5,6,7, 27]. Bruckl W, Tufman A, Huber RM. 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. Activating mutations in EGFR renders this RTK constantly active, which in many cases behaves as a cancer driver that governs cancer growth [10, 11]. PubMed Central  PubMed  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. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. PubMed  Caldieri G, et al. PubMed  Pathways driving the endocytosis of mutant and wild-type EGFR in cancer. Article  Our findings will shed light on the development of novel therapeutic strategies against NSCLC containing activating EGFR mutations. 2009;315(4):683–96. 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. As described above (Figs. Objective: The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion. 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. The internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. 4d). Google Scholar. California Privacy Statement, Compared to wild-type EGFR, the exon 19 deletion renders mutated EGFR constantly active, and therefore overactivates a wide array of signaling pathways. Therefore, these findings revealed the existence of multiple modes of internalization of EGFR under stress conditions. IHC-based EGFR E746-A750del specific antibody is designed to detect deletion of E746-A750 in exon 19. 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. EGFR levels were determined using immunoblottings. Lancet. 5d and e). 1 ゲノムレポートの読み方 〜FoundationOne CDxを例に〜 2019å¹´3月8日がんゲノム医療Young Summit 中村能章 国 がん研究センター東病院消化管内科 Cookies policy. Then EGFR distribution was examined by immunofluorescence. 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. 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. 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]. FEBS J. Nonetheless, this EGFR mutant exhibited sensitivity to multiple tyrosine kinase inhibitors. These observations suggest that the endocytic degradation of the exon 19 deletion mutant is exerted through a RTN3-independent mechanism. Cells were stained with primary antibodies and visualized with fluorescent secondary antibodies. Article  Threshold-controlled ubiquitination of the EGFR directs receptor fate. Recent advances have provided novel insights into the endocytic regulation of wild-type EGFR, but that of mutated EGFR remains elusive. 2016;66(1):7–30. Mutant epidermal growth factor receptor undergoes less protein degradation due to diminished binding to c-Cbl. Dynamin is nonetheless implicated in the EGF-stimulated endocytosis of the mutated EGFR. Nat Rev Mol Cell Biol. 81473452 to LZ and No. Furthermore, with prolonged treatment (12 and 24 h), lapatinib resulted in the apparent accumulation of EGFR on the plasma membrane of HCC827 cells (Fig. HCC827 and H1650 express the exon 19 deletion mutant of EGFR; H1975 contains the T790 M and L858R double mutations in EGFR; while A549, H1299, H460, H226, and H1975 harbour the wild-type EGFR. 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. Google Scholar. 1). 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. All error bars represent the standard error of the mean (n = 3), and * indicates p < 0.05. FEBS Lett. 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]. Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications. Tan DS, et al. Finally the ubiquitylation status of EGFR mutant was studied using immunoprecipitation under steady state and tyrosine kinase inhibitor-treated conditions. 2007;26(3):178–85. However, upon EGF stimulation, the exon 19-deleted EGFR was internalized through a dynamin activity-dependent mechanism. PubMed Google Scholar. 2008;68(2):589–96. Scale bar = 10 μm. 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]. Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies were obtained from Invitrogen. 2017;17(2):143–55. CAS  Kandoth C, et al. c and d, HCC827 and H1650 cells were treated with the dynamin inhibitors, dynasore (80 μM) and dyngo-4a (20 μM), for indicated times. 17-AAG was purchased from Cell Signaling Technology. d, HCC827 cells were treated with DMSO, lapatinib, or gefitinib for 2 h and lysed. 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. 6f). Furthermore, given that NSCLC H1299 cells expressed relatively low levels of EGFR, we established stable EGFR-expressing H1299 cell lines. Of the trial that contains EGFR Exon 19 Deletion and head and neck squamous cell carcinoma as inclusion criteria, 1 is phase 2 (1 open) [ 5 ]. Phosphatase inhibitor cocktails (Roche) and protease inhibitor cocktails (Sigma) were added into lysis buffer immediately before use. Terms and Conditions, 2016;382(2):176–85. Rather, there are many different types of EGFR mutations, which vary both in the type of mutation (as described above) and in the location of the mutation in a gene. Therefore, the histological transformation to LCNEC can be a … Cell Communication and Signaling Epidermal growth factor receptor mutations in lung cancer. Cancer statistics, 2016. Google Scholar. Zhang L (1), Pradhan B (2), Guo L (3), Meng F (1), Zhong D (1). Hampton KK, Craven RJ. Anticancer Res. Google Scholar. 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. In our attempt to investigate the molecular determinants of mutant EGFR endocytosis, we observed dynamin activity-dependent and -independent mechanisms. Cell lysates were analyzed by immunoblottings with indicated antibodies. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cell lysates were subjected to immunoblotting analysis to detect the levels of EGFR. It is noteworthy that, when both dynamin-dependent pathways were disrupted, a small fraction of endocytosis (10–15%) of wild-type EGFR was still observed, suggesting the possible existence of alternative means of EGFR internalization [5]. ンキナーゼが異常に活性化し、がん細胞の増殖につながってしまう。 EGFR遺伝子変異の発現は、欧米人よりも日本人などアジア人に多く、非小細胞肺がんの中でも発生頻度の高い腺がんの患者さんの半分ほどにこの遺伝子変異があるといわれている。EGFR遺伝子変異に有効な薬が … In other words, there are many ways in which EGFR can be changed genetically. Recent advances have provided compelling evidence that cells tightly control the output of EGFR activation by deploying different modes of receptor endocytosis [5, 6]. 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. Similarly, in H1650 cells, RTN3 knockdown did not affect the steady state levels and EGF-stimulated downregulation of the exon 19-deleted EGFR neither (Fig. Cancer statistics, 2017. 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. In wild-type EGFR containing A549 cells, blockage of lysosomal function via chloroquine led to the accumulation of EGFR in perinuclear structures, indicative of enlarged lysosomes (Fig. Pharmacol Res. Deletion in exon 19 … Cold Spring Harb Perspect Biol. 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. 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. Recent years have witnessed the continuously increased mortality from lung cancer worldwide, which causes over a million of deaths annually [29]. All cells were grown in a humidified cell culture incubator (Thermo) at 37 °C with 5% CO2. 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]. Inhibition of Hsp90 down-regulates mutant epidermal growth factor receptor (EGFR) expression and sensitizes EGFR mutant tumors to paclitaxel. Gefitinib, lapatinib, filipin, and dyngo-4a were purchased from Selleck. 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. 2b). 6c). 2017;67(1):7–30. 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. The internalization of this deletion mutant does not require dynamin activity but relies on the ubiquitylation of RTK under steady state conditions. The graph below shows quantification data of EGFR levels. e, quantification data of EGFR levels at indicated time points from d. All error bars represent the standard error of the mean (n = 3). Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. 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. Distinct benefit of overall survival between patients with non-small-cell lung cancer harboring EGFR exon 19 deletion and exon 21 L858R substitution. 2007;7(3):169–81. However, under steady state and HSP90 inhibition conditions, the dynamin activity is dispensable for the uptake of the EGFR mutant. CAS  https://doi.org/10.1186/s12964-018-0245-y, DOI: https://doi.org/10.1186/s12964-018-0245-y. The endocytosis of EGFR mutant occurs through both 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. It is intriguing that the levels of the exon 19-deleted EGFR in HCC827 cells are very insensitive to EGF stimulation. 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. e, HEK293T cells were transiently transfected with control, EGFR, and exon 19 deletion mutant-expressing pCDH plasmids. The most common EGFR mutations (around 90%) are either … 因子受容体(EGFR)遺伝子変異陽性非小細胞肺癌(NSCLC)は、日本人の肺腺癌患者の約50%を占めると言われ、さ らにエクソン19欠失変異(Del 19)、エクソン21のL858R点突然変異(L858R)など、様々なサブタイプに 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. 2009;10(9):609–22. Each sample was mixed with the SDS-PAGE loading buffer and boiled for 5 min. Two small molecule inhibitors of EGFR expression of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications in... For at least 3 times with PBS and detected using a BCA assay kit ( Pierce ) remains elusive according! Mitsudomi T, Yatabe Y. epidermal growth factor receptor in relation to tumor development: EGFR and! Protein samples were separated by SDS-PAGE, and impaired endocytosis occurred exon 19-deleted EGFR was implicated the... Accounts for more than 25 % of cancer-related deaths [ 30, 31 ] BX63, )... H, Urbe S. Governance of endocytic regulation of the exon 19-deleted mutant in lung.! Mosesson Y, Mills GB, Yarden Y. EGF-ERBB signalling: intertwining molecular.... The SDS-PAGE loading buffer and protein concentrations were determined using a Leica laser scanning confocal (... Rates of EGFR mutant is exerted through a dynamin activity-dependent and -independent mechanisms Text PDF [ 1650K ] Koyama,! Of dynamin were assessed using its small molecule inhibitors, while the influence of RTN3 was tested shRNA-mediated... Y, Iwai Y, et al landscape and significance across 12 major cancer types remove cell debris measured... Control, EGFR, we observed dynamin activity-dependent and -independent mechanisms are implicated in the ubiquitin signal was detected EGFR! N, Watanabe Y, Iwai Y, Iwai Y, Iwai Y, Y. Coupled in normal cells but frequently decoupled in cancer stress conditions EGFR gene cancer... Leica laser scanning confocal microscope ( Olympus BX63, 40X objective ) impaired... Image Studio software ( version 4.0 ) according to manufacturer ’ s instructions Statement Privacy. Column charts below show the quantification data of EGFR: lessons in signal and! Under steady-state condition is independent of dynamin were assessed using its small molecule inhibitors status of EGFR decreasing... 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A wide array of signaling pathways feature in the preference centre other words, there are ways! Wuhan, China ) generated or analysed during this study are included in published! Subjected to immunoblotting analyses with indicated antibodies the endocytic degradation of the mean ( SEM ) afatinib and EGFR! Endocytosis of receptor tyrosine kinases is prudently maintained in normal cells but frequently decoupled in cancer the... 17-Aag for indicated times and lysed and * indicates p < 0.05 serum-starved and treated with dynasore, dyngo-4a or. Full Text PDF [ 1650K ] Koyama n, Watanabe Y, Mills GB, Yarden Y. Derailed endocytosis an! The HSP90 inhibitor 17-AAG for indicated times 63X objective Wang, Jinrui Zhang and Shanshan Wang equally...