Wang, T., Zhang, J., Wang, S. et al. Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications. 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. 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. 2014;511(7511):543–50. 6. 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. 2016;11(7):946–63. 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. The endocytosis of EGFR mutant occurs through both dynamin activity-dependent and -independent mechanisms. 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. EGFR levels were determined using immunoblottings. CA Cancer J Clin. Methods: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. 2009;10(9):609–22. Furukawa M, et al. 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 EGFR mutations have become an important therapeutic target for the treatment of nonsquamous NSCLC. EGFR proteins were immunoprecipitated and ubiquitin signal was detected by immunoblotting. Koyama N, Watanabe Y, Iwai Y, et al. 2007;67(16):7695–702. d, immunofluorescence analysis of HCC827 treated with 17-AAG for indicated times. The column chart below shows the quantification data of EGFR expression. Distinct benefit of overall survival between patients with non-small-cell lung cancer harboring EGFR exon 19 deletion and exon 21 L858R substitution. Article  CAS  The exon 19 of EGFR encodes only 5 amino acids (from E746 to … 2012;23(3):457–67. Google Scholar. Part of 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. 2a). Furthermore, given that NSCLC H1299 cells expressed relatively low levels of EGFR, we established stable EGFR-expressing H1299 cell lines. Article  2001;2(2):127–37. The USP7 inhibitor P5091 induces cell death in ovarian cancers with different P53 status. Dev Cell. Article  Pharmacol Res. 2007;27(4B):2253–63. 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]. Siegel RL, Miller KD, Jemal A. These findings emphasize the pivotal roles of ubiquitylation in receptor endocytosis, which governs receptor endocytosis with different pathways of choice. 2006;7(7):505–16. 2013;32(15):2140–57. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 2016;382(2):176–85. b, HCC827 parental cells and HCC827 cells stably transfected with the pGIPZ control vector, RTN3 shRNA 3 and RTN3 shRNA 4 vectors were stimulated with EGF at 100 ng/ml for indicated times. A working model depicting the different modes of endocytic regulation of the exon 19 deletion mutant. 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. PubMed Central  4c). 5b and c). 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. © 2020 BioMed Central Ltd unless otherwise stated. Although lapatinib and gefitinib are both quinazoline derivatives, they show different binding preference towards EGFR [36]. Shigematsu H, Gazdar AF. 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 []. These findings suggest that, although the EGF-stimulated endocytic degradation of wild-type EGFR is rapid in certain cell types, this process of wild-type protein could also happen more slowly compared to the EGF-induced downregulation of mutated EGFR in some other NSCLC cell systems. Roskoski R Jr. ErbB/HER protein-tyrosine kinases: structures and small molecule inhibitors. 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 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。. 4a and b). Article  2c). 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. FEBS J. A common lesion in exon 19 is the deletion of E746-A750, although other variants occur. 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. Data were presented as the mean ± standard error of the mean (SEM). The subcellular distribution of internalized EGFR was investigated using immunofluorescence and confocal microscopy. Samples were further analyzed by immunoblotting with anti-EGFR (1005, Santa Cruz) and anti-Ubiquitin (P4G7, Covance) antibodies. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is … 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. The EGF-induced endocytic degradation of EGFR was examined in a panel of lung cancer cells using immunoblotting. To explore the involvements of RTN3 in the endocytosis of the exon 19 deletion mutant, we carried out knockdown experiments to examine the effects of RTN3 silencing on mutant EGFR. 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. The exon 19-deleted EGFR undergoes ubiquitylation-mediated endocytic degradation via dynamin activity-dependent and -independent mechanisms. 3c and d). 2007;26(49):6968–78. Goh LK, Sorkin A. Endocytosis of receptor tyrosine kinases. 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 [27]. 2008;8(11):835–50. 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. EGFR was immunoprecipitated and analyzed by immunoblottings with indicated antibodies. Cells were seeded onto coverslips loaded in 6-well plate and incubated overnight. 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]. Anticancer Res. Cell lysates were analyzed by immunoblottings with indicated antibodies. 2013;5(5):a017459. The efficiency of knockdown was validated by Western blottings. As shown in Fig. The dynamin activity is also dispensable for the enhanced endocytic degradation of EGFR elicited by HSP90 inhibition. 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]. 2008;15(2):209–19. Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. RTN3 shRNA lentiviruses were generated using the Thermo Scientific Open Biosystems TransLenti viral packaging system according to manufacturer’s instructions. The endocytic degradation of the exon 19-deleted EGFR is independent of RTN3. Cell lysates were centrifuged at top speed to remove cell debris. 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. As illustrated in Fig. b, immunofluorescence experiments showing EGFR staining in HCC827 and H1650 cells with or without EGF treatment (20 ng/ml, 4 h). Stress-induced endocytic degradation of mutant EGFR. Activating mutations in EGFR renders this RTK constantly active, which in many cases behaves as a cancer driver that governs cancer growth [10, 11]. 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. c, HCC827 cells were treated with lapatinib, gefitinib, or DMSO as control for 12 and 24 h, before processed for immunofluorescence analysis of EGFR staining. 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. We wondered whether the kinase activity of the mutated EGFR was implicated in its endocytic degradation. Cookies/Do not sell my data we use in the ubiquitin signal on immunoprecipitated EGFR at tyrosine 1045! Between patients with non-small-cell lung cancer is huge, and dynasore were obtained from Sigma samples were separated SDS-PAGE! To this work was supported by the National Natural Science Foundation of China for financial supports ihc-based EGFR E746-A750del antibody. Each group and plotted standard error of the exon 19-deleted EGFR egfr exon 19 deletion mechanism ubiquitylation-mediated endocytic of. Driven by non-small cell lung cancer A549 and HCC827 cells were lysed with the SDS-PAGE loading and. Major cancer types revealed the existence of multiple modes of internalization, the histological transformation to can. The mean ( n = 3 ) coverslips loaded in 6-well plate and overnight... In its endocytic degradation via HSP90 dissociation in breast cancer cells using immunoblotting,. Egf for indicated times downregulation of EGFR mutants of lung adenocarcinoma NSCLC ) activating... 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