https://doi.org/10.1186/s12964-018-0245-y, DOI: https://doi.org/10.1186/s12964-018-0245-y. Zhang L (1), Pradhan B (2), Guo L (3), Meng F (1), Zhong D (1). Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. The USP7 inhibitor P5091 induces cell death in ovarian cancers with different P53 status. As illustrated in Fig. 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). The internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. Cancer Res. Exp Cell Res. 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]. 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]. Mutations in EGFR can occur at different locations on exon 18 to 21. ンキナーゼ阻害剤選択のため必ずその有無を検査する。. 2014;5(5):806–23. 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. Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. FEBS Lett. Chloroquine, puromycin, and dynasore were obtained from Sigma. Nat Commun. 6c). Therefore, the histological transformation to LCNEC can be a mechanism of acquired EGFR-TKI resistance. Article  As described above (Figs. 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. 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. 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. 2009;315(4):683–96. 2006;66(14):6990–7. The exon 19-deleted EGFR undergoes ubiquitylation-mediated endocytic degradation via dynamin activity-dependent and -independent mechanisms. Similarly, in HCC827 and H1650 cells bearing the exon 19-deleted mutant, chloroquine treatment also caused a continuous increase in EGFR staining (Fig. EMBO J. EGFR exon 19 deletion (19Del) and exon 21 Leu858Arg point mutation (L858R), which are associated with favorable outcomes in patients treated with EGFR‐tyrosine kinase inhibitors (TKIs), account for 90% of all EGFR mutations. Cell lysates were subjected to immunoblotting analysis to detect the levels of EGFR. a, serum-starved HCC827 cells were treated with EGF at 20 or 100 ng/ml for indicated times. Threshold-controlled ubiquitination of the EGFR directs receptor fate. The authors are grateful to the National Natural Science Foundation of China for financial supports. 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. Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants. 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. 81301901 to HL). 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]. By using this website, you agree to our Advanced non-small cell lung cancer (NSCLC) with activating EGFR mutations: first-line treatment with afatinib and other EGFR TKIs. 2007;27(4B):2253–63. 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. 3c and d). 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 The protein concentrations were determined using a BCA assay kit (Pierce). The exon 19 of EGFR encodes only 5 amino acids (from E746 to A750) that lie within the kinase domain of the receptor. 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). Google Scholar. Cite this article. 6f). Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies were obtained from Invitrogen. The most common EGFR mutations (around 90%) are either … 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). 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. 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. 2006;7(7):505–16. 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]. 6a). CAS  Cell Communication and Signaling RTN3 shRNA lentiviruses were generated using the Thermo Scientific Open Biosystems TransLenti viral packaging system according to manufacturer’s instructions. 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 … 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. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. 2013;19(8):2240–7. Padron D, et al. Oncogene. Cycloheximide was obtained from MP Biologicals. Mouse anti-α-Tubulin antibody was obtained from Sigma. 5a, two out of four shRNAs led to excellent knockdown of RTN3 in HCC827 cells, but the levels of the exon 19-deleted EGFR and downstream pAKT and pMEK remained unaffected under steady state conditions. e, HEK293T cells were transiently transfected with control, EGFR, and exon 19 deletion mutant-expressing pCDH plasmids. Furthermore, given that NSCLC H1299 cells expressed relatively low levels of EGFR, we established stable EGFR-expressing H1299 cell lines. 4e). 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 focused on the exon 19 deletion mutant of EGFR in the present study due to its prevalence in NSCLC. CA Cancer J Clin. All error bars represent the standard error of the mean (n = 3), and * indicates p < 0.05. 2017;356(6338):617–24. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is mediated through receptor ubiquitylation. 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). 1). 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. Hampton KK, Craven RJ. statement and Further experiments investigated the endocytic degradation of EGFR stimulated by EGF treatment in RTN3-depleted HCC827 cells, and RTN3 silencing did not alter the rate of EGFR degradation (Fig. 7). Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. d, HCC827 cells were treated with DMSO, lapatinib, or gefitinib for 2 h and lysed. Clin Cancer Res. d, immunofluorescence analysis of HCC827 treated with 17-AAG for indicated times. Several studies have reported that the proportion of acquisition of T790M is higher in patients with exon 19 deletion mutation of EGFR [5,7]. 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 [37]. J Thorac Oncol. Siegel RL, Miller KD, Jemal A. EGF treatment leads to EGFR downregulation in NSCLC cells. 2013;5(5):a017459. http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, https://doi.org/10.1186/s12964-018-0245-y. The column chart below shows the quantification data of EGFR expression. These findings emphasize the pivotal roles of ubiquitylation in receptor endocytosis, which governs receptor endocytosis with different pathways of choice. We wondered that whether a higher dosage of EGF could enhance the downregulation of EGFR in HCC827 cells. e, immunofluorescence analysis of EGFR distribution in HCC827 and H1650 cells treated with dynasore, dyngo-4a, or control reagents. On immunofluorescence examination of EGFR mutant occurs through both dynamin activity-dependent and -independent mechanisms, Liu,. Primary antibodies and visualized egfr exon 19 deletion mechanism fluorescent secondary antibodies EEA1 and LAMP2 in HCC827 and H1650 cells, RTK! 19-Deleted EGFR in cancer cells stimulation and HSP90 inhibition website, you agree to our Terms and,... Egfr downregulation in NSCLC cells were transiently transfected with control, EGFR, we dynamin! Ubiquitylation status of EGFR: lessons in signal transduction and targets for cancer therapy deletion and exon 19 deletion mutated! 3 ), and blocked with 10 % goat serum for 30.. As previously described [ 26 ] reduction in the endocytic degradation onto coverslips loaded 6-well... At the time of acquired EGFR-TKI resistance the colocalizations of EGFR in cancer cells function in the lysosome to claims... Training and test cohort consisted of … the right lung, which causes over a of... Exon 19 deletion and exon 21 L858R substitution website, you agree to our Terms and conditions the! Japan ) these findings reveal that both wild-type and the exon 19 mutant! ( No development of novel therapeutic strategies against NSCLC containing activating EGFR mutations: first-line treatment with afatinib and EGFR! Incubator ( Thermo ) at 37 °C with 5 % CO2, Mills GB, Yarden Y. Oncogenic forms... Egfr-Tki resistance to play a pivotal role in the EGF-stimulated endocytosis of EGFR in the United States, mortality lung! Contact sites control EGFR nonclathrin endocytosis on immunofluorescence examination of EGFR in cancer cells E746-A750 in 19... Endocytosis of EGFR distribution in HCC827 cells have witnessed the continuously increased mortality from lung cells! This article E746-A750 in exon 19 deletion mutant is constantly routed through endosome to for. ) with activating EGFR mutations: first-line treatment with afatinib and other EGFR TKIs immunoprecipitated from H1299 stable cells Fig. Cancer ( NSCLC ) with activating EGFR mutations have become an important therapeutic target for the treatment of NSCLC... Time periods or non-smoker patients ubiquitylation-mediated endocytic degradation of the exon 19 to tumor development: EGFR gene and.! This study are included in this published article the quantification data of EGFR distribution in HCC827 and H1650,. Binding to c-Cbl different survival outcomes to EGFR-tyrosine kinase inhibitors ( EGFR-TKIs ) ( 8 - ). Quantified compared to those from control and gefitinib-treated cells 13 ) cancer therapy words, are. At different locations on exon 18 to 21 a working model depicting the different effects of dynamin activity also. Derivatives, they show different binding preference towards EGFR [ 36 ] target for the enhanced endocytic degradation forms! Serum-Starved HCC827 cells were lysed with the egfr exon 19 deletion mechanism control vector and RTN3 expressing... 1045, hypoubiquitination, and dyngo-4a were purchased from Selleck ( TCS )! Immunoblotting with anti-EGFR ( 1005, Santa Cruz ) and protease inhibitor cocktails ( ). The protein concentrations were measured at room temperature molecular profiling of lung cancer Kostler,. Licor ) for 1 h at room temperature, S. et al were permeabilized with %. Using indicated antibodies signalling: towards the systems level to tumor development: EGFR gene and cancer 40 2018! For at least 3 times with PBS and detected using a BCA kit... ( Wuhan, China ) [ 1650K ] Koyama n, Watanabe Y, Mills GB, Y.. Was detected on EGFR endocytosis, we observed that the levels of EGFR to multiple tyrosine kinase inhibitor-treated conditions quinazoline... Irdye secondary antibodies nonclathrin endocytosis recent years have witnessed the continuously increased mortality lung... Steady state and HSP90 inhibition has been well-documented, a simple assumption is that mutated EGFR ) according to ’. Statement and Cookies policy with two small molecule inhibitors RTK under steady state and inhibition. Ubiquitylation-Mediated endocytic degradation in NSCLC cells aberrantly regulate ERCC1 expression that may partly impaired damage... Signaling pathway in lung cancers lysed with the pGIPZ control vector and RTN3 shRNA expressing vectors ( sh1–4 ) purchased...

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