Label-free定量蛋白质组学揭示GATA6调控胃癌细胞对曲妥珠单抗耐药的信号通路

引用本文: 刘文虎, 袁江北, 常晋霞. Label-free定量蛋白质组学揭示GATA6调控胃癌细胞对曲妥珠单抗耐药的信号通路[J]. 分析化学, 2020, 48(2): 187-196. doi: 10.19756/j.issn.0253-3820.191612 shu

Citation: LIU Wen-Hu, YUAN Jiang-Bei, CHANG Jin-Xia. Label-free Quantitative Proteomics for Investigation of Signaling Pathways of GATA6 Regulating Trastuzumab Resistance in Gastric Cancer Cells[J]. Chinese Journal of Analytical Chemistry, 2020, 48(2): 187-196. doi: 10.19756/j.issn.0253-3820.191612 shu

Label-free定量蛋白质组学揭示GATA6调控胃癌细胞对曲妥珠单抗耐药的信号通路

1.
中南大学湘雅医院分子代谢组学研究中心, 长沙 410008;
2.
川北医学院药学院, 南充 637100;
3.
川北医学院基础医学院, 南充 637100;
4.
重庆大学药学院, 重庆 401331
基金项目:
本文系四川省科技厅应用基础科研项目（No.2019YJ0378）、四川省教育厅项目（No.17ZB0170）、南充市市校合作项目（No.18SXHZ0402）和川北医学院博士科研项目（No.CBY17-QD05）资助

摘要: 肿瘤细胞对曲妥珠单抗耐药是导致其化疗失败的主要原因。前期研究表明，在曲妥珠单抗耐药胃癌细胞（NCI N87/R）中，转录因子GATA6与DNA的结合活性显著增强，这与其耐药是否有关尚不清楚。本研究以NCI N87/R为研究对象，采用Crispr/Cas9技术构建GATA6敲除细胞系（NCI N87R/GATA6）。在此基础上，基于Label-free定量蛋白质组学技术并结合生物信息学分析GATA6调控曲妥珠单抗耐药的信号通路。蛋白样品经还原烷基化，FASP酶切，肽段经液相色谱分离，LC-MS/MS定量分析，通过差异倍数及t检验筛选差异表达蛋白；采用WebGestalt数据库进行基因本体分析，利用GeneAnalytics数据库进行通路富集分析。结果表明，敲除GATA6增强了曲妥珠单抗对NCI N87/R的抑制作用，降低其侵袭。采用质谱定量检测出5792种蛋白质，其中305种在NCI N87R/GATA6细胞中表达上调，182种表达下调。通路富集分析显示，线粒体转运、细胞凋亡、DNA损伤、葡萄糖代谢、丙酮酸代谢及TCA循环、Wnt/β-catenin降解通路在NCI N87R/GATA6细胞中显著改变。蛋白免疫印迹实验（Western blot）表明，线粒体动力蛋白OPA1和DNM1L、凋亡蛋白Caspase-9、TCA循环代谢酶SUCLG2和MDH1、糖原代谢酶PYGL在NCI N87R/GATA6中显著改变，表明GATA6敲除导致NCI N87/R细胞线粒体功能障碍、能量代谢异常，进而诱导其凋亡，提示抑制GATA6转录活性可能是逆转胃癌曲妥珠单抗耐药的有效途径。

关键词:

English

Label-free Quantitative Proteomics for Investigation of Signaling Pathways of GATA6 Regulating Trastuzumab Resistance in Gastric Cancer Cells

1.
Research Center of Molecular Metabolomics, Xiangya Hospital Central South University, Changsha 410008, China;
2.
Department of Pharmacy, North Sichuan Medical College, Nanchong 637100, China;
3.
School of Basic Medical Sciences, North Sichuan Medical College, Nanchong 637100, China;
4.
School of Pharmaceutical Sciences and Innovative Drug Research Center, Chongqing University, Chongqing 401331, China
Received Date: 17 October 2019
Revised Date: 07 November 2019
Fund Project:
This work was supported by the Applied Basic Research Program of Science and Technology of Sichuan Province, China (No. 2019YJ0378), the Project of Sichuan Province Education Department (No. 17ZB0170), the Doctoral Program of North Sichuan Medical College (No. CBY17-QD05), and the Program of Science and Technology of Nanchong, China (No. 18SXHZ0402).

Abstract: Trastuzumab resistance is one of the principal causes of failure in tumor chemotherapy. Previous studies show that the DNA-binding activity of GATA6, a transcription factor, has a remarkable enhancement in trastuzumab resistant gastric cancer cells (NCI N87/R), while its correlation to resistance remains unclear. In this study, Crispr/Cas9 was employed to establish a GATA6 knock-out cell line (NCI N87R/GATA6). The signaling pathways regulated by GATA6 and related to trastuzumab resistance were investigated based on label-free quantitative proteomics combined with bioinformatics. The extracted proteins were alkylated, and digested using filter aided sample preparation (FASP), and the peptides were separated via high performance liquid chromatography and thereafter quantified by LC-MS/MS. Differentially expressed proteins were screened by fold change and student's t-test between NCI N87/R and NCI N87R/GATA6 cells. WebGestalt website was adopted for Gene ontology analysis, and GeneAnalytics was utilized for pathway enrichment analysis. The results demonstrated that GATA6 knock-out enhanced the antiproliferative effect of trastuzumab on NCI N87/R cells and suppressed their invasion ability. A total of 5792 proteins were quantified by LC-MS/MS, among which 305 proteins were up-regulated in NCI N87R/GATA6 cells while 182 ones down-regulated. Pathway enrichment analysis revealed that mitochondrial transport, apoptosis, DNA damage, glucose metabolism, pyruvate metabolism and TCA cycle and Wnt/β-catenin degradation pathways exhibited significant changes. Western blot manifested that the expression of mitochondrial dyneins OPA1 and DNM1L, apoptosis protein caspase-9, TCA metabolic enzymes SUCLG2 and MDH1, and glycogen metabolic enzyme PYGL changed significantly in NCI N87R/GATA6 cells, manifesting that GATA6 knock-out gave rise to mitochondrial dysfunction and abnormal energy metabolism, and therefore inducing the apoptosis of NCI N87R/GATA6 cells. The result implicated that inhibiting the transcriptional activity of GATA6 could be an effective strategy to reverse trastuzumab resistance in gastric cancer.

Key words:

1. [1]
  Bray F,Ferlay J, Soerjomataram I, Siegel R L, Torre L A, Jemal A. CA Cancer J. Clin.,2018,68(6):394-424
2. [2]
  Boku N. Gastric Cancer, 2014,17(1):1-12
3. [3]
  Liu T S, Qin Y R, Li J, Xu R H, Xu J M, Yang S J, Qin S K, Bai Y X, Wu C P, Mao Y X, Wu H Y, Ge Y L, Shen L. Cancer Commun.,2019,39:38
4. [4]
  ZHOU Ye, LIU Zhe-Yi, WANG Fang-Jun. Chinese Journal of Chromatography,2019,37(8):788-797 周烨, 刘哲益, 王方军.色谱, 2019,37(8):788-797
5. [5]
  CHAI Shuang-Shuang, MA You-Ning, GAO Huan-Huan, QIN Mei-Ling, YANG Huan, ZHANG Han-Tong, HE Qiao, LIN Xiao-Yan. Chinese Journal of Chromatography,2018,36(2):107-113 柴爽爽, 马有宁, 高欢欢, 秦美玲, 杨欢, 张涵彤, 何巧, 林晓燕.色谱, 2018,36(2):107-113
6. [6]
  Martinelli P, Carrillo-de Santa Pau E, Cox T, Sainz B, Dusetti N, Greenhalf W, Rinaldi L, Costello E, Ghaneh P, Malats N, Büchler M, Pajic M, Biankin AV, Iovanna J, Neoptolemos J, Real F X. Gut,2017,66(9):1665-1676
7. [7]
  Belaguli N S, Aftab M, Rigi M, Zhang M, Albo D, Berger D H. Neoplasia,2010,12(11):856-865
8. [8]
  Lin L, Bass A J, Lockwood WW, Wang Z, Silvers A L, Thomas D G, Chang A C, Lin J, Orringer M B, Li W, Glover T W, Giordano T J, Lam W L, Meyerson M, Beer D G. Proc. Natl. Acad. Sci. USA,2012,109(11):4251-4257
9. [9]
  Shen W W, Niu N, Lawson B, Qi L S, Zhang J, Li T, Zhang H L, Liu J S. Hum. Pathol.,2019,86:163-169
10. [10]
  Kamnasaran D, Qian B P, Hawkins C, Stanford W L, Guha A. Proc. Natl. Acad. Sci. USA,2007,104(19):8053-8058
11. [11]
  Song Y C, Tian T, Fu X, Wang W J, Li S N, Shi T T, Suo A L, Ruan Z P, Guo H, Yao Y. Exp. Mol. Pathol.,2015,99(3):617-627
12. [12]
  Ma R S, Li X, Liu H, Jiang R, Yang M P, Zhang M H, Wang Y, Zhao Y B, Li H L. Cancer Biol. Ther.,2019,20(9):1206-1212
13. [13]
  Lu Y Y, Zhao X D, Liu Q, Li C X, Graves-Deal R, Cao Z, Singh B, Franklin J L, Wang J, Hu H Y, Wei T Y, Yang M L, Yeatman T J, Lee E, Saito-Diaz K, Hinger S, Patton J G, Chung C H, Emmrich S, Klusmann J H, Fan D M, Coffey R J. Nat. Med., 2017,23(11):1331-1341
14. [14]
  CHANG Jin-Xia, WANG Yi, ZHANG Fan, LIU Wen-Hu. Chinese J. Anal. Chem.,2019,47(7):1035-1044 常晋霞, 汪宜, 张帆, 刘文虎.分析化学,2019,47(7):1035-1044
15. [15]
  Liu W H, Yuan J B, Liu Z Z, Zhang J W, Chang J X. Int. J. Mol. Sci.,2018,19(7):1981
16. [16]
  Liu W H, Chang J X, Liu M W, Yuan J B, Zhang J Q, Qin J, Xia X F, Wang Y. Oncotarget,2017,8(28):45793-45806
17. [17]
  Wi-niewski J R, Zougman A, Nagaraj N, Mann M. Nat. Methods, 2009,6(5):359-362
18. [18]
  Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M. Nature,2011,473(7347):337-342
19. [19]
  Lai M, Liang L Z, Chen J W, Qiu N Q, Ge S, Ji S H, Shi T L, Zhen B, Liu M W, Ding C, Wang Y, Qin J. Mol. Cell. Proteomics,2016,15(7):2263-2278
20. [20]
  Li Y Z, Liu X H. J. Cell. Physiol.,2018,233(8):5589-5597
21. [21]
  Kiriyama Y, Nochi H. Cells, 2017,7(1):1
22. [22]
  Tilokani L, Nagashima S, Paupe V, Prudent J. Essays Biochem.,2018,62(3):341-360
23. [23]
  Lee H, Smith S B, Yoon Y. J. Biol. Chem., 2017,292(17):7115-7130
24. [24]
  El-Hattab A W, Suleiman J, Almannai M, Scaglia F. Mol. Genet. Metab.,2018,125(4):315-321
25. [25]
  Puchalska P, Crawford P A. Cell Metab.,2017,25(2):262-284
26. [26]
  Favaro E, Bensaad K, Chong M G, Tennant D A, Ferguson D J, Snell C, Steers G, Turley H, Li J L, Günther U L, Buffa F M, McIntyre A, Harris A L. Cell Metab.,2012,16(6):751-764
27. [27]
  Heim S, Lage H. In Vivo,2005,19(3):583-590

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Label-free定量蛋白质组学揭示GATA6调控胃癌细胞对曲妥珠单抗耐药的信号通路

English

Label-free Quantitative Proteomics for Investigation of Signaling Pathways of GATA6 Regulating Trastuzumab Resistance in Gastric Cancer Cells

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