粒状头样蛋白2下调诱导上皮间质转化促进肿瘤细胞对吉非替尼耐药
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摘要
目的探讨粒状头样蛋白2(GRHL2)在肿瘤靶向治疗药物表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼获得性耐药中的作用和可能机制。方法利用吉非替尼浓度逐步递增的体外诱导方法培养人结直肠癌细胞DiFi和人肺腺癌细胞HCC4006,获得吉非替尼耐药细胞株。通过RNA测序方法筛选在耐药细胞株与其亲代细胞中差异表达的基因并进行实时荧光定量PCR验证。使用pcDNA3.1-GRHL2质粒转染耐药细胞株使GRHL2过表达,用CCK-8法检测细胞对吉非替尼的敏感性。采用蛋白质印迹法检测耐药细胞株中上皮标志物E-cadherin和间质标志物Vimentin的表达变化,并通过CellMinerTM数据库分析60株人肿瘤细胞系中GRHL2表达与E-cadherin和Vimentin表达的关系。结果成功获得DiFi和HCC4006的耐药细胞株。通过RNA测序和实时荧光定量PCR验证发现GRHL2在耐药细胞株中表达下调,而在耐药细胞株中过表达GRHL2后细胞恢复了对吉非替尼的敏感性。蛋白质印迹分析表明耐药细胞株中上皮标志物E-cadherin表达下调,而间质标志物Vimentin表达上调;CellMinerTM数据库分析表明GRHL2的表达与E-cadherin/Vimentin比值高度一致。结论 GRHL2表达下调通过诱导上皮间质转化介导肿瘤细胞对吉非替尼的耐药。
Objective To explore the role and possible mechanism of grainyhead-like protein 2(GRHL2) downregulation in acquired drug resistance to tumor-targeted therapeutic drug gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI). Methods Human colon cancer cell line DiFi and human lung adenocarcinoma cell line HCC4006 were cultured in a stepwise increasing concentration of gefitinib to obtain gefitinib-resistant cell lines. The differentially expressed genes between gefitinib-resistant cell lines and parent cells were selected by RNA sequencing and verified by real-time fluorescent quantitative PCR(qRT-PCR). The pcDNA3.1-GRHL2 plasmid was transfected into the gefitinib-resistant cell lines to overexpress GRHL2, and the sensitivity of the cells to gefitinib was detected by CCK-8 method. The expression of epithelial marker(E-cadherin) and mesenchymal marker(Vimentin) in the gefitinib-resistant cells was detected by Western blotting. The relationship between GRHL2 expression and expression of E-cadherin and Vimentin in 60 human tumor cell lines was analyzed by CellMinerTM database. Results We successfully obtained two gefitinib-resistant cell lines. RNA sequencing and qRT-PCR confirmed that the expression of GRHL2 in the gefitinib-resistant cells was decreased, while the sensitivity of the cells to gefitinib was restored after overexpressing GRHL2 in the gefitinib-resistant cells. Western blotting analysis showed that the E-cadherin expression was decreased and the Vimentin expression was increased in the gefitinib-resistant cell line. CellMinerTM database analysis showed that the expression of GRHL2 was highly consistent with the ratio of E-cadherin to Vimentin. Conclusion Down-regulation of GRHL2 mediates drug resistance of tumor cell to gefitinib by inducing epithelial-mesenchymal transition.
Objective To explore the role and possible mechanism of grainyhead-like protein 2(GRHL2) downregulation in acquired drug resistance to tumor-targeted therapeutic drug gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI). Methods Human colon cancer cell line DiFi and human lung adenocarcinoma cell line HCC4006 were cultured in a stepwise increasing concentration of gefitinib to obtain gefitinib-resistant cell lines. The differentially expressed genes between gefitinib-resistant cell lines and parent cells were selected by RNA sequencing and verified by real-time fluorescent quantitative PCR(qRT-PCR). The pcDNA3.1-GRHL2 plasmid was transfected into the gefitinib-resistant cell lines to overexpress GRHL2, and the sensitivity of the cells to gefitinib was detected by CCK-8 method. The expression of epithelial marker(E-cadherin) and mesenchymal marker(Vimentin) in the gefitinib-resistant cells was detected by Western blotting. The relationship between GRHL2 expression and expression of E-cadherin and Vimentin in 60 human tumor cell lines was analyzed by CellMinerTM database. Results We successfully obtained two gefitinib-resistant cell lines. RNA sequencing and qRT-PCR confirmed that the expression of GRHL2 in the gefitinib-resistant cells was decreased, while the sensitivity of the cells to gefitinib was restored after overexpressing GRHL2 in the gefitinib-resistant cells. Western blotting analysis showed that the E-cadherin expression was decreased and the Vimentin expression was increased in the gefitinib-resistant cell line. CellMinerTM database analysis showed that the expression of GRHL2 was highly consistent with the ratio of E-cadherin to Vimentin. Conclusion Down-regulation of GRHL2 mediates drug resistance of tumor cell to gefitinib by inducing epithelial-mesenchymal transition.
引文
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[2]HABER D A,GRAY N S,BASELGA J.The evolving war on cancer[J].Cell,2011,145:19-24.
[3]REDMOND K L,PAPAFILI A,LAWLER M,VANSCHAEYBROECK S.Overcoming resistance to targeted therapies in cancer[J].Semin Oncol,2015,42:896-908.
[4]OHASHI K,SEQUIST L V,ARCILA M E,MORANT,CHMIELECKI J,LIN Y L,et al.Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS,NRAS,or MEK1[J].Proc Natl Acad Sci USA,2012,109:E2127-E2133.
[5]FADDAOUI A,SHETA R,BACHVAROVA M,PLANTEM,GREGOIRE J,RENAUD M C,et al.Suppression of the grainyhead transcription factor 2 gene(GRHL2)inhibits the proliferation,migration,invasion and mediates cell cycle arrest of ovarian cancer cells[J].Cell Cycle,2017,16:693-706.
[6]MISALE S,YAEGER R,HOBOR S,SCALA E,JANAKIRAMAN M,LISKA D,et al.Emergence of KRASmutations and acquired resistance to anti-EGFR therapy in colorectal cancer[J].Nature,2012,486:532-536.
[7]C H M I E L E C K I J,F O O J,O X N A R D G R,HUTCHINSON K,OHASHI K,SOMWAR R,et al.Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling[J/OL].Sci Transl Med,2011,3:90ra59.doi:10.1126/scitranslmed.3002356.
[8]REINHOLD W C,SUNSHINE M,LIU H,VARMA S,KOHN K W,MORRIS J,et al.CellMiner:a web-based suite of genomic and pharmacologic tools to explore transcript and drug patterns in the NCI-60 cell line set[J].Cancer Res,2012,72:3499-3511.
[9]PARK S M,GAUR A B,LENGYEL E,PETER M E.The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1and ZEB2[J].Genes Dev,2008,22:894-907.
[10]HATA A,KATAKAMI N,YOSHIOKA H,KAJI R,MASAGO K,FUJITA S,et al.Spatiotemporal T790Mheterogeneity in individual patients with EGFR-mutant non-small-cell lung cancer after acquired resistance to EGFR-TKI[J].J Thorac Oncol,2015,10:1553-1559.
[11]PRESUTTI D,SANTINI S,CARDINALI B,PAPOFF G,LALLI C,SAMPERNA S,et al.MET gene amplification and MET receptor activation are not sufficient to predict efficacy of combined MET and EGFR inhibitors in EGFRTKI-resistant NSCLC cells[J/OL].PLoS One,2015,10:e0143333.doi:10.1371/journal.pone.0143333.
[12]LI X,XU Y,DING Y,LI C,ZHAO H,WANG J,et al.Posttranscriptional upregulation of HER3 by HER2 mRNAinduces trastuzumab resistance in breast cancer[J/OL].Mol Cancer,2018,17:113.doi:10.1186/s12943-018-0862-5.
[13]WARE K E,HINZ T K,KLECZKO E,SINGLETON KR,MAREK L A,HELFRICH B A,et al.A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1autocrine growth loop[J/OL].Oncogenesis,2013,2:e39.doi:10.1038/oncsis.2013.4.
[14]MITRA A,MISHRA L,LI S.EMT,CTCs and CSCs in tumor relapse and drug-resistance[J].Oncotarget,2015,6:10697-10711.
[15]FUCHS B C,FUJII T,DORFMAN J D,GOODWIN J M,ZHU A X,LANUTI M,et al.Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells[J].Cancer Res,2008,68:2391-2399.
[16]BASU D,NGUYEN T T,MONTONE K T,ZHANGG,WANG L P,DIEHL J A,et al.Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity[J].Oncogene,2010,29:4170-4182.
[17]FREDERICK B A,HELFRICH B A,COLDREN C D,ZHENG D,CHAN D,BUNN P A Jr,et al.Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non-small cell lung carcinoma[J].Mol Cancer Ther,2007,6:1683-1691.
[18]唐冠楠,颜宏利,王玉招,孙树汉.粒状头样2(GRHL2)与肿瘤[J].第二军医大学学报,2013,34:1243-1247.TANG G N,YAN H L,WANG Y Z,SUN S H.Grainyheadlike-2(GRHL2)and carcinogenesis:an advance[J].Acad JSec Mil Med Univ,2013,34:1243-1247.
[19]WERTH M,WALENTIN K,AUE A,SCHHEITJ,WUEBKEN A,PODE-SHAKKED N,et al.The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex[J].Development,2010,137:3835-3845.
[20]KALLURI R.EMT:when epithelial cells decide to become mesenchymal-like cells[J].J Clin Invest,2009,119:1417-1419.
[21]CIEPLY B,RILEY P 4th,PIFER P M,WIDMEYER J,ADDISON J B,IVANOV A V,et al.Suppression of the epithelial-mesenchymal transition by grainyhead-like-2[J].Cancer Res,2012,72:2440-2453.