Establishment and biological characteristics of acquired gefitinib resistance in cell line NCI-H1975/gefinitib-resistant with epidermal growth factor receptor T790M mutation

Zhao,Bao‑Xia; Wang,Jing; Song,Bo; Wei,Hong; Lv,Wei‑Peng; Tian,Li‑Min; Li,Mei; Lv,Shen

doi:10.3892/mmr.2014.3058

Establishment and biological characteristics of acquired gefitinib resistance in cell line NCI-H1975/gefinitib-resistant with epidermal growth factor receptor T790M mutation

Authors:
- Bao‑Xia Zhao
- Jing Wang
- Bo Song
- Hong Wei
- Wei‑Peng Lv
- Li‑Min Tian
- Mei Li
- Shen Lv
View Affiliations

Affiliations: Laboratory Center, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116027, P.R. China, Department of Pathology, College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning 116004, P.R. China
Published online on: December 8, 2014 https://doi.org/10.3892/mmr.2014.3058
Pages: 2767-2774

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Non‑small cell lung cancer (NSCLC) cells harboring mutations in the epidermal growth factor receptor (EGFR) gene initially respond well to EGFR tyrosine kinase inhibitors (TKI), including gefitinib. However the tumor cells will invariably develop acquired resistance to the drug. The EGFR T790M mutation is generally considered to be the molecular genetic basis of acquired TKI resistance. The present study aimed to explore how the T790M mutation induces tumor cells to escape inhibition by TKI treatment. An acquired gefitinib‑resistant cell line (NCI‑H1975/GR) was generated from the NCI‑H1975 human NSCLC cell line, which harbors the sensitive L858R and resistant T790M mutations of EGFR. The resistant cell line was established by exposing the cells intermittently to increasing concentrations of gefitinib. The mechanisms by which NSCLC acquires resistance to TKIs based on the T790M mutation, were investigated by detecting the protein expression levels of the EGFR/Kirsten rat sarcoma viral oncogene homolog (KRAS)/v‑Raf murine sarcoma viral oncogene homolog B (BRAF) transduction pathway, and epithelial‑mesenchymal transition (EMT) with immunocytochemistry. The resistance of the NCI‑H1975/GR cells to gefitinib was 2.009‑fold, as compared with the parent cells; however, the protein expression levels of EGFR, KRAS and BRAF were lower in the resistant cells. Some mesenchymal morphology was observed in the NCI‑H1975/GR cells, alongside a decreasing E‑cadherin expression and increasing vimentin expression. These results suggest that the reactivation of the EGFR/KRAS/BRAF transduction pathway was not detected in the NCI‑H1975/GR cells. EMT may have an important role in the development of acquired resistance to EGFR‑TKIs in NSCLC cells with sensitivity and resistance mutations.

View Figures

View References

1	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI
2	Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI
3	Paez JG, Jänne PA, Lee JC, et al: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497–1500. 2004. View Article : Google Scholar : PubMed/NCBI
4	Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from ‘neversmokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101:13306–13311. 2004. View Article : Google Scholar
5	Kobayashi S, Boggon TJ, Dayaram T, et al: EGFR mutation and resistance of non-small cell lung cancer to gefitinib. N Engl J Med. 352:786–792. 2005. View Article : Google Scholar : PubMed/NCBI
6	Pao W, Miller VA, Politi KA, et al: Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2:e732005. View Article : Google Scholar : PubMed/NCBI
7	Ohashi K, Sequist LV, Arcila ME, et al: Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1. Proc Natl Acad Sci USA. 109:E2127–E2133. 2012. View Article : Google Scholar : PubMed/NCBI
8	Ercan D, Xu C, Yanagita M, et al: Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors. Cancer Discov. 2:934–947. 2012. View Article : Google Scholar : PubMed/NCBI
9	Faber AC, Corcoran RB, Ebi H, et al: BIM expression in treatment-naïve cancers predicts responsiveness to kinase inhibitors. Cancer Discov. 1:352–365. 2011. View Article : Google Scholar : PubMed/NCBI
10	Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar : PubMed/NCBI
11	Bean J, Brennan C, Shih JY, et al: MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA. 104:20932–20937. 2007. View Article : Google Scholar : PubMed/NCBI
12	Yauch RL, Januario T, Eberhard DA, et al: Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res. 11:8686–8698. 2005. View Article : Google Scholar : PubMed/NCBI
13	Suda K, Tomizawa K, Fujii M, et al: Epithelial to mesenchymal transition in an epidermal growth factor receptor-mutant lung cancer cell line with acquired resistance to erlotinib. J Thorac Oncol. 6:1152–1161. 2011. View Article : Google Scholar : PubMed/NCBI
14	Mok TS, Wu YL, Thongprasert S, et al: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 361:947–957. 2009. View Article : Google Scholar : PubMed/NCBI
15	Mitsudomi T, Morita S, Yatabe Y, et al: Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harboring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomized phase 3 trial. Lancet Oncol. 11:121–128. 2010. View Article : Google Scholar
16	Rosell R, Moran T, Queralt C, et al: Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 361:958–967. 2009. View Article : Google Scholar : PubMed/NCBI
17	Yun CH, Mengwasser KE, Toms AV, et al: The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA. 105:2070–2075. 2008. View Article : Google Scholar : PubMed/NCBI
18	Costa DB, Kobayashi S, Tenen DG and Huberman MS: Pooled analysis of the prospective trials of gefitinib monotherapy for EGFR-mutant non-small cell lung cancers. Lung Cancer. 58:95–103. 2007. View Article : Google Scholar : PubMed/NCBI
19	Nguyen KS, Kobayashi S and Costa DB: Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancers dependent on the epidermal growth factor receptor pathway. Clin Lung Cancer. 10:281–289. 2009. View Article : Google Scholar : PubMed/NCBI
20	Sequist LV, Bell DW, Lynch TJ and Haber DA: Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol. 25:587–595. 2007. View Article : Google Scholar : PubMed/NCBI
21	Inukai M, Toyooka S, Ito S, et al: Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res. 66:7854–7858. 2006. View Article : Google Scholar : PubMed/NCBI
22	Sequist LV, Martins RG, Spigel D, et al: First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations. J Clin Oncol. 26:2442–2449. 2008. View Article : Google Scholar : PubMed/NCBI
23	Toyooka S, Kiura K and Mitsudomi T: EGFR mutation and response of lung cancer to gefitinib. N Engl J Med. 352:21362005. View Article : Google Scholar : PubMed/NCBI
24	Bell DW, Gore I, Okimoto RA, et al: Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet. 37:1315–1316. 2005. View Article : Google Scholar : PubMed/NCBI
25	Koizumi F, Shimoyama T, Taguchi F, Saijo N and Nishio K: Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int J Cance. 116:36–44. 2005. View Article : Google Scholar
26	Engelman JA, Mukohara T, Zejnullahu K, et al: Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J Clin Invest. 116:2695–2706. 2006. View Article : Google Scholar : PubMed/NCBI
27	Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar : PubMed/NCBI
28	Adjei AA: K-ras as a target for lung cancer therapy. J Thorac Oncol. 3:S160–S163. 2008. View Article : Google Scholar : PubMed/NCBI
29	Suda K, Tomizawa K, Fujii M, et al: Epithelial to mesenchymal transition in an epidermal growth factor receptor-mutant lung cancer cell line with acquired resistance to erlotinib. J Thorac Oncol. 6:1152–1161. 2011. View Article : Google Scholar : PubMed/NCBI
30	Rho JK, Choi YJ, Lee JK, et al: Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line. Lung Cancer. 63:219–226. 2009. View Article : Google Scholar