Advertisement

Overcoming acquired resistance of gefitinib in lung cancer cells without T790M by AZD9291 or Twist1 knockdown in vitro and in vivo

  • Zhongwei Liu
  • Weimin GaoEmail author
Molecular Toxicology
  • 7 Downloads

Abstract

The T790M mutation is recognized as a typical mechanism of acquired resistance to first generation of epithermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib in non-small cell lung cancer (NSCLC) patients who are commonly treated by third generation of EGFR-TKI AZD9291 (osimertinib). However, the therapeutic strategy for overcoming acquired resistance to EGFR-TKIs in NSCLC patients without T790M remains to be definitively determined. In the present study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing NSCLC cell line H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11 months. The cytotoxic effects of gefitinib or AZD9291 in vitro were evaluated via the half maximal inhibitory concentrations (IC50s) determined by the MTT assay. IC50 of gefitinib in H1650GR (50.0 ± 3.0 µM) significantly increased compared with H1650 (31.0 ± 1.0 µM) (p < 0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3 ± 0.9 µM) significantly increased compared with H1975 (5.5 ± 0.6 µM) (p < 0.05). However, IC50 of AZD9291 on H1650GR (8.5 ± 0.5 µM) did not increase compared with H1650 (9.7 ± 0.7 µM). On the other hand, IC50 of AZD9291 on gefitinib-resistant A549 (A549GR established in our previous study) (12.7 ± 0.8 µM) was significantly increased compared with A549 (7.0 ± 1.0 µM) (p < 0.05). AZD9291 induced caspase 3/7 activation in A549, H1650, and H1650GR, but not in A549GR. Western blot analyses showed that p-Akt played a key role in determining the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291. Additionally, increased expression of Twist1 was observed in all cells with acquired EGFR-TKI resistance and knockdown of Twist1 by shRNA was found to significantly enhance the sensitivity of A549GR to gefitinib or AZD9291 via reversing epithelial–mesenchymal transition and downregulating p-Akt, but not of H1975AR to AZD9291. The enhanced cytotoxic effect of AZD9291 on A549GR by Twist1 knockdown in vitro was further validated by in vivo studies which showed that Twist1 knockdown could lead to significantly delayed tumor growth of A549GR xenograft with increased sensitivity to AZD9291 treatment in nude mice without any observed side toxic effects. In summary, our study demonstrated that the mechanisms of acquired resistance in different NSCLC cell lines treated by even the same EGFR-TKI might be quite different, which provide a rationale for adopting different therapeutic strategies for those NSCLC patients with acquired EGFR-TKI resistance based on different status of heterogeneous mutations.

Keywords

Gefitinib AZD9291 EGFR-TKI Drug resistance p-AKT Twist1 

Notes

Acknowledgements

This research was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R15ES026789. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethics approval

All procedures performed in animal studies were in accordance with the ethical standards and guidelines of Institutional Animal Care and Use Committee.

Supplementary material

204_2019_2453_MOESM1_ESM.pdf (421 kb)
Supplementary material 1 (PDF 421 kb)

References

  1. Ansieau S, Bastid J, Doreau A et al (2008) Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer Cell 14(1):79–89Google Scholar
  2. Brozovic A (2017) The relationship between platinum drug resistance and epithelial–mesenchymal transition. Arch Toxicol 91(2):605–619Google Scholar
  3. Burns TF, Dobromilskaya I, Murphy SC et al (2013) Inhibition of TWIST1 leads to activation of oncogene-induced senescence in oncogene-driven non–small cell lung cancer. Mol Cancer Res 11(4):329–338Google Scholar
  4. Caiola E, Frapolli R, Tomanelli M et al (2018) Wee1 inhibitor MK1775 sensitizes KRAS mutated NSCLC cells to sorafenib. Sci Rep 8(1):948Google Scholar
  5. Chen ZY, Zhong WZ, Zhang XC et al (2012) EGFR mutation heterogeneity and the mixed response to EGFR tyrosine kinase inhibitors of lung adenocarcinomas. Oncologist 17(7):978–985Google Scholar
  6. Choi YJ, Rho JK, Jeon BS et al (2010) Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors. Cancer Chemother Pharmacol 66(2):381–388Google Scholar
  7. Conde E, Angulo B, Tang M et al (2006) Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6 K signaling. Clin Cancer Res 12(3):710–717Google Scholar
  8. Cross DA, Ashton SE, Ghiorghiu S et al (2014) AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov 4(9):1046–1061Google Scholar
  9. de Freitas Silva BS, Yamamoto FP, Pontes FSC et al (2012) TWIST and p-Akt immunoexpression in normal oral epithelium oral dysplasia and in oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 17(1):e29–e34Google Scholar
  10. El Khoury F, Corcos L, Durand S, Simon B, Jossic C (2016) Acquisition of anticancer drug resistance is partially associated with cancer stemness in human colon cancer cells. Int J Oncol 49(6):2558–2568Google Scholar
  11. Gao W, Lu C, Chen L, Keohavong P (2015) Overexpression of CRM1: a characteristic feature in a transformed phenotype of lung carcinogenesis and a molecular target for lung cancer adjuvant therapy. J Thorac Oncol 10(5):815–825Google Scholar
  12. Gavert N, Ben-Ze’ev A (2008) Epithelial–mesenchymal transition and the invasive potential of tumors. Trends Mol Med 14(5):199–209Google Scholar
  13. Ghosh G, Lian X, Kron SJ, Palecek SP (2012) Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors. BMC Cancer 12:95Google Scholar
  14. Han F, He J, Li F et al (2015) Emerging roles of microRNAs in EGFR-targeted therapies for lung cancer. Biomed Res Int 2015:672759Google Scholar
  15. Huang L, Fu L (2015) Mechanisms of resistance to EGFR tyrosine kinase inhibitors. Acta Pharm Sin B 5(5):390–401Google Scholar
  16. Hwang W, Chiu YF, Kuo MH et al (2017) Expression of neuroendocrine factor VGF in lung cancer cells confers resistance to EGFR kinase inhibitors and triggers epithelial-to-mesenchymal transition. Cancer Res 77(11):3013–3026Google Scholar
  17. Jacobsen K, Bertran-Alamillo J, Molina MA et al (2017) Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat Commun 8(1):410Google Scholar
  18. Janjigian YY, Smit EF, Groen HJ et al (2014) Dual inhibition of EGFR with afatinib and cetuximab in kinase inhibitor–resistant EGFR-mutant lung cancer with and without T790M mutations. Cancer Discov 4(9):1036–1045Google Scholar
  19. Jänne P, Ahn M, Kim D et al (2015) LBA3 A phase I study of AZD9291 in patients with EGFR-TKI-resistant advanced NSCLC–updated progression free survival and duration of response data. Ann Oncol 26(1):60Google Scholar
  20. Jin H, Hong S, Woo S et al (2012) Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition. Cell Death Dis 3(6):e319Google Scholar
  21. Kazandjian D, Blumenthal GM, Yuan W et al (2016) FDA approval of gefitinib for the treatment of patients with metastatic EGFR mutation–positive non-small cell lung cancer. Clin Cancer Res 22(6):1307–1312Google Scholar
  22. Khan MA, Chen HC, Zhang D, Fu J (2013) Twist: a molecular target in cancer therapeutics. Tumor Biol 34(5):2497–2506Google Scholar
  23. Koizumi F, Shimoyama T, Taguchi F, Saijo N, Nishio K (2005) Establishment of a human non small cell lung cancer cell line resistant to gefitinib. Int J Cancer 116(1):36–44Google Scholar
  24. Kwak EL, Sordella R, Bell DW et al (2005) Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci USA 102(21):7665–7670Google Scholar
  25. Lantermann AB, Chen D, McCutcheon K et al (2015) Inhibition of casein kinase 1 alpha prevents acquired drug resistance to Erlotinib in EGFR-mutant non-small cell lung cancer. Cancer Res 75(22):4937–4948Google Scholar
  26. Lito P, Solomon M, Li LS, Hansen R, Rosen N (2016) Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science 351(6273):604–608Google Scholar
  27. Liu Z, Gao W (2017) Leptomycin B reduces primary and acquired resistance of gefitinib in lung cancer cells. Toxicol Appl Pharmacol 335:16–27Google Scholar
  28. Lu C, Shao C, Cobos E, Singh KP, Gao W (2012) Chemotherapeutic sensitization of leptomycin B resistant lung cancer cells by pretreatment with doxorubicin. PLoS One 7(3):e32895Google Scholar
  29. Lv T, Wang Q, Cromie M et al (2015) Twist1-mediated 4E-BP1 regulation through mTOR in non-small cell lung cancer. Oncotarget 6(32):33006–33018Google Scholar
  30. Marchetti A, Milella M, Felicioni L et al (2009) Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones. Neoplasia 11(10):1084–1092Google Scholar
  31. Massarelli E, Varella-Garcia M, Tang X et al (2007) KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. Clin Cancer Res 13(10):2890–2896Google Scholar
  32. Moore N, Lyle S (2011) Quiescent, slow-cycling stem cell populations in cancer: a review of the evidence and discussion of significance. J Oncol 2011:396076Google Scholar
  33. Nairismägi ML, Füchtbauer A, Labouriau R, Bramsen JB, Füchtbauer EM (2013) The proto-oncogene TWIST1 is regulated by microRNAs. PLoS One 8(5):e66070Google Scholar
  34. Niederst MJ, Sequist LV, Poirier JT et al (2015) RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer. Nat Commun 6:6377Google Scholar
  35. O’Connor L, Strasser A, O’Reilly LA et al (1998) Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J 17(2):384–395Google Scholar
  36. Ou SI, Agarwal N, Ali SM (2016) High MET amplification level as a resistance mechanism to osimertinib (AZD9291) in a patient that symptomatically responded to crizotinib treatment post-osimertinib progression. Lung Cancer 98:59–61Google Scholar
  37. Pallier K, Cessot A, Côté JF et al (2012) TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma. PLoS One 7(1):e29954Google Scholar
  38. Park MY, Jung MH, Eo EY et al (2017) Generation of lung cancer cell lines harboring EGFR T790M mutation by CRISPR/Cas9-mediated genome editing. Oncotarget 8(22):36331–36338Google Scholar
  39. Polosukhina D, Love HD, Correa H et al (2017) Functional KRAS mutations and a potential role for PI 3 K/AKT activation in Wilms tumors. Mol Oncol 11(4):405–421Google Scholar
  40. Puisieux A, Valsesia-Wittmann S, Ansieau S (2006) A twist for survival and cancer progression. Br J Cancer 94(1):13–17Google Scholar
  41. Rho JK, Choi YJ, Lee JK et al (2009) 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(2):219–226Google Scholar
  42. Ricciuti B, Mecca C, Cenci M et al (2015) miRNAs and resistance to EGFR—TKIs in EGFR-mutant non-small cell lung cancer: beyond ‘traditional mechanisms’ of resistance. Ecancermedicalscience 9:569Google Scholar
  43. Sato H, Shien K, Tomida S et al (2017) Targeting the miR-200c/LIN28B axis in acquired EGFR-TKI resistance non-small cell lung cancer cells harboring EMT features. Sci Rep 7:40847Google Scholar
  44. Seto T, Kato T, Nishio M et al (2014) Erlotinib alone or with bevacizumab as first-line therapy in patients with advanced non-squamous non-small-cell lung cancer harbouring EGFR mutations (JO25567): an open-label, randomised, multicentre, phase 2 study. Lancet Oncol 15(11):1236–1244Google Scholar
  45. Shao C, Lu C, Chen L et al (2011) p53-Dependent anticancer effects of leptomycin B on lung adenocarcinoma. Cancer Chemother Pharmacol 67(6):1369–1380Google Scholar
  46. Sharma SV, Lee DY, Li B et al (2010) A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141(1):69–80Google Scholar
  47. Soucheray M, Capelletti M, Pulido I et al (2015) Intratumoral heterogeneity in EGFR mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition. Cancer Res 75(20):4372–4383Google Scholar
  48. Suda K, Mitsudomi T (2015) Role of EGFR mutations in lung cancers: prognosis and tumor chemosensitivity. Arch Toxicol 89(8):1227–1240Google Scholar
  49. Suda K, Bunn PA Jr, Rivard CJ, Mitsudomi T, Hirsch FR (2017a) Primary double-strike therapy for cancers to overcome EGFR kinase inhibitor resistance: proposal from the bench. J Thorac Oncol 12(1):27–35Google Scholar
  50. Suda K, Rivard CJ, Mitsudomi T, Hirsch FR (2017b) Overcoming resistance to EGFR tyrosine kinase inhibitors in lung cancer, focusing on non-T790M mechanisms. Expert Rev Anticancer Ther 17(9):779–786Google Scholar
  51. Takezawa K, Pirazzoli V, Arcila ME et al (2012) HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discov 2(10):922–933Google Scholar
  52. Tang ZH, Jiang XM, Guo X et al (2016) Characterization of osimertinib (AZD9291)-resistant non-small cell lung cancer NCI-H1975/OSIR cell line. Oncotarget 7(49):81598–81610Google Scholar
  53. Thress KS, Paweletz CP, Felip E et al (2015) Acquired EGFR C797S mutation mediates resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M. Nat Med 21(6):560–562Google Scholar
  54. Tomasini P, Walia P, Labbe C, Jao K, Leighl N (2016) Targeting the KRAS pathway in non-small cell lung cancer. Oncologist 21(12):1450–1460Google Scholar
  55. Watanabe S, Sone T, Matsui T et al (2013) Transformation to small-cell lung cancer following treatment with EGFR tyrosine kinase inhibitors in a patient with lung adenocarcinoma. Lung Cancer 82(2):370–372Google Scholar
  56. Way TD, Huang JT, Chou CH et al (2014) Emodin represses TWIST1-induced epithelial–mesenchymal transitions in head and neck squamous cell carcinoma cells by inhibiting the β-catenin and Akt pathways. Eur J Cancer 50(2):366–378Google Scholar
  57. Wu SG, Shih JY (2018) Management of acquired resistance to EGFR TKI–targeted therapy in advanced non-small cell lung cancer. Mol Cancer 17(1):38Google Scholar
  58. Xia J, Bai H, Yan B et al (2017) Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer. Oncotarget 8(65):108522–108533Google Scholar
  59. Xu W, Yang Z, Lu N (2015) A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition. Cell Adh Migr 9(4):317–324Google Scholar
  60. Xue G, Restuccia DF, Lan Q et al (2012) Akt/PKB-mediated phosphorylation of Twist1 promotes tumor metastasis via mediating cross-talk between PI3K/Akt and TGF-β signaling axes. Cancer Discov 2(3):248–259Google Scholar
  61. Yochum ZA, Cades J, Mazzacurati L et al (2017) A first-in-class TWIST1 inhibitor with activity in oncogene-driven lung cancer. Mol Cancer Res 15(12):1764–1776Google Scholar
  62. Yochum ZA, Cades J, Wang H et al (2019) Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer. Oncogene 38:656–670Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Occupational and Environmental Health Sciences, School of Public HealthWest Virginia UniversityMorgantownUSA

Personalised recommendations