Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer

Abstract

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial–mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

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References

  1. 1.

    The Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–50.

  2. 2.

    Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ, Iafrate AJ, Wistuba II. et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014;311:1998–2006.

  3. 3.

    Hirsch FR, Scagliotti GV, Mulshine JL, Kwon R, Curran WJ Jr., Wu YL, et al. Lung cancer: current therapies and new targeted treatments. Lancet. 2017;389:299–311.

  4. 4.

    Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361:947–57.

  5. 5.

    Rosell R, Molina MA, Costa C, Simonetti S, Gimenez-Capitan A, Bertran-Alamillo J, et al. Pretreatment EGFR T790M mutation and BRCA1 mRNA expression in erlotinib-treated advanced non-small-cell lung cancer patients with EGFR mutations. Clin Cancer Res. 2011;17:1160–8.

  6. 6.

    Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13:239–46.

  7. 7.

    Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3:75ra26.

  8. 8.

    Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat Genet. 2012;44:852–60.

  9. 9.

    Song KA, Niederst MJ, Lochmann TL, Hata AN, Kitai H, Ham J, et al. Epithelial-to-mesenchymal transition antagonizes response to targeted therapies in lung cancer by suppressing BIM. Clin Cancer Res. 2017;24:197–208.

  10. 10.

    McGowan M, Kleinberg L, Halvorsen AR, Helland A, Brustugun OT. NSCLC depend upon YAP expression and nuclear localization after acquiring resistance to EGFR inhibitors. Genes Cancer. 2017;8:497–504.

  11. 11.

    Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8.

  12. 12.

    Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90.

  13. 13.

    Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res. 2013;19:279–90.

  14. 14.

    Suda K, Tomizawa K, Fujii M, Murakami H, Osada H, Maehara Y, 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. 2011;6:1152–61.

  15. 15.

    Witta SE, Gemmill RM, Hirsch FR, Coldren CD, Hedman K, Ravdel L, et al. Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res. 2006;66:944–50.

  16. 16.

    Izumchenko E, Chang X, Michailidi C, Kagohara L, Ravi R, Paz K, et al. The TGFβ-miR200-MIG6 pathway orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors. Cancer Res. 2014;74:3995–4005.

  17. 17.

    Jakobsen KR, Demuth C, Sorensen BS, Nielsen AL. The role of epithelial to mesenchymal transition in resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Transl Lung Cancer Res. 2016;5:172–82.

  18. 18.

    Vazquez-Martin A, Cufi S, Oliveras-Ferraros C, Torres-Garcia VZ, Corominas-Faja B, Cuyas E, et al. IGF-1R/epithelial-to-mesenchymal transition (EMT) crosstalk suppresses the erlotinib-sensitizing effect of EGFR exon 19 deletion mutations. Sci Rep. 2013;3:2560.

  19. 19.

    Yochum ZA, Socinski MA, Burns TF. Paradoxical functions of ZEB1 in EGFR-mutant lung cancer: tumor suppressor and driver of therapeutic resistance. J Thorac Dis. 2016;8:E1528–E31.

  20. 20.

    Yoshida T, Song L, Bai Y, Kinose F, Li J, Ohaegbulam KC, et al. ZEB1 mediates acquired resistance to the epidermal growth factor receptor-tyrosine kinase inhibitors in non-small cell lung cancer. PLoS ONE. 2016;11:e0147344.

  21. 21.

    Zhang T, Guo L, Creighton CJ, Lu Q, Gibbons DL, Yi ES, et al. A genetic cell context-dependent role for ZEB1 in lung cancer. Nat Commun. 2016;7:12231.

  22. 22.

    Chang TH, Tsai MF, Su KY, Wu SG, Huang CP, Yu SL, et al. Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor. Am J Respir Crit Care Med. 2011;183:1071–9.

  23. 23.

    Burns TF, Dobromilskaya I, Murphy SC, Gajula RP, Thiyagarajan S, Chatley SN, et al. Inhibition of TWIST1 leads to activation of oncogene-induced senescence in oncogene-driven non-small cell lung cancer. Mol Cancer Res. 2013;11:329–38.

  24. 24.

    Tran PT, Shroff EH, Burns TF, Thiyagarajan S, Das ST, Zabuawala T, et al. Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis. PLoS Genet. 2012;8:e1002650.

  25. 25.

    Yochum ZA, Cades J, Mazzacurati L, Neumann NM, Khetarpal SK, Chatterjee S, et al. A first-in-class TWIST1 inhibitor with activity in oncogene-driven lung cancer. Mol Cancer Res. 2017;15:1764–76.

  26. 26.

    Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117:927–39.

  27. 27.

    Pham CG, Bubici C, Zazzeroni F, Knabb JR, Papa S, Kuntzen C, et al. Upregulation of Twist-1 by NF-kappaB blocks cytotoxicity induced by chemotherapeutic drugs. Mol Cell Biol. 2007;27:3920–35.

  28. 28.

    Cheng GZ, Chan J, Wang Q, Zhang W, Sun CD, Wang LH. Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res. 2007;67:1979–87.

  29. 29.

    Ansieau S, Bastid J, Doreau A, Morel AP, Bouchet BP, Thomas C, et al. Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer Cell. 2008;14:79–89.

  30. 30.

    Sos ML, Koker M, Weir BA, Heynck S, Rabinovsky R, Zander T, et al. PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res. 2009;69:3256–61.

  31. 31.

    Hwang W, Chiu YF, Kuo MH, Lee KL, Lee AC, Yu CC, et al. Expression of neuroendocrine factor VGF in lung cancer cells confers resistance to EGFR kinase inhibitors and triggers epithelial-to-mesenchymal transition. Cancer Res. 2017;77:3013–26.

  32. 32.

    Jin HO, Hong SE, Woo SH, Lee JH, Choe TB, Kim EK, et al. Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition. Cell Death Dis. 2012;3:e319.

  33. 33.

    Roberts CM, Tran MA, Pitruzzello MC, Wen W, Loeza J, Dellinger TH, et al. TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6, L1CAM, and Akt signalling. Sci Rep. 2016;6:37652.

  34. 34.

    Zhuo WL, Wang Y, Zhuo XL, Zhang YS, Chen ZT. Short interfering RNA directed against TWIST, a novel zinc finger transcription factor, increases A549 cell sensitivity to cisplatin via MAPK/mitochondrial pathway. Biochem Biophys Res Commun. 2008;369:1098–102.

  35. 35.

    Faber AC, Corcoran RB, Ebi H, Sequist LV, Waltman BA, Chung E, et al. BIM expression in treatment-naive cancers predicts responsiveness to kinase inhibitors. Cancer Discov. 2011;1:352–65.

  36. 36.

    Ohashi K, Sequist LV, Arcila ME, Moran T, Chmielecki J, Lin YL, 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. 2012;109:E2127–33.

  37. 37.

    Shi P, Oh YT, Deng L, Zhang G, Qian G, Zhang S, et al. Overcoming acquired resistance to AZD9291, a third generation EGFR inhibitor, through modulation of MEK/ERK-dependent Bim and Mcl-1 degradation. Clin Cancer Res 2017;23:6567–79.

  38. 38.

    Costa DB, Halmos B, Kumar A, Schumer ST, Huberman MS, Boggon TJ, et al. BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med. 2007;4:1669–79. discussion 1680.

  39. 39.

    Deng J, Shimamura T, Perera S, Carlson NE, Cai D, Shapiro GI, et al. Proapoptotic BH3-only BCL-2 family protein BIM connects death signaling from epidermal growth factor receptor inhibition to the mitochondrion. Cancer Res. 2007;67:11867–75.

  40. 40.

    Sionov RV, Vlahopoulos SA, Granot Z. Regulation of Bim in health and disease. Oncotarget. 2015;6:23058–134.

  41. 41.

    Chang AT, Liu Y, Ayyanathan K, Benner C, Jiang Y, Prokop JW, et al. An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors. Genes Dev. 2015;29:603–16.

  42. 42.

    Politi K, Zakowski MF, Fan PD, Schonfeld EA, Pao W, Varmus HE. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev. 2006;20:1496–510.

  43. 43.

    Zheng H, Kang Y. Multilayer control of the EMT master regulators. Oncogene. 2014;33:1755–63.

  44. 44.

    Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, et al. Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer. N Engl J Med. 2018;378:113–25.

  45. 45.

    Ramalingam SS, Yang JC, Lee CK, Kurata T, Kim DW, John T, et al. Osimertinib as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer. J Clin Oncol. 2018;36:841–9.

  46. 46.

    Ricordel C, Llamas-Gutierrez F, Chiforeanu D, Lena H, Corre R. Large cell neuroendocrine lung carcinoma transformation as an acquired resistance mechanism to osimertinib. J Thorac Oncol. 2017;12:e184–6.

  47. 47.

    Kim TM, Song A, Kim DW, Kim S, Ahn YO, Keam B, et al. Mechanisms of acquired resistance to AZD9291: a mutation-selective, irreversible EGFR inhibitor. J Thorac Oncol. 2015;10:1736–44.

  48. 48.

    Eberlein CA, Stetson D, Markovets AA, Al-Kadhimi KJ, Lai Z, Fisher PR, et al. Acquired resistance to the mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models. Cancer Res. 2015;75:2489–500.

  49. 49.

    Planchard D, Loriot Y, Andre F, Gobert A, Auger N, Lacroix L, et al. EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients. Ann Oncol. 2015;26:2073–8.

  50. 50.

    Ou SI, Agarwal N, Ali SM. 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. 2016;98:59–61.

  51. 51.

    Li L, Wang H, Li C, Wang Z, Zhang P, Yan X. Transformation to small-cell carcinoma as an acquired resistance mechanism to AZD9291: a case report. Oncotarget. 2017;8:18609–14.

  52. 52.

    Moloudizargari M, Mikaili P, Aghajanshakeri S, Asghari MH, Shayegh J. Pharmacological and therapeutic effects of Peganum harmala and its main alkaloids. Pharmacogn Rev. 2013;7:199–212.

  53. 53.

    Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med. 2015;373:1627–39.

  54. 54.

    Tanimoto A, Takeuchi S, Arai S, Fukuda K, Yamada T, Roca X, et al. Histone deacetylase 3 inhibition overcomes BIM Deletion polymorphism-mediated osimertinib resistance in EGFR-mutant lung cancer. Clin Cancer Res. 2017;23:3139–49.

  55. 55.

    Soh SX, Siddiqui FJ, Allen JC, Kim GW, Lee JC, Yatabe Y, et al. A systematic review and meta-analysis of individual patient data on the impact of the BIM deletion polymorphism on treatment outcomes in epidermal growth factor receptor mutant lung cancer. Oncotarget. 2017;8:41474–86.

  56. 56.

    Jacqueroud L, Bouard C, Richard G, Payen L, Devouassoux-Shisheboran M, Spicer DB, et al. The heterodimeric TWIST1-E12 complex drives the oncogenic potential of TWIST1 in human mammary epithelial cells. Neoplasia. 2016;18:317–27.

  57. 57.

    Pan D, Fujimoto M, Lopes A, Wang YX. Twist-1 is a PPARdelta-inducible, negative-feedback regulator of PGC-1alpha in brown fat metabolism. Cell. 2009;137:73–86.

  58. 58.

    Chen YT, Akinwunmi PO, Deng JM, Tam OH, Behringer RR. Generation of a Twist1 conditional null allele in the mouse. Genesis. 2007;45:588–92.

  59. 59.

    Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell. 2006;124:1283–98.

  60. 60.

    Tran PT, Bendapudi PK, Lin HJ, Choi P, Koh S, Chen J, et al. Survival and death signals can predict tumor response to therapy after oncogene inactivation. Sci Transl Med. 2011;3:103ra99.

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Acknowledgements

This work was supported by the following funding sources: HW has received research funding from the Uniting Against Lung Cancer Foundation. TFB was supported for this project from a V Foundation Scholar Award, American Lung Association Award (LCD 257864), Sidney Kimmel Foundation (SKF-15-099) and a Doris Duke Charitable Foundation Clinical Scientist Award (2015097), a Pittsburgh LUNG SPORE CDA P50CA090440 and generous support from the We Wish Foundation. This project used the Hillman Cancer Center Animal, Flow Cytometry and Biostatistics Facilities that are supported in part by award P30CA047904. ZAY has received research funding for this project from the Howard Hughes Medical Institute Medical Fellowship, T32GM008421-22 and a 1F30CA213765-01. PTT has received research funding for this project from the Nesbitt Family; Movember-Prostate Cancer Foundation; Uniting Against Lung Cancer Foundation; American Lung Association (LCD-339465); Commonwealth Foundation; Sidney Kimmel Foundation (SKF-13-021); ACS (122688-RSG-12-196-01-TBG); DoD (W81XWH-13-1-0182); NIH/NCI (R01CA166348). We Laura Stabile, PhD, James G. Herman, MD, and Frank P. Vendetti PhD at the University of Pittsburgh for the discussion, advice regarding experimental design and supply of reagents when applicable. In addition, we would like to acknowledge the helpful discussion and comments provided by the Burns, Rudin, and Tran laboratory members.

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Correspondence to Phuoc T. Tran or Timothy F. Burns.

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These authors contributed equally: Zachary A. Yochum, Jessica Cades and Hailun Wang.

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Yochum, Z.A., Cades, J., Wang, H. et al. Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer. Oncogene 38, 656–670 (2019). https://doi.org/10.1038/s41388-018-0482-y

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