Abstract
We previously demonstrated that the transcription factor NF-E2-related factor2 (Nrf2), expressed abundantly in non-small-cell lung cancer (NSCLC) cells, plays a pivotal role in the proliferation and chemoresistance of NSCLC. Here we show that Nrf2-mediated NSCLC cell proliferation is dually regulated by epidermal growth factor receptor (EGFR) signaling and an Nrf2 repressor protein Keap1 (Kelch-like ECH-associated protein-1). NSCLC cells expressing wild-type EGFR and Keap1 genes show enhanced proliferation on stimulation with EGFR ligand under non-stress conditions. Exposure to cigarette smoke extract (CSE) enhanced cell proliferation by modification of the Nrf2/Keap1 interaction. Although EGFR-tyrosine kinase inhibitor (TKI) inhibited the proliferation of these cells, exposure to CSE attenuated its efficacy. In NSCLC cells with Keap1 gene mutations, Nrf2 was constitutively activated owing to dysfunction of Keap1 and cells proliferated independently of EGFR signaling. Furthermore, EGFR-TKI was unable to inhibit their proliferation. In NSCLC cells with EGFR gene mutations, Nrf2 was constitutively activated by EGFR signaling. In these cells, proliferation was largely dependent on the EGFR signaling pathway. Although these cells were highly sensitive to EGFR-TKI, exposure to CSE or knockdown of Keap1 mRNA reduced sensitivity to EGFR-TKI. We found a case of NSCLC showing resistance to EGFR-TKI despite having EGFR-TKI-sensitive EGFR gene mutation because of dysfunctional mutation in Keap1 gene. Results indicate that oxidative stress reduces the anticancer effects of EGFR-TKI in wild-type Keap1 NSCLC cells. Analysis of Keap1 dysfunction may become a novel molecular marker to predict resistance to EGFR-TKI in NSCLC cells having EGFR-TKI-sensitive EGFR mutations. Finally, as the downstream molecule of both EGFR and Keap1 signaling, Nrf2 is an important molecular target for the treatment of NSCLC, where cells have mutations in EGFR, KRAS or Keap1 genes.
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References
Espey DK, Wu XC, Swan J, Wiggins C, Jim MA, Ward E et al. Annual report to the nation on the status of cancer, 1975–2004, featuring cancer in American Indians and Alaska Natives. Cancer 2007; 110: 2119–2152.
Zhang DD . Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug Metab Rev 2006; 38: 769–789.
Ohta T, Iijima K, Miyamoto M, Nakahara I, Tanaka H, Ohtsuji M et al. Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. Cancer Res 2008; 68: 1303–1309.
Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO et al. Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLOS Med 2006; 3: 1865–1876.
Homma S, Ishii Y, Morishima Y, Yamadori T, Matsuno Y, Haraguchi N et al. Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer. Clin Cancer Res 2009; 15: 3423–3432.
Singh A, Boldin-Adamsky S, Thimmulappa RK, Rath SK, Ashush H, Coulter J et al. RNAi-mediated silencing of nuclear factor erythroid-2-related factor-2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy. Cancer Res 2008; 68: 7975–7984.
Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 2004; 24: 7130–7139.
Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M . Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol 2004; 24: 10941–10953.
Solis LM, Behrens C, Dong W, Suraokar M, Ozburn NC, Moran CA et al. Nrf2 and Keap1 abnormalities in non-small cell lung carcinoma and association with clinicopathologic features. Clin Cancer Res 2010; 16: 3743–3753.
Kim KC, Kang KA, Zhang R, Piao MJ, Kim GY, Kang MY et al. Up-regulation of Nrf2-mediated heme oxygenase-1 expression by eckol, a phlorotannin compound, through activation of Erk and PI3K/Akt. Int J Biochem Cell Biol 2009; 42: 297–305.
Scaltriti M, Baselga J . The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 2006; 12: 5268–5272.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350: 2129–2139.
Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S et al. EGFR mutation in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–1500.
Sakai K, Arao T, Shimoyama T, Murofushi K, Sekijima M, Kaji N et al. Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor. FASEB J 2006; 20: 311–313.
Okabe T, Okamoto I, Tamura K, Terashima M, Yoshida T, Satoh T et al. Differential constitutive activation of the epidermal growth factor receptor in non-small cell lung cancer cells bearing EGFR gene mutation and amplification. Cancer Res 2007; 67: 2046–2053.
Itoh K, Tong KI, Yamamoto M . Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med 2004; 36: 1208–1213.
Kode A, Rajendrasozhan S, Caito S, Yang SR, Megson IL, Rahman I et al. Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2008; 294: L478–L488.
Yageta Y, Ishii Y, Morishima Y, Masuko H, Ano S, Yamadori T et al. Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice. J Virol 2011; 85: 4679–4690.
Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 2010; 362: 2380–2388.
Jackman DM, Yeap BY, Sequist LV, Lindeman N, Holmes AJ, Joshi VA et al. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin Cancer Res 2006; 12: 3908–3914.
Riely GJ, Pao W, Pham D, Li AR, Rizvi N, Venkatraman ES et al. Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res 2006; 12: 839–844.
Massarelli E, Varella-Garcia M, Tang X, Xavier AC, Ozburn NC, Liu DD et al. 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 2007; 13: 2890–2896.
Nguyen KS, Kobayashi S, 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 2009; 10: 281–289.
Xu Y, Liu H, Chen J, Zhou Q . Acquired resistance of lung adenocarcinoma to EGFR-tyrosine kinase inhibitors gefitinib and erlotinib. Cancer Biol Ther 2010; 9: 572–582.
Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005; 352: 786–792.
Muscarella LA, Parrella P, D’Alessandro V, la Torre A, Barbano R, Fontana A et al. Frequent epigenetics inactivation of KEAP1 gene in non-small cell lung cancer. Epigenetics 2011; 6: 710–719.
Kreuzer KA, Le Coutre P, Landt O, Na IK, Schwarz M, Schultheis K et al. Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique. Ann Hematol 2003; 82: 284–289.
Mitchell CE, Belinsky SA, Lechner JF . Detection and quantitation of mutant K-ras codon 12 restriction fragments by capillary electrophoresis. Anal Biochem 1995; 224: 148–153.
Janmaat ML, Rodriguez JA, Gallegos-Ruiz M, Kruyt FA, Giaccone G . Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells. Int J Cancer 2006; 118: 209–214.
Huber RM, Stratakis DF . Molecular oncology—perspectives in lung cancer. Lung Cancer 2004; 45: S209–S213.
DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011; 475: 106–109.
Ren D, Villeneuve NF, Jiang T, Wu T, Lau A, Toppin HA et al. Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc Natl Acad Sci USA 2011; 108: 1433–1438.
Yao H, Edirisinghe I, Yang SR, Rajendrasozhan S, Kode A, Caito S et al. Genetic ablation of NADPH oxidase enhances susceptibility to cigarette smoke-induced lung inflammation and emphysema in mice. Am J Pathol 2008; 172: 1222–1237.
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This work was partially supported by a grant from the Smoking Research Foundation.
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Yamadori, T., Ishii, Y., Homma, S. et al. Molecular mechanisms for the regulation of Nrf2-mediated cell proliferation in non-small-cell lung cancers. Oncogene 31, 4768–4777 (2012). https://doi.org/10.1038/onc.2011.628
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DOI: https://doi.org/10.1038/onc.2011.628
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