Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with tyrosine kinase inhibitors or cetuximab

BMC Medicine

Jacques De Grève²  Ijeoma Adaku Umelo²  Erik Teugels²  Peter Kronenberger¹  Gang Chen³ 

[1] Laboratory for Biotechnology, Department of Gezondheidszorg, Erasmushogeschool Brussel, Laarbeeklaan 125, 1090, Brussels, Belgium;Laboratory of Medical and Molecular Oncology and Department of Medical Oncology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090, Brussels, Belgium;Department of Pathology, First Affiliated Hospital, Guangxi Medical University, Shuangyong Road 6, 530021, Nanning Guangxi, People's Republic of China

关键词: lung cancer;    apoptosis;    proliferation;    anti-EGFR monoclonal antibodies (mAbs);    tyrosine kinase inhibitors (TKIs);    RNA interference;    EGFR;   

Others  :  1222512
DOI  :  10.1186/1741-7015-10-28

 received in 2011-11-27, accepted in 2012-03-21,  发布年份 2012

【 摘 要 】

Background

The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab.

Methods

NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index.

Results

EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA.

Conclusions

EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.

【 授权许可】

   
2012 Chen et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Pfister DG, Johnson DH, Azzoli CG, Sause W, Smith TJ, Baker S Jr, Olak J, Stover D, Strawn JR, Turrisi AT, Somerfield MR: American Society of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 2004, 22:330-353.
• [2]Rosell R, Gervais R, Vergnenegre A, Massuti B, Felip E, Cardenal F, Garcia Gomez R, Pallares C, Sanchez JM, Porta R, Cobo M, Di Seri M, Garrido Lopez P, Insa A, De Marinis F, Corre R, Carreras M, Carcereny E, Taron M, Paz-Ares LG: Erlotinib versus chemotherapy (CT) in advanced non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations: Interim results of the European Erlotinib Versus Chemotherapy (EURTAC) phase III randomized trial. J Clin Oncol 2011, 29:s1.
• [3]Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, Nishiwaki Y, Ohe Y, Yang JJ, Chewaskulyong B, Jiang H, Duffield EL, Watkins CL, Armour AA, Fukuoka M: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009, 361:947-957.
• [4]Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H: Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005, 2:e73.
• [5]Harari PM, Allen GW, Bonner JA: Biology of interactions: antiepidermal growth factor receptor agents. J Clin Oncol 2007, 25:4057-4065.
• [6]Mendelsohn J, Baselga J: Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003, 21:2787-2799.
• [7]Zhou S, Ren S, Yan L, Zhang L, Tang L, Zhang J, Zhou C: Clinical efficacy of erlotinib in patients previously treated for advanced non-small cell lung cancer. Respirology 2009, 14:709-715.
• [8]Felip E, Rojo F, Reck M, Heller A, Klughammer B, Sala G, Cedres S, Peralta S, Maacke H, Foernzler D, Parera M, Möcks J, Saura C, Gatzemeier U, Baselga J: A phase II pharmacodynamic study of erlotinib in patients with advanced non-small cell lung cancer previously treated with platinum-based chemotherapy. Clin Cancer Res 2008, 14:3867-3874.
• [9]Milton DT, Azzoli CG, Heelan RT, Venkatraman E, Gomez JE, Kris MG, Krug LM, Pao W, Rizvi NA, Dunne M, Miller VA: A phase I/II study of weekly high-dose erlotinib in previously treated patients with nonsmall cell lung cancer. Cancer 2006, 107:1034-1041.
• [10]Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, Maoleekoonpiroj S, Smylie M, Martins R, van Kooten M, Dediu M, Findlay B, Tu D, Johnston D, Bezjak A, Clark G, Santabárbara P, Seymour L, National Cancer Institute of Canada Clinical Trials Group: Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005, 353:123-132.
• [11]Kelly K, Chansky K, Gaspar LE, Albain KS, Jett J, Ung YC, Lau DH, Crowley JJ, Gandara DR: Phase III trial of maintenance gefitinib or placebo after concurrent chemoradiotherapy and docetaxel consolidation in inoperable stage III non-small-cell lung cancer: SWOG S0023. J Clin Oncol 2008, 26:2450-2456.
• [12]Giaccone G, Wang Y: Strategies for overcoming resistance to EGFR family tyrosine kinase inhibitors. Cancer Treat Rev 2011, 37:456-464.
• [13]Yap TA, Vidal L, Adam J, Stephens P, Spicer J, Shaw H, Ang J, Temple G, Bell S, Shahidi M, Uttenreuther-Fischer M, Stopfer P, Futreal A, Calvert H, de Bono JS, Plummer R: Phase I trial of the irreversible EGFR and HER2 kinase inhibitor BIBW 2992 in patients with advanced solid tumors. J Clin Oncol 2010, 28:3965-3972.
• [14]Spicer JF, Rudman SM: EGFR inhibitors in non-small cell lung cancer (NSCLC): the emerging role of the dual irreversible EGFR/HER2 inhibitor BIBW 2992. Target Oncol 2010.
• [15]Minkovsky N, Berezov A: BIBW-2992, a dual receptor tyrosine kinase inhibitor for the treatment of solid tumors. Curr Opin Investig Drugs 2008, 9:1336-1346.
• [16]Eskens FA, Mom CH, Planting AS, Gietema JA, Amelsberg A, Huisman H, van Doorn L, Burger H, Stopfer P, Verweij J, de Vries EG: A phase I dose escalation study of BIBW 2992, an irreversible dual inhibitor of epidermal growth factor receptor 1 (EGFR) and 2 (HER2) tyrosine kinase in a 2-week on, 2-week off schedule in patients with advanced solid tumours. Br J Cancer 2008, 98:80-85.
• [17]Schutze C, Dorfler A, Eicheler W, Zips D, Hering S, Solca F, Baumann M, Krause M: Combination of EGFR/HER2 tyrosine kinase inhibition by BIBW 2992 and BIBW 2669 with irradiation in FaDu human squamous cell carcinoma. Strahlenther Onkol 2007, 183:256-264.
• [18]Griffin S, Walker S, Sculpher M, White S, Erhorn S, Brent S, Dyker A, Ferrie L, Gilfillan C, Horsley W, Macfarlane K, Thomas S: Cetuximab plus radiotherapy for the treatment of locally advanced squamous cell carcinoma of the head and neck. Health Technol Assess 2009, 13:s49-s54.
• [19]Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, Sur R, Raben D, Jassem J, Ove R, Kies MS, Baselga J, Youssoufian H, Amellal N, Rowinsky EK, Ang KK: Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006, 354:567-578.
• [20]Hanna N, Lilenbaum R, Ansari R, Lynch T, Govindan R, Janne PA, Bonomi P: Phase II trial of cetuximab in patients with previously treated non-small-cell lung cancer. J Clin Oncol 2006, 24:5253-5258.
• [21]Pirker R, Pereira JR, Szczesna A, von Pawel J, Krzakowski M, Ramlau R, Vynnychenko I, Park K, Yu CT, Ganul V, Roh JK, Bajetta E, O'Byrne K, de Marinis F, Eberhardt W, Goddemeier T, Emig M, Gatzemeier U, FLEX Study Team: Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an open-label randomised phase III trial. Lancet 2009, 373:1525-1531.
• [22]Burnett JC, Rossi JJ, Tiemann K: Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol J 2011, 6:1130-46.
• [23]Hickerson RP, Leachman SA, Pho LN, Gonzalez-Gonzalez E, Smith FJ, McLean WH, Contag CH, Leake D, Milstone LM, Kaspar RL: Development of quantitative molecular clinical end points for siRNA clinical trials. J Invest Dermatol 2011, 131:1029-1036.
• [24]Giovannetti E, Lemos C, Tekle C, Smid K, Nannizzi S, Rodriguez JA, Ricciardi S, Danesi R, Giaccone G, Peters GJ: Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells. Mol Pharmacol 2008, 73:1290-1300.
• [25]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.
• [26]Alemany R, Ruan S, Kataoka M, Koch PE, Mukhopadhyay T, Cristiano RJ, Roth JA, Zhang WW: Growth inhibitory effect of anti-K-ras adenovirus on lung cancer cells. Cancer Gene Ther 1996, 3:296-301.
• [27]Chenau J, Michelland S, de Fraipont F, Josserand V, Coll JL, Favrot MC, Seve M: The cell line secretome, a suitable tool for investigating proteins released in vivo by tumors: application to the study of p53-modulated proteins secreted in lung cancer cells. J Proteome Res 2009, 8:4579-4591.
• [28]Sos ML, Koker M, Weir BA, Heynck S, Rabinovsky R, Zander T, Seeger JM, Weiss J, Fischer F, Frommolt P, Michel K, Peifer M, Mermel C, Girard L, Peyton M, Gazdar AF, Minna JD, Garraway LA, Kashkar H, Pao W, Meyerson M, Thomas RK: PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res 2009, 69:3256-3261.
• [29]Choi YJ, Rho JK, Jeon BS, Choi SJ, Park SC, Lee SS, Kim HR, Kim CH, Lee JC: 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 2010, 66:381-388.
• [30]Shimamura T, Lowell AM, Engelman JA, Shapiro GI: Epidermal growth factor receptors harboring kinase domain mutations associate with the heat shock protein 90 chaperone and are destabilized following exposure to geldanamycins. Cancer Res 2005, 65:6401-6408.
• [31]Tang Z, Du R, Jiang S, Wu C, Barkauskas DS, Richey J, Molter J, Lam M, Flask C, Gerson S, Dowlati A, Liu L, Lee Z, Halmos B, Wang Y, Kern JA, Ma PC: Dual MET-EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer. Br J Cancer 2008, 99:911-922.
• [32]Chen G, Kronenberger P, Teugels E, De Greve J: Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells. Biol Proced Online 2011, 13:1. BioMed Central Full Text
• [33]Chen G, Kronenberger P, Umelo IA, Teugels E, De Greve J: Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction. Anal Biochem 2010, 398:266-268.
• [34]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402-408.
• [35]Suhnel J: Zero interaction response surfaces for combined-action assessment. Food Chem Toxicol 1996, 34:1151-1153.
• [36]Koivusalo R, Krausz E, Helenius H, Hietanen S: Chemotherapy compounds in cervical cancer cells primed by reconstitution of p53 function after short interfering RNA-mediated degradation of human papillomavirus 18 E6 mRNA: opposite effect of siRNA in combination with different drugs. Mol Pharmacol 2005, 68:372-382.
• [37]Chou TC: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006, 58:621-681.
• [38]Tabernero J, Ciardiello F, Rivera F, Rodriguez-Braun E, Ramos FJ, Martinelli E, Vega-Villegas ME, Roselló S, Liebscher S, Kisker O, Macarulla T, Baselga J, Cervantes A: Cetuximab administered once every second week to patients with metastatic colorectal cancer: a two-part pharmacokinetic/pharmacodynamic phase I dose-escalation study. Ann Oncol 2010, 21:1537-1545.
• [39]Kobayashi S, Ji H, Yuza Y, Meyerson M, Wong KK, Tenen DG, Halmos B: An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res 2005, 65:7096-7101.
• [40]Regales L, Gong Y, Shen R, de Stanchina E, Vivanco I, Goel A, Koutcher JA, Spassova M, Ouerfelli O, Mellinghoff IK, Zakowski MF, Politi KA, Pao W: Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer. J Clin Invest 2009, 119:3000-3010.
• [41]Janjigian YY, Groen HJ, Horn L, Smit EF, Fu Y, Wang F, Shahidi M, Denis LJ, Pao W, Miller VA: Activity and tolerability of afatinib (BIBW 2992) and cetuximab in NSCLC patients with acquired resistance to erlotinib or gefitinib. J Clin Oncol 2011, 29:s7525^.
• [42]Engelman JA, Mukohara T, Zejnullahu K, Lifshits E, Borrás AM, Gale CM, Naumov GN, Yeap BY, Jarrell E, Sun J, Tracy S, Zhao X, Heymach JV, Johnson BE, Cantley LC, Jänne PA: Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J Clin Invest 2006, 116:2695-2706.
• [43]Rothenberg SM, Engelman JA, Le S, Riese DJ, Haber DA, Settleman J: Modeling oncogene addiction using RNA interference. Proc Natl Acad Sci USA 2008, 105:12480-12484.
• [44]Sordella R, Bell DW, Haber DA, Settleman J: Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004, 305:1163-1167.
• [45]Yamanaka S, Gu Z, Sato M, Fujisaki R, Inomata K, Sakurada A, Inoue A, Nukiwa T, Kondo T, Horii A: siRNA targeting against EGFR, a promising candidate for a novel therapeutic application to lung adenocarcinoma. Pathobiology 2008, 75:2-8.
• [46]Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M, Zakowski MF, Heelan RT, Kris MG, Varmus HE: KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2005, 2:e17.
• [47]Massarelli E, Varella-Garcia M, Tang X, Xavier AC, Ozburn NC, Liu DD, Bekele BN, Herbst RS, Wistuba II: 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.
• [48]Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, Settleman J: A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival. Cancer Cell 2009, 15:489-500.
• [49]Sunaga N, Shames DS, Girard L, Peyton M, Larsen JE, Imai H, Soh J, Sato M, Yanagitani N, Kaira K, Xie Y, Gazdar AF, Mori M, Minna JD: Knockdown of oncogenic KRAS in non-small cell lung cancers suppresses tumor growth and sensitizes tumor cells to targeted therapy. Mol Cancer Ther 2011, 10:336-346.
• [50]Boehrer S, Adès L, Braun T, Galluzzi L, Grosjean J, Fabre C, Le Roux G, Gardin C, Martin A, de Botton S, Fenaux P, Kroemer G: Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study. Blood 2008, 111:2170-2180.
• [51]Reid T, Jin X, Song H, Tang HJ, Reynolds RK, Lin J: Modulation of Janus kinase 2 by p53 in ovarian cancer cells. Biochem Biophys Res Commun 2004, 321:441-447.
• [52]Busser B, Sancey L, Josserand V, Niang C, Favrot MC, Coll JL, Hurbin A: Amphiregulin promotes BAX inhibition and resistance to gefitinib in non-small-cell lung cancers. Mol Ther 2010, 18:528-535.
• [53]Tam IY, Leung EL, Tin VP, Chua DT, Sihoe AD, Cheng LC, Chung LP, Wong MP: Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations. Mol Cancer Ther 2009, 8:2142-2151.
• [54]Rice SD, Bush JE, Brower SL: Assessment of erlotinib in chemoresponse assay. Anticancer Res 2009, 29:1993-1997.
• [55]Gadgeel SM, Ali S, Philip PA, Wozniak A, Sarkar FH: Genistein enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nuclear factor kappa B in nonsmall cell lung cancer cell lines. Cancer 2009, 115:2165-2176.
• [56]Yun CH, Boggon TJ, Li Y, Woo MS, Greulich H, Meyerson M, Eck MJ: Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 2007, 11:217-227.
• [57]Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR, Padera RF, Shapiro GI, Baum A, Himmelsbach F, Rettig WJ, Meyerson M, Solca F, Greulich H, Wong KK: BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 2008, 27:4702-4711.
• [58]Takezawa K, Okamoto I, Tanizaki J, Kuwata K, Yamaguchi H, Fukuoka M, Nishio K, Nakagawa K: Enhanced anticancer effect of the combination of BIBW2992 and thymidylate synthase-targeted agents in non-small cell lung cancer with the T790M mutation of epidermal growth factor receptor. Mol Cancer Ther 2010, 9:1647-1656.
• [59]Marshall J: Clinical implications of the mechanism of epidermal growth factor receptor inhibitors. Cancer 2006, 107:1207-1218.
• [60]Huang S, Armstrong EA, Benavente S, Chinnaiyan P, Harari PM: Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor. Cancer Res 2004, 64:5355-5362.
• [61]Matar P, Rojo F, Cassia R, Moreno-Bueno G, Di Cosimo S, Tabernero J, Guzman M, Rodriguez S, Arribas J, Palacios J, Baselga J: Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting. Clin Cancer Res 2004, 10:6487-6501.
• [62]Ramalingam S, Forster J, Naret C, Evans T, Sulecki M, Lu H, Teegarden P, Weber MR, Belani CP: Dual inhibition of the epidermal growth factor receptor with cetuximab, an IgG1 monoclonal antibody, and gefitinib, a tyrosine kinase inhibitor, in patients with refractory non-small cell lung cancer (NSCLC): a phase I study. J Thorac Oncol 2008, 3:258-264.
• [63]Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A: Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464:1067-1070.