联合抑制IGF-1R和NF-κB信号通路逆转吉非替尼耐药的人肺癌细胞株H1650耐药的研究
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摘要
第一部分靶向IGF-1R的siRNA逆转吉非替尼耐药的人非小细胞肺癌细胞株H1975和H1650耐药的研究
目的：研究IGF-1R特异性的siRNA对逆转吉非替尼耐药的人非小细胞肺癌细胞H1975和H1650耐药的作用,并探讨其可能存在的机制。
方法：不同浓度的吉非替尼分别处理吉非替尼耐药的肺癌细胞株(H1975和H1650),吉非替尼敏感的肺癌细胞株(HCC827),以及RNAi的表达载体分别转染后的H1975和H1650细胞,采用MTT法检测吉非替尼对各处理组细胞的生长抑制作用;AnnexinV-FITC和PI双标法标记细胞后,在流式细胞仪上检测吉非替尼浓度为0、0.01、0.1和1uM的条件下,处理24h和48h后各组细胞的凋亡水平；Western blot法检测细胞内EGFR、IGF-1R通路相关蛋白和凋亡相关蛋白的表达。
结果：HCC827、H1975和H1650细胞株的IC50值分别为0.006±0.0003uM、24.62±0.3uM和18.39±0.2uM。经1uM吉非替尼处理48h后,H1975和H1650细胞中均检出了p-IGF-1R的高表达,而HCC827细胞中则未见明显的p-IGF-1R。当用siRNA技术分别沉默H1975和H1650细胞中的IGF-1R后,吉非替尼在两种细胞中的ICso值分别下降至7.94±0.1uM和9.42±0.4uM,明显低于IGF-1R未沉默的相应细胞(P<0.05)。作用24h和48h时,吉非替尼在IGF-1R沉默后的H1975和H1650细胞中诱导的凋亡比例均明显高于IGF-1R未沉默的相应细胞株(P<0.05)。IGF-1RsiRNA联合吉非替尼作用时明显抑制了H1975和H1650细胞中的p-IGF-1R(?)(?)p-Akt的表达。
结论：IGF-1R siRNA能有效增强吉非替尼在耐药的人肺癌细胞株H1975和H1650中诱导的生长抑制和凋亡效应。IGF-1R siRNA技术或可成为逆转EGFR-TKIs耐药的新策略。
第二部分
联合抑制IGF-1R和NF-KB信号通路逆转吉非替尼耐药的人肺癌细胞株H1650耐药的研究
目的：在第一部分研究中我们证明,ⅠGF-1R siRNA能够在EGFR-TKIs耐药的肿癌细胞中提高吉非替尼诱导的生长抑制和凋亡水平。然而,临床试验显示,联合使用IGF-1R印制剂(?)(?)EGFR-TKIs并未能在肺癌治疗中取得显效。新近发现,NF-KB通路是影响肺癌细胞对EGFR-TKIs敏感性的因素。我们旨在探索联合抑制(?)IGF-1R和NF-KB通路能否提高耐药细胞对吉非替尼的敏感性,并探讨其可能的机制。
方法：分别用吉非替尼和NF-KB特异性抑制齐(?)JPDTC处理IGF-1R未沉默和沉默的细胞H1650-sictrl和H1650-siIGF-1R。用MTT实验检测(?)PDTC和吉非替尼对各组细胞的生长抑制作用,流式细胞术检测细胞的凋亡水平,EMSA实验检测细胞内NF-kB的DNA结合能力,Western blot法检测细胞内EGFR、IGF-1R和NF-kB信号通路的相关蛋白表达,并检测凋亡相关蛋白PARPcl的水平。
结果：IkB在吉非替尼敏感的肺癌细胞HCC827中的水平高于吉非替尼耐药的H1975和H1650细胞,其表达因吉非替尼的作用而上调。在IGF-1R未沉默的H1650-sictrl细胞中,IKB的表达水平低于其在H1650-siIGF-1R细胞中的表达,且两种细胞中IKB水平均随吉非替尼浓度增加而出现上调。与H1650-sictrl细胞相比,PDTC在H1650-siIGF-1R细胞中明显增强了吉非替尼诱导的生长抑制和凋亡效应(P<0.05)。IGF-1R的沉默降低了NF-KB p65在H1650-siIGF-1R细胞核中的累积,NF-κB的DNA结合能力随着IGF-1R和EGFR同时受到抑制而减弱,但仍然具有活性。PDTC对EGFR、IGF-1R、Akt、ERK、p-EGFR、p-IGF-1R、p-Akt和p-ERK等蛋白的表达均无明显影响。
结论：在IGF-1R激活的吉非替尼耐药的肺癌细胞H1650中,NF-KB信号通路也处于活化的状态,NF-KB作为下游因子其活性部分受到IGF-1R的调控。联合抑制IGF-1R和NF-kB信号通路能够有效提高吉非替尼在H1650细胞中所诱导的生长抑制和促凋亡作用。
Part One IGF-1R siRNA enhances the effects of gefitinib in H1650 and H1975: Non small cell lung cancer cell lines with acquried resistance to EGFR-TK inhibitors
Purpose:H1975 and H1650 are non small cell lung cancer (NSCLC) cells lines which display resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). We investigated the effect of silencing insulin like growth factor 1 receptor (IGF-1R) on the sensitivity of H1650 and H1975 to Gefitinib by siRNA considering that IGF1R signaling has been involved in the development of EGFR-TKIs acquired resistance in lung cancer.
Methods:Three human NSCLC cell lines with EGFR mutations of HCC827, H1975 and H1650 were used for this part of experiment. Cell viability and proliferative activity were assessed by MTT. The expression of EGFR- and IGFR-related proteins was examined by western blots.
Results:H1650 and H1975 cells were much more resistant to gefitinib than HCC827 cells, respectively. In spite of gefitinib treatment, phosphatase and tensin homolog loss and sustained phosphorylation of Akt were evident in H1650 and H1975 cells, but not in HCC827 cells. When the tumor cells were treated with gefitinib, IGF-1R phosphorylation, which was decreased in HCC827 cells, maintained in H1650 and H1975 cells. Combined treatment with IGF1R siRNA enhanced growth inhibition and apoptosis induced by gefitinib, and down-regulated phosphorylated Akt, EGFR and IGF1R.
Conclusion:Combined inhibition of IGF1R signaling by si RNA enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the aquired resistance to EGFR-TKIs in lung cancer.
Part Two Combined inhibition of IGF-1R and NF-κB enhances the effects of gefitinib in EGFR-TK inhibitors resistant lung cancer cells line H1650
Purpose:We have previously demonstrated that IGF-1R siRNA is a crucial regulator in mediating gefitinib-induced cell death, which upregulates apoptosis-related molecules. Nevertheless, the clinical trials that evaluated the addition of IGF-1R inhibition to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer have not demonstrated improvements in outcomes. There is increasing evidence suggests that NF-κB pathway is a determinant of sensitivity of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. Since the interactions of IGF-1R and NF-κB pathways were demonstrated in types of cells and tissues, we try to explore the effect of combined inhibition of NF-κB and IGF-1R on EGFR-TKIs resistant lung cancer cell line H1650.
Methods:The expression of IGF-1R in H1650 was silenced using an IGF-1R specific siRNA. PDTC was taken as NF-κB specifitic inhibitor in this study. Cell viability was assessed by MTT. The apoptosis of cells was measured and analysed by flow cytometry. The expression and activity of EGFR-, IGFR-and NF-κB-related proteins were detected by western blots and EMSA.
Results:We found that H1650, with resistance to Gefitinib exhibited a high level activity of both IGF-1R and NF-κB. The DNA binding activity of NF-κB in H1650 was partly reduced by the silencing of IGF-1R, suggesting the activation of NF-κB was not totally IGF-1R dependent in lung cancer. In addition, the silencing of IGF-1R expression together with NF-κB inhibition made the lung cancer cells more sensitive to gefitinib-mediated apoptosis than it did alone, suggesting the involvement of both IGF-1R and NF-κB pathways in gefitinib acquired resistance in H1650 cells.
Conclusion:These results suggest that NF-κB is potentially involved in promoting gefitinib resistance even when the IGF-1R pathway was inhibited in lung cancer cell line H1650. Our findings also suggest that NF-κB activation was partially IGF-1R dependent, thus, combined inhibition of both NF-κB and IGF-1R may provide an effective way to overcome EGFR-TKIs resistance.
目的：研究IGF-1R特异性的siRNA对逆转吉非替尼耐药的人非小细胞肺癌细胞H1975和H1650耐药的作用,并探讨其可能存在的机制。
方法：不同浓度的吉非替尼分别处理吉非替尼耐药的肺癌细胞株(H1975和H1650),吉非替尼敏感的肺癌细胞株(HCC827),以及RNAi的表达载体分别转染后的H1975和H1650细胞,采用MTT法检测吉非替尼对各处理组细胞的生长抑制作用;AnnexinV-FITC和PI双标法标记细胞后,在流式细胞仪上检测吉非替尼浓度为0、0.01、0.1和1uM的条件下,处理24h和48h后各组细胞的凋亡水平；Western blot法检测细胞内EGFR、IGF-1R通路相关蛋白和凋亡相关蛋白的表达。
结果：HCC827、H1975和H1650细胞株的IC50值分别为0.006±0.0003uM、24.62±0.3uM和18.39±0.2uM。经1uM吉非替尼处理48h后,H1975和H1650细胞中均检出了p-IGF-1R的高表达,而HCC827细胞中则未见明显的p-IGF-1R。当用siRNA技术分别沉默H1975和H1650细胞中的IGF-1R后,吉非替尼在两种细胞中的ICso值分别下降至7.94±0.1uM和9.42±0.4uM,明显低于IGF-1R未沉默的相应细胞(P<0.05)。作用24h和48h时,吉非替尼在IGF-1R沉默后的H1975和H1650细胞中诱导的凋亡比例均明显高于IGF-1R未沉默的相应细胞株(P<0.05)。IGF-1RsiRNA联合吉非替尼作用时明显抑制了H1975和H1650细胞中的p-IGF-1R(?)(?)p-Akt的表达。
结论：IGF-1R siRNA能有效增强吉非替尼在耐药的人肺癌细胞株H1975和H1650中诱导的生长抑制和凋亡效应。IGF-1R siRNA技术或可成为逆转EGFR-TKIs耐药的新策略。
第二部分
联合抑制IGF-1R和NF-KB信号通路逆转吉非替尼耐药的人肺癌细胞株H1650耐药的研究
目的：在第一部分研究中我们证明,ⅠGF-1R siRNA能够在EGFR-TKIs耐药的肿癌细胞中提高吉非替尼诱导的生长抑制和凋亡水平。然而,临床试验显示,联合使用IGF-1R印制剂(?)(?)EGFR-TKIs并未能在肺癌治疗中取得显效。新近发现,NF-KB通路是影响肺癌细胞对EGFR-TKIs敏感性的因素。我们旨在探索联合抑制(?)IGF-1R和NF-KB通路能否提高耐药细胞对吉非替尼的敏感性,并探讨其可能的机制。
方法：分别用吉非替尼和NF-KB特异性抑制齐(?)JPDTC处理IGF-1R未沉默和沉默的细胞H1650-sictrl和H1650-siIGF-1R。用MTT实验检测(?)PDTC和吉非替尼对各组细胞的生长抑制作用,流式细胞术检测细胞的凋亡水平,EMSA实验检测细胞内NF-kB的DNA结合能力,Western blot法检测细胞内EGFR、IGF-1R和NF-kB信号通路的相关蛋白表达,并检测凋亡相关蛋白PARPcl的水平。
结果：IkB在吉非替尼敏感的肺癌细胞HCC827中的水平高于吉非替尼耐药的H1975和H1650细胞,其表达因吉非替尼的作用而上调。在IGF-1R未沉默的H1650-sictrl细胞中,IKB的表达水平低于其在H1650-siIGF-1R细胞中的表达,且两种细胞中IKB水平均随吉非替尼浓度增加而出现上调。与H1650-sictrl细胞相比,PDTC在H1650-siIGF-1R细胞中明显增强了吉非替尼诱导的生长抑制和凋亡效应(P<0.05)。IGF-1R的沉默降低了NF-KB p65在H1650-siIGF-1R细胞核中的累积,NF-κB的DNA结合能力随着IGF-1R和EGFR同时受到抑制而减弱,但仍然具有活性。PDTC对EGFR、IGF-1R、Akt、ERK、p-EGFR、p-IGF-1R、p-Akt和p-ERK等蛋白的表达均无明显影响。
结论：在IGF-1R激活的吉非替尼耐药的肺癌细胞H1650中,NF-KB信号通路也处于活化的状态,NF-KB作为下游因子其活性部分受到IGF-1R的调控。联合抑制IGF-1R和NF-kB信号通路能够有效提高吉非替尼在H1650细胞中所诱导的生长抑制和促凋亡作用。
Part One IGF-1R siRNA enhances the effects of gefitinib in H1650 and H1975: Non small cell lung cancer cell lines with acquried resistance to EGFR-TK inhibitors
Purpose:H1975 and H1650 are non small cell lung cancer (NSCLC) cells lines which display resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). We investigated the effect of silencing insulin like growth factor 1 receptor (IGF-1R) on the sensitivity of H1650 and H1975 to Gefitinib by siRNA considering that IGF1R signaling has been involved in the development of EGFR-TKIs acquired resistance in lung cancer.
Methods:Three human NSCLC cell lines with EGFR mutations of HCC827, H1975 and H1650 were used for this part of experiment. Cell viability and proliferative activity were assessed by MTT. The expression of EGFR- and IGFR-related proteins was examined by western blots.
Results:H1650 and H1975 cells were much more resistant to gefitinib than HCC827 cells, respectively. In spite of gefitinib treatment, phosphatase and tensin homolog loss and sustained phosphorylation of Akt were evident in H1650 and H1975 cells, but not in HCC827 cells. When the tumor cells were treated with gefitinib, IGF-1R phosphorylation, which was decreased in HCC827 cells, maintained in H1650 and H1975 cells. Combined treatment with IGF1R siRNA enhanced growth inhibition and apoptosis induced by gefitinib, and down-regulated phosphorylated Akt, EGFR and IGF1R.
Conclusion:Combined inhibition of IGF1R signaling by si RNA enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the aquired resistance to EGFR-TKIs in lung cancer.
Part Two Combined inhibition of IGF-1R and NF-κB enhances the effects of gefitinib in EGFR-TK inhibitors resistant lung cancer cells line H1650
Purpose:We have previously demonstrated that IGF-1R siRNA is a crucial regulator in mediating gefitinib-induced cell death, which upregulates apoptosis-related molecules. Nevertheless, the clinical trials that evaluated the addition of IGF-1R inhibition to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer have not demonstrated improvements in outcomes. There is increasing evidence suggests that NF-κB pathway is a determinant of sensitivity of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. Since the interactions of IGF-1R and NF-κB pathways were demonstrated in types of cells and tissues, we try to explore the effect of combined inhibition of NF-κB and IGF-1R on EGFR-TKIs resistant lung cancer cell line H1650.
Methods:The expression of IGF-1R in H1650 was silenced using an IGF-1R specific siRNA. PDTC was taken as NF-κB specifitic inhibitor in this study. Cell viability was assessed by MTT. The apoptosis of cells was measured and analysed by flow cytometry. The expression and activity of EGFR-, IGFR-and NF-κB-related proteins were detected by western blots and EMSA.
Results:We found that H1650, with resistance to Gefitinib exhibited a high level activity of both IGF-1R and NF-κB. The DNA binding activity of NF-κB in H1650 was partly reduced by the silencing of IGF-1R, suggesting the activation of NF-κB was not totally IGF-1R dependent in lung cancer. In addition, the silencing of IGF-1R expression together with NF-κB inhibition made the lung cancer cells more sensitive to gefitinib-mediated apoptosis than it did alone, suggesting the involvement of both IGF-1R and NF-κB pathways in gefitinib acquired resistance in H1650 cells.
Conclusion:These results suggest that NF-κB is potentially involved in promoting gefitinib resistance even when the IGF-1R pathway was inhibited in lung cancer cell line H1650. Our findings also suggest that NF-κB activation was partially IGF-1R dependent, thus, combined inhibition of both NF-κB and IGF-1R may provide an effective way to overcome EGFR-TKIs resistance.
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6. Jackman D, William P, Gregory JR, et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol2010;28:357-360.
7. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol2001;2:127-137.
8. Bazley LA, Gullick WJ. The epidermal growth factor receptor family. Endocr Relat Cancer2005;12:S17-27.
9. Olayioye MA, Neve RM, Lane HA, et al. The ErbB signaling network:receptor heterodimerization in development and cancer. EMBO J2000; 19:3159-67.
10. Ciardiello F, Tortora G EGFR antagonists in cancer treatment. N Engl J Med 2008;358:1160-74.
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1. National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncologyTM. Non-Small Cell Lung Cancer. V.3.2011. Available at: http://www.nccn.org/professionals/phvsician_gls/PDF/nscl.pdf.
2. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med2008;359: 1367-1380.
3. Surveillance Epidemiology and End Results, National Cancer Institute. SEER stat fact sheets:Lung and Bronchus.2010. Available at:http://seer.cancer.gov/ statfacts/html/lungb.html.
4. AstraZeneca. IRESSA (Gefitinib) receives marketing authorisation for the treatment of non-small cell lung cancer in Europe. Available at:http://www. astrazeneca.com/Media/Press-releases/Article/20090701--IRESSA-Gefitinib-Receives-Marketing-Authorisation-f.
5. Roche's Tarceva gains positive CHMP opinion for first-line use in genetically distinct type of lung cancer. Available at:http://www.roche.com/media/media_releases/med-cor-2011-07-22.htm.
6. Jackman D, William P, Gregory JR, et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol2010;28:357-360.
7. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol2001;2:127-137.
8. Bazley LA, Gullick WJ. The epidermal growth factor receptor family. Endocr Relat Cancer2005;12:S17-27.
9. Olayioye MA, Neve RM, Lane HA, et al. The ErbB signaling network:receptor heterodimerization in development and cancer. EMBO J2000; 19:3159-67.
10. Ciardiello F, Tortora G EGFR antagonists in cancer treatment. N Engl J Med 2008;358:1160-74.
11. Klapper LN, Glathe S, Vaisman N, et al. The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci USA1999;96:4995-5000.
12. Hynes NE, Lane HA. ERBB receptors and cancer:the complexity of targeted inhibitors. Nat Rev Cancer2005;5:341-354.
13. Baselga J, Swain SM. Novel anticancer targets:revisiting ERBB2 and discovering ERBB3. Nat Rev Cancer2009;9:463-475.
14. Guy PM, Platko JV, Cantley LC, et al. Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc Natl Acad Sci USA1994;91:8132-8136.
15. Wallasch C, Weiss FU, Niederfellner G, et al. Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3. EMBO J1995; 14: 4267-4275.
16. Marmor MD, Skaria KB, Yarden Y. Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys2004;58:903-913.
17. Blackhall F, Ranson M, Thatcher N. Where next for gefitinib in patients with lung cancer? Lancet Oncol2006;7:499-507.
18. Suda K, Onozato R, Yatabe Y, et al. EGFR T790M mutation:a double role in lung cancer cell survival? J Thorac Oncol2009;4:1-4.
19. Sharma SV, Bell DW, Settleman J, et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer2007;7:169-181.
20. Giaccone G, Herbst RS, Manegold C, et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer:a phase III trial-INTACT 1. J Clin Oncol2004;22:777-784.
21. Herbst RS, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced nonsmall-cell lung cancer:a phase III trial-INTACT 2. J Clin Oncol2004;22:785-794.
22. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet2005; 366:1527-1537.
23. Iressa(?) (gefitinib tablets) [package insert]. Wilmington, DE:AstraZeneca Pharmaceuticals LP 2005.
24. Johnson JR, Cohen M, Sridhara R, et al. Approval summary for erlotinib for treatment of patients with locally advanced or metastatic non-small cell lung cancer after failure of at least one prior chemotherapy regimen. Clin Cancer Res2005;11: 6414-6421.
25. Erlotinib (Tarceva) for advanced non-small cell lung cancer. Med Lett Drugs Ther 2005; 47:25-26.
26. Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management:A Multidisciplinary Approach.11 ed.2008.
27. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected].J Clin Oncol2003;21:2237-2246.
28. Perez-Soler R, Chachoua A, Hammond LA, et al. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol 2004;22:3238-3247.
29. Hepherd FA, Pereira JR, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med2005;353:123-132.
30. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer:a randomized trial. JAMA2003;290:2149-2158.
31.Kim ES, Hirsh V, Mok T, et al. Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST):a randomised phase III trial. Lancet 2008;372:1809-1818.
32. Jackman D, Pao W, Riely GJ, et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol2010; 28:357-60.
33. Kumar A, Petri ET, Halmos B, et al. Structure and clinical relevance of the epidermal growth factor receptor in human cancer. J Clin Oncol2008;26:1742-1751.
34. Costa DB, Kobayashi S, Tenen DG, et al. Pooled analysis of the prospective trials of gefitinib monotherapy for EGFR-mutant non-small cell lung cancers. Lung Cancer 2007;58:95-103.
35. 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 Oncol2008;26:2442-2449.
36. Asahina H, Yamazaki K, Kinoshita I, et al. A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung, cancer with epidermal growth factor receptor mutations. Br J Cancer2006;95:998-1004.
37. Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med2009;361:958-967.
38. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst2005;97:339-346.
39. Tokumo M, Toyooka S, Kiura K, et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res2005;11:1167-1173.
40. 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 Med2005;2:e73.
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