就全球來說，肺癌是最常見的惡性腫瘤之一，它的高死亡率主要歸咎於它容易併發轉移以及它對於治療的抗藥性。其中，非小細胞肺癌為其最常見的種類。艾瑞莎是種專門治療那些具有上皮細胞生長因子接受體突變的病人的標靶藥物，它能夠阻斷上皮細胞生長因子接受體的訊息傳遞，以此達到治療效果，儘管艾瑞莎能夠改善病患的生活品質，但是，依然存在著在艾瑞莎的治療之下沒有效果的病患。這樣的臨床結果顯示出，對於上皮細胞生長因子接受體的酪胺酸激酶抑制劑有抗藥性的原因，可能在於有其他額外的基因損傷或是其他的訊息傳遞路徑被調控。因此，為了去探討這潛在的機制是否能改善對EGFR TKI有抗藥性的病患的治療結果。在我們先前的研究中，我們得到了兩株對於艾瑞莎具有抗藥性的非小細胞肺癌細胞株，並且利用了核糖核酸干擾的資料庫，去探討這兩株細胞為何會具有上皮細胞生長因子的標靶藥物的抗藥性。因此，我們發現了在伴隨著某些基因被表現被調降之後，艾瑞莎的治療效果得到提升。其中我們在這些基因中挑選出BTK這基因做深入的研究，在先前的研究中指出BTK這蛋白質是重要的訊息傳遞酪氨酸激酶，它參與了B細胞惡性腫瘤的存活以及增生，我們發現利用特殊的shRNA去減弱BTK基因的表現量時，會增加抗藥性細胞對於艾瑞莎的敏感度，另外Ibrutinib是已被開發出來作為BTK的小分子抑制劑，因此，當我們將ibrutinib與艾瑞莎共同作用在具有抗藥性的肺癌細胞後，看到了有更佳的毒殺效果的結果，此現象跟BTK以及表皮生長因子受器的訊息傳遞有關 (AKT，ERK1/2，NF-κB)，我們也發現在共同處理下，非小細胞肺癌細胞的凋亡有著明顯的上升 (Cleaved-PARP表現量較高)。最後，藉由在更加了解BTK基因的情況下，對於治療那些具有抗藥性的病患時，或許能發展出一套新的或是搭配其他藥物使用的治療方式。

SUMMARY
In our previous study, we identified two NSCLC cell lines, which have partial response to Gefitinib. Then, to explore the mechanisms of EGFR TKI primary resistance by genome-wide RNAi library screening, we identified some genes which, following their knockdown can induce cell death and enhance the therapeutic efficacy of Gefitinib. Specifically, we looked at the gene Bruton tyrosine kinase (BTK) in greater detail. BTK is a critical signaling kinase, which is involved in the survival and progression of mature B-cell malignancies. We found that silencing of BTK by specific shRNAs against BTK increases the sensitivity of Gefitinib in NSCLC resistant cell lines. Ibrutinib (PCI-32765) is a small-molecule irreversible inhibitor of BTK. When we treated the resistant cells with Gefitinib plus ibrutinib, we observed that that there was synergistic cell toxicity against the drug resistant cells. The phenomenon relied on BTK and EGFR signaling (AKT, ERK1/2, and NF-κB). Moreover, we also detected the cleaved-PARP, which plays a significant role in apoptosis. Through a greater understanding of the BTK gene, we can search for possible mono-or combine strategies for antitumor therapy.

Key word: BTK, Gefitinib, Ibrutinib

Asami K. (2014). Treatment strategy for activating EGFR-mutated non-small cell lung cancer after failure of first-generation EGFR-TKIs. Gan To Kagaku Ryoho. 41(5):533-8.
Baeuerle PA, Baltimore D. (1996). NF-kappa B: ten years after. Cell. 87:13-20.
Cano E, Mahadevan LC. (1995). Parallel signal processing among mammalian MAPKs. Trends Biochem Sci. Mar;20:117-22
Chou TC. (2010). Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 15;70:440-6.
Chou TY, Chiu CH, Li LH et al. (2005). Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non–small cell lung cancer. Clin Cancer Res. 11: 3750-57
Davids MS, Brown JR. (2014). Ibrutinib: a first in class covalent inhibitor of Bruton's tyrosine kinase. Future Oncol. 10:957-67.

De Martel C, Ferlay J, Franceschi S et al. (2012). Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. The Lancet Oncology. 13: 607-615.
Desnoyers LR, Vasiljeva O, Richardson JH et al. (2013). Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index Sci Transl Med. 5: 207ra144
De Vuyst P, Dumortier P, Jacobovitz D et al. (1994). Environmental asbestosis complicated by lung cancer. Chest. 105:1593-5.
Franke TF, Hornik CP, Segev L et al. (2003). PI3K/Akt and apoptosis: size matters. Oncogene. 22:8983-98.
Fukuoka M, Yano S, Giaccone G, et al. (2003). Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial). J Clin Oncol. 21:2237-2246.
Giaccone G. (2005). Selection of patients with non-small cell lung cancer for epidermal growth factor receptor (EGFR) inhibitors: The post-EGFR mutation era. Am Soc Clin Oncol. Ed Book 684-88.

Glaysher S, Bolton LM, Johnson P et al. (2014). Activity of EGFR, mTOR and PI3K inhibitors in an isogenic breast cell line model. BMC Res Notes. 25:397.

Grewal SS, York RD, Stork PJ. (1999). Extracellular-signal-regulated kinase signalling in neurons. Curr Opin Neurobiol. 9:544-53

Grilli M, Goffi F, Memo M, Spano P. (1996). Interleukin-1beta and glutamate activate the NF-kappaB/Rel binding site from the regulatory region of the amyloid precursor protein gene in primary neuronal cultures. J Biol Chem. 271:15002-7.
Han SW, Kim TY, Hwang PG, et al. (2005). Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol. 23: 2493-501.
Herman SE, Gordon AL, Hertlein E,et al. (2011). Bruton's tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 117:6287-96
Honma H. (1966). Classification of lung cancer by disease stage, symptom type and histological type. Naika. 18:832-6.
Hsu JH, Shi Y, Frost P et al. (2004). Interleukin-6 activates phosphoinositol-3' kinase in multiple myeloma tumor cells by signaling through RAS-dependent and, separately, through p85-dependent pathways. Oncogene. 23:3368-75.
Huang SF, Liu HP, Li LH et al. (2004). High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res. 10: 8195-203.
Johnson GL, Lapadat R. (2002). Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 298:1911-2.
Kaltschmidt B, Uherek M, Wellmann H et al. (1999). Inhibition of NF-kappaB potentiates amyloid beta-mediated neuronal apoptosis. Proc Natl Acad Sci U S A. 96:9409-14.
Kastan MB. (2007). Wild-type p53: tumor can’t stand it. Cell. 128:837-40.
Koga F, Xu W, Karpova TS et al. (2006). Hsp90 inhibition transiently activates Src kinase and promotes Src-dependent Akt and Erk activation. Proc Natl Acad Sci U S A. 103:11318-22.
Kobayashi S, Boggon TJ, Dayaram T, et al. (2005). EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 352: 786-792.
Krewski D, Burnett R, Jerrett M et al. (2005). Mortality and long-term exposure to ambient air pollution: ongoing analyses based on the American Cancer Society cohort. J Toxicol Environ Health A. 68: 1093–109.
Kyriakis JM and Avruch J. (2001). Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 81:807-69.
Lai SW, Ho CL, Dai MS et al. (2014). Gefitinib as first-line treatment for patients with epidermal growth factor receptor-mutated advanced lung adenocarcinoma: A single institution experience in Taiwan. J BUON. 19:459-65.
Lewis TS, Shapiro PS, Ahn NG. (1998). Signal transduction through MAP kinase cascades. Adv Cancer Res. 74:49-139.
Lin CH, Lin MT, Kuo YW, et al. (2014). Afatinib combined with cetuximab for lung adenocarcinoma with leptomeningeal carcinomatosis. Lung Cancer. pii: S0169-5002(14)00260-8.
Lindop PJ, Rotblat J. (1966). Induction of lung tumours by the action of radiation and urethane. Nature. 210:1392-3.
Lynch TJ, Bell DW, Sordella R et al. (2004). Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129-2139
Maemondo M, Inoue A, Kobayashi K, et al. (2010). Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 362:2380-8
Mattson MP, Camandola S. (2001). NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest. 107:247-54.
Mattsson, P. T., Vihinen, M. and Smith, C. I. E. (1995). X-linked agam- maglobulinemia (XLA): a genetic tyrosine kinase (Btk) disease. Bioessays. 18: 825–834. ;
Mayo LD and Donner DB. (2001). A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A. 98:11598-603
McCubrey JA, Steelman LS, Franklin RA et al. (2007). Targeting the RAF/MEK/ERK, PI3K/AKT and p53 pathways in hematopoietic drug resistance. Adv Enzyme Regul. 47:64-103.
Meng Q, Xia C, Fang J et al. (2006). Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway. Cell Signal. 18:2262-71
Minna JD, Peyton MJ, Gazdar AF. (2005). Gefitinib versus cetuximab in lung cancer: round one. J Natl Cancer Inst. 97:1168-9
Mitsudomi T, Kosaka T, Endoh H et al. (2005). Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol. 23: 2513-20.
Ohashi K, Maruvka YE, Michor F et al. (2013). Epidermal Growth Factor Receptor Tyrosine KinaseInhibitor–Resistant Disease. J Clin Oncol. 31:1070-80.
Ono M, Hirata A, Kometani T et al. (2004). Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol Cancer Ther. 3(4):465-72.
Paez JG, Janne PA, Lee JC et al.(2004). EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304: 1497-500.
Pao W, Miller V, Zakowski M et al. (2004). EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 101: 13306-11.
Pope CA 3rd, Burnett RT, Thun MJ et al. (2002). Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution.. Journal of the American Medical Association. 287: 1132–1141.

Puglisi M, Thavasu P, Stewart A et al. (2014). AKT inhibition synergistically enhances growth-inhibitory effects of gefitinib and increases apoptosis in non-small cell lung cancer cell lines. Lung Cancer. 85:141-6.

Rahman I. (2000). Regulation of nuclear factor-kappa B, activator protein-1, and glutathione levels by tumor necrosis factor-alpha and dexamethasone in alveolar epithelial cells. Biochem Pharmacol. 60:1041-9
Rawlings DJ, and Witte ON. (1995). The Btk subfamily of cytoplas- mic tyrosine kinases: structure, regulation and function. Semin. Immunol. 7: 237–246.

Roskoski R Jr. (2014). ErbB/HER protein-tyrosine kinases: Structures and small molecule inhibitors. Pharmacol Res. 87:42-59.
Samet JM. (2004). Environmental causes of lung cancer: what do we know in 2003. Chest. 125:80S-3S.
Sasaki M, Sugio K, Kuwabara Y et al. (2003) Alterations of tumor suppressor genes (Rb, p16, p27 and p53) and an increased FDG uptake in lung cancer. Ann Nucl Med. 17:189-96.
Sequist LV et al. (2013). Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. J Clin Oncol. 31:3327-34
Shigematsu H, Gazdar A. (2005). The epidemiology of EGFR mutations. Signal. 6: 4-8.
Sozzi G, Pastorino U, Moiraghi L et al. (1998). Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res. 58:5032-7.
Tammemagi CM, Neslund-Dudas C, Simoff M et al. (2004). Smoking and lung cancer survival: the role of comorbidity and treatment. Chest. 125:27-37.
Tian B, Lessan K, Kahm J et al. (2002). beta 1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway. J Biol Chem. 277:24667-75.
Thatcher T, Chang A, Parikh P et al. (2005). ISEL: a phase III survival study comparing gefitinib (Iressa) plus best supportive care (BSC) with placebo plus BSC, in patients with advanced NSCLC (NSCLC) who had received one or two prior chemotherapy regimens. Lung Cancer. 49(suppl 2):S4.
Tong L, Perez-Polo JR. (1995). Transcription factor DNA binding activity in PC12 cells undergoing apoptosis after glucose deprivation. Neurosci Lett. 191:137-40.
Van der Wal AM, Huizinga E, Orie NG et al. (1966). Cancer and chronic non-specific lung disease (C.N.S.L.D). Scand J Respir Dis. 47:161-72

Vencevicius V. (2004). Surgical approach in treatment of associated lung pathology - lung cancer at tuberculosis]. Medicina (Kaunas). 1:149-51.
Verheul HM, Pinedo HM. (2000). The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors. Clin Breast Cancer. 1:S80-4
Vivanco I and Sawyers LS. (2002). The phosphatidylinositol 3-Kinase–AKT pathway in human cancer. Nature Reviews Cancer. 2: 489-501.
Wagoner JK, Archer VE, Lundin FE, Jr et al. (1965). Radiation as the cause of lung cancer among uranium miners. N Engl J Med.273:181-8.
Wang YC, Lu YP, Tseng RC et al. (2003). Inactivation of hMLH1 and hMSH2 by promoter methylation in primary non-small cell lung tumors and matched sputum samples. J Clin Invest. 111:887-95.
Whirl-Carrillo M, McDonagh EM, Hebert JM, et al. (2012). Pharmacogenomics Knowledge for Personalized Medicine. Clin Pharmacol Ther. 92: 414-417.
Wislez M, Malka D, Bennouna J et al. (2014). A new perspective in the treatment of non-small-cell lung cancer (NSCLC). Role of afatinib: An oral and irreversible ErbB family blocker. Bull Cancer. 101:647-652.
Woyach JA, Bojnik E, Ruppert AS et al . Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood. 123:1207-1213)
Yi HK, Kim SY, Hwang PH et al. (2005). Impact of PTEN on the expression of insulin-like growth factors (IGFs) and IGF-binding proteins in human gastric adenocarcinoma cells. Biochem Biophys Res Commun. 330:760-7.
Yun Qiu and Hsing-Jien Kung. (2000). Signaling network of the Btk family kinases. Oncogene. 19: 5651 – 5661