SOX2是一種被細胞激素所調控的幹細胞特性因子，進而控制細胞的自我新生(self-renewal)和分化(differentiation)。表皮生長因子受體(epidermal growth factor receptor)的活化性突變對於肺腺癌患者使用酪胺酸激酶抑制劑(tyrosine kinase inhibitors)是重要的治療標記，但不可避免地伴隨抗藥性的產生。然而，哪一種幹細胞特性和分化訊息能影響肺癌細胞對TKI產生耐受性和肺癌細胞的轉移還未被闡明。在此研究中，我們指出SOX2和TGF-β signaling會影響肺癌細胞的塑性(plasticity)和TKI耐受性。我們發現TKI治療有利於篩選出一群具有間質細胞型態的肺癌細胞，且此細胞伴隨SOX2表現量的缺乏，然而SOX2的表現會促進肺癌細胞對TKI的敏感性和抑制間質細胞型態。具有間質細胞型態的EGFR-mutant肺癌細胞，會伴隨SOX2的表現和TKI敏感性的減少，且SOX2表現減弱會促進VIM、抑制BCL2L11表現和促進TKI耐受性。TGF-β刺激會抑制SOX2的表現和誘導上皮間質轉化(epithelial-to-mesenchymal transdifferentiation)，並伴隨增加TKI耐受性，然而此現象會被SOX2過度表達所干擾。我們發現比起SOX2低表現的肺癌細胞，SOX2高表現的肺癌細胞展現低轉移能力。免疫組織化學染色法揭示在EGFR突變的病人上，腫瘤具有SOX2低表現和VIM高表現的蛋白質表達特徵時，病人有較差的存活率。綜合上述，我們的研究結果發現，SOX2和TGF-β signaling會影響EGFR-TKI耐受性和肺癌細胞的轉移，且我們的發現對於癌細胞塑性如何影響癌症的進展提供重要的見解。

SOX2, a stemness factor, is regulated by cytokine stimuli to control self-renewal and differentiation in cells. Activating mutations of epidermal growth factor receptor (EGFR) are critical therapeutic targets for tyrosine kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKI inevitably occurs. However, the mechanism by which stemness and differentiation signaling emerge in lung cancers to affect TKI tolerance and lung cancer dissemination has yet to be elucidated. In this study, we report that crosstalk between SOX2 and transforming growth factor-β (TGF-β) signaling affects lung cancer cell plasticity and TKI tolerance. We found that TKI treatment selected in favor of lung cancer cells displaying mesenchymal morphology with deficient SOX2 expression, whereas SOX2 expression promoted TKI sensitivity and inhibited the mesenchymal phenotype. Preselection of EGFR-mutant lung cancer cells with the mesenchymal phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2 silencing induced Vimentin but suppressed BCL2L11 expression and promoted TKI tolerance. TGF-β stimulation downregulated SOX2 and induced epithelial-to-mesenchymal transdifferentiation (EMT) accompanied by increased TKI tolerance, which can interfere with ectopic SOX2 expression. We observed that SOX2-positive lung cancer cells exhibited a lower dissemination capacity than their SOX2-negative counterparts. Immunohistochemistry analysis revealed that tumors expressing low SOX2 and high Vimentin signature are associated with worse survival outcomes in patients with EGFR mutations. In our study, we identify the involvement of SOX2 and TGF-β signaling affected EGFR–TKI tolerance and lung cancer dissemination, providing insights for cancer cell plasticity.

中文摘要----------------------ii
英文摘要----------------------iii
Acknowledgement--------------iv-v
Table of Contents-------------vi
List of Figures--------------vii-ix
List of Tables-----------------x
List of Abbreviations---------xi
Introduction------------------1-3
Materials and Methods---------4-7
Results-----------------------8-16
Discussion--------------------17-20
Figures-----------------------21-64
Tables------------------------65-70
References--------------------71-76

1. Youlden DR, Cramb SM, Baade PD. The International Epidemiology of Lung Cancer: geographical distribution and secular trends. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2008;3:819-31
2. Sobol RE, Astarita RW, Hofeditz C, Masui H, Fairshter R, Royston I, et al. Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. Journal of the National Cancer Institute 1987;79:403-7
3. Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. The Lancet Oncology 2010;11:121-8
4. 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. New Engl J Med 2010;362:2380-8
5. Janne PA, Yang JC, Kim DW, Planchard D, Ohe Y, Ramalingam SS, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. The New England journal of medicine 2015;372:1689-99
6. Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 2015;21:560-2
7. Chen Y, Shi L, Zhang L, Li R, Liang J, Yu W, et al. The molecular mechanism governing the oncogenic potential of SOX2 in breast cancer. J Biol Chem 2008;283:17969-78
8. Lee CJ, Sung PL, Kuo MH, Tsai MH, Wang CK, Pan ST, et al. Crosstalk between SOX2 and cytokine signaling in endometrial carcinoma. Sci Rep-Uk 2018;8
9. Bass AJ, Watanabe H, Mermel CH, Yu SY, Perner S, Verhaak RG, et al. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nat Genet 2009;41:1238-U105
10. Sholl LM, Long KB, Hornick JL. Sox2 Expression in Pulmonary Non-small Cell and Neuroendocrine Carcinomas. Appl Immunohisto M M 2010;18:55-61
11. Lu Y, Futtner C, Rock JR, Xu X, Whitworth W, Hogan BLM, et al. Evidence That SOX2 Overexpression Is Oncogenic in the Lung. Plos One 2010;5
12. Bora-Singhal N, Perumal D, Nguyen J, Chellappan S. Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer. Neoplasia 2015;17:538-51
13. Fong H, Hohenstein KA, Donovan PJ. Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem cells 2008;26:1931-8
14. Que J, Luo X, Schwartz RJ, Hogan BL. Multiple roles for Sox2 in the developing and adult mouse trachea. Development 2009;136:1899-907
15. Tompkins DH, Besnard V, Lange AW, Wert SE, Keiser AR, Smith AN, et al. Sox2 Is Required for Maintenance and Differentiation of Bronchiolar Clara, Ciliated, and Goblet Cells. Plos One 2009;4
16. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76
17. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-72
18. Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, et al. A Small-Molecule Inhibitor of Tgf-beta Signaling Replaces Sox2 in Reprogramming by Inducing Nanog. Cell Stem Cell 2009;5:491-503
19. Maherali N, Hochedlinger K. Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Current biology : CB 2009;19:1718-23
20. Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, Yao J, et al. Molecular definition of breast tumor heterogeneity. Cancer Cell 2007;11:259-73
21. Lin SC, Chou YT, Jiang SS, Chang JL, Chung CH, Kao YR. Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell Plasticity (vol 76, pg 7036, 2016). Cancer Res 2017;77:3720-
22. Ferone G, Song JY, Sutherland KD, Bhaskaran R, Monkhorst K, Lambooij JP, et al. SOX2 Is the Determining Oncogenic Switch in Promoting Lung Squamous Cell Carcinoma from Different Cells of Origin. Cancer Cell 2016;30:519-32
23. Chou YT, Lee CC, Hsiao SH, Lin SE, Lin SC, Chung CH, et al. The Emerging Role of SOX2 in Cell Proliferation and Survival and Its Crosstalk with Oncogenic Signaling in Lung Cancer. Stem cells 2013;31:2607-19
24. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. New Engl J Med 2009;361:947-57
25. Zhou CC, Wu YL, Chen GY, Feng JF, Liu XQ, Wang CL, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncology 2011;12:735-42
26. Sequist LV, Yang JCH, Yamamoto N, O'Byrne K, Hirsh V, Mok T, et al. Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. J Clin Oncol 2013;31:3327-+
27. Wu YL, Zhou CC, Hu CP, Feng JF, Lu S, Huang YC, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncology 2014;15:213-22
28. 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 Oncology 2012;13:239-46
29. Arcila ME, Oxnard GR, Nafa K, Riely GJ, Solomon SB, Zakowski MF, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res 2011;17:1169-80
30. Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005;352:786-92
31. 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
32. Piotrowska Z, Niederst MJ, Karlovich CA, Wakelee HA, Neal JW, Mino-Kenudson M, et al. Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor. Cancer discovery 2015;5:713-22
33. Antonicelli A, Cafarotti S, Indini A, Galli A, Russo A, Cesario A, et al. EGFR-targeted therapy for non-small cell lung cancer: focus on EGFR oncogenic mutation. International journal of medical sciences 2013;10:320-30
34. Uramoto H, Yano S, Tanaka F. T790M is associated with a favorable prognosis in Japanese patients treated with an EGFR-TKI. Lung Cancer 2012;76:129-30
35. Kuiper JL, Heideman DA, Thunnissen E, Paul MA, van Wijk AW, Postmus PE, et al. Incidence of T790M mutation in (sequential) rebiopsies in EGFR-mutated NSCLC-patients. Lung Cancer 2014;85:19-24
36. Li W, Ren S, Li J, Li A, Fan L, Li X, et al. T790M mutation is associated with better efficacy of treatment beyond progression with EGFR-TKI in advanced NSCLC patients. Lung Cancer 2014;84:295-300
37. Thomson S, Buck E, Petti F, Griffin G, Brown E, Ramnarine N, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:9455-62
38. Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 2005;11:8686-98
39. 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
40. 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
41. Hata AN, Niederst MJ, Archibald HL, Gomez-Caraballo M, Siddiqui FM, Mulvey HE, et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat Med 2016;22:262-9
42. 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 2018;24:197-208
43. Chou SJ, Tseng WL, Chen CT, Lai YF, Chien CS, Chang YL, et al. Impaired ROS Scavenging System in Human Induced Pluripotent Stem Cells Generated from Patients with MERRF Syndrome. Sci Rep-Uk 2016;6
44. Zhang ZF, Lee JC, Lin LP, 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-+
45. Kaichi S, Hasegawa K, Takaya T, Yokoo N, Mima T, Kawamura T, et al. Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice. Cardiovasc Res 2010;88:314-23
46. VanderMolen KM, McCulloch W, Pearce CJ, Oberlies NH. Romidepsin (Istodax, NSC 630176, FR901228, FK228, depsipeptide): a natural product recently approved for cutaneous T-cell lymphoma. J Antibiot 2011;64:525-31
47. Li RH, Liang JL, Ni S, Zhou T, Qing XB, Li HP, et al. A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts. Cell Stem Cell 2010;7:51-63
48. Bagnaninchi PO, Drummond N. Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing. P Natl Acad Sci USA 2011;108:6462-7
49. Kamachi Y, Kondoh H. Sox proteins: regulators of cell fate specification and differentiation. Development 2013;140:4129-44
50. Sarkar A, Hochedlinger K. The Sox Family of Transcription Factors: Versatile Regulators of Stem and Progenitor Cell Fate. Cell Stem Cell 2013;12:15-30
51. Shaffer SM, Dunagin MC, Torborg SR, Torre EA, Emert B, Krepler C, et al. Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance (vol 546, pg 431, 2017). Nature 2018;555:274-
52. Ennen M, Keime C, Gambi G, Kieny A, Coassolo S, Thibault-Carpentier C, et al. MITF-High and MITF-Low Cells and a Novel Subpopulation Expressing Genes of Both Cell States Contribute to Intra- and Intertumoral Heterogeneity of Primary Melanoma. Clin Cancer Res 2017;23:7097-107
53. Vallette FM, Olivier C, Lezot F, Oliver L, Cochonneau D, Lalier L, et al. Dormant, quiescent, tolerant and persister cells: Four synonyms for the same target in cancer. Biochem Pharmacol 2019;162:169-76
54. Kochanowski K, Morinishi L, Altschuler S, Wu L. Drug persistence - from antibiotics to cancer therapies. Current opinion in systems biology 2018;10:1-8
55. Hu Q, Zhang L, Wen J, Wang S, Li M, Feng R, et al. The EGF receptor-sox2-EGF receptor feedback loop positively regulates the self-renewal of neural precursor cells. Stem cells 2010;28:279-86
56. Dogan I, Kawabata S, Bergbower E, Gills JJ, Ekmekci A, Wilson W, et al. SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines. Lung Cancer 2014;85:1-6
57. Rothenberg SM, Concannon K, Cullen S, Boulay G, Turke AB, Faber AC, et al. Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6 dependent survival pathways. Elife 2015;4
58. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. P Natl Acad Sci USA 2010;107:21931-6
59. Benayoun BA, Pollina EA, Ucar D, Mahmoudi S, Karra K, Wong ED, et al. H3K4me3 Breadth Is Linked to Cell Identity and Transcriptional Consistency. Cell 2014;158:673-88
60. Akhurst RJ. Targeting TGF-beta Signaling for Therapeutic Gain. Cold Spring Harbor perspectives in biology 2017;9
61. Viswanathan VS, Ryan MJ, Dhruv HD, Gill S, Eichhoff OM, Seashore-Ludlow B, et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 2017;547:453-7