激活性的表皮生長因子受體（EGFR）突變，啟動癌化的發生，並且也促進腫瘤針對突變EGFR的EGFR酪氨酸激酶抑製劑（TKIs）效果增加。 SOX2在肺前驅細胞中決定細胞命運決定因子的作用。然而，SOX2表達如何影響TKI治療和肺癌中的腫瘤侵襲尚未了解。在這篇研究中我們報導了突變的EGFR與SOX2相互作用以維持肺癌細胞的增殖，其中一些通過關閉SOX2表達來產生癌細胞異質性，由此促進TKI抗性和侵襲性。通過shRNA對SOX2誘導的上皮 - 間質轉化（EMT）下調SOX2表達並賦予細胞對TKI的抗性。 TKI的治療能夠篩選出具有EMT並且SOX2低表達的EGFR突變肺癌細胞。相反的，增強內源性SOX2表達可逆轉EMT並增加對TKIs的敏感性。免疫組織化學分析顯示低SOX2表達能夠在帶有EGFR突變的患者中預測較差的存活。綜上所述，我們的研究結果顯示了透過表觀遺傳介導的SOX2表達和TKI選擇引發的癌症可塑性如何產生不同的癌細胞異質性和TKI敏感性，並且也為EGFR突變的肺癌腫瘤進展提供了重要見解。

Activating mutations in the epidermal growth factor receptor (EGFR), while initiating lung tumorigenesis, render tumors susceptible to EGFR-tyrosine kinase inhibitors (TKIs). SOX2 functions as a cell fate determination factor in lung progenitor cells. However, how SOX2 expression affects TKI treatment and tumor invasion in lung cancer is obscure. Here we report that EGFR mutations crosstalk with SOX2 to maintain cell proliferation and dissemination in lung cancer cells, some of which generate heterogeneity by switching off SOX2 expression, thus promoting TKI resistance and invasiveness. Downregulation of SOX2 expression by shRNA against SOX2 induced epithelial-to-mesenchymal transition (EMT) and endowed cell resistant to TKIs. TKI treatment selected EGFR-mutated lung cancer cells harboring low SOX2 expression with the EMT signature. Conversely, enhancing endogenous SOX2 expression reverses EMT and increases sensitivity to TKIs. Immunohistochemistry analysis revealed that low SOX2 expression predicts a poor survival in patients harboring EGFR mutations. Taken together, our findings show how cancer plasticity elicited by epigenetically mediated SOX2 expression and TKI selection generates distinct oncogenic properties and TKI sensitivity, providing critical insights into tumor progression in EGFR-mutated lung cancer.

1. Torre, L.A., et al., Global cancer statistics, 2012. CA Cancer J Clin, 2015. 65(2):
p. 87-108.
2. Molina, J.R., et al., Non-small cell lung cancer: epidemiology, risk factors,
treatment, and survivorship. Mayo Clin Proc, 2008. 83(5): p. 584-94.
3. Midha, A., S. Dearden, and R. McCormack, EGFR mutation incidence in nonsmall-
cell lung cancer of adenocarcinoma histology: a systematic review and
global map by ethnicity (mutMapII). Am J Cancer Res, 2015. 5(9): p. 2892-911.
4. Mok, T.S., et al., Gefitinib or carboplatin-paclitaxel in pulmonary
adenocarcinoma. N Engl J Med, 2009. 361(10): p. 947-57.
5. Paez, J.G., et al., EGFR mutations in lung cancer: correlation with clinical
response to gefitinib therapy. Science, 2004. 304(5676): p. 1497-500.
6. Zhou, C., 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 Oncol, 2011. 12(8): p. 735-42.
7. Sequist, L.V., 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(27): p. 3327-34.
8. Maemondo, M., et al., Gefitinib or chemotherapy for non-small-cell lung cancer
with mutated EGFR. N Engl J Med, 2010. 362(25): p. 2380-8.
9. Gasinska, A., et al., Clinical significance of biological differences between
cavitated and solid form of squamous cell lung cancer. Lung Cancer, 2005. 49(2):
p. 171-9.
10. Sharma, S.V., et al., Epidermal growth factor receptor mutations in lung cancer.
Nat Rev Cancer, 2007. 7(3): p. 169-81.
11. Mulloy, R., et al., Epidermal growth factor receptor mutants from human lung
cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib.
Cancer Res, 2007. 67(5): p. 2325-30.
12. Mitsudomi, T., 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. Lancet
Oncol, 2010. 11(2): p. 121-8.
13. Rosell, R., et al., Screening for epidermal growth factor receptor mutations in
lung cancer. N Engl J Med, 2009. 361(10): p. 958-67.
14. Arcila, M.E., et al., Rebiopsy of lung cancer patients with acquired resistance to
EGFR inhibitors and enhanced detection of the T790M mutation using a locked
47
nucleic acid-based assay. Clin Cancer Res, 2011. 17(5): p. 1169-80.
15. Kobayashi, S., et al., EGFR mutation and resistance of non-small-cell lung cancer
to gefitinib. N Engl J Med, 2005. 352(8): p. 786-92.
16. Sequist, L.V., et al., Genotypic and histological evolution of lung cancers
acquiring resistance to EGFR inhibitors. Sci Transl Med, 2011. 3(75): p. 75ra26.
17. Pao, W., et al., Acquired resistance of lung adenocarcinomas to gefitinib or
erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS
Med, 2005. 2(3): p. e73.
18. Yun, C.H., et al., The T790M mutation in EGFR kinase causes drug resistance by
increasing the affinity for ATP. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2070-
5.
19. Cross, D.A., et al., AZD9291, an irreversible EGFR TKI, overcomes T790Mmediated
resistance to EGFR inhibitors in lung cancer. Cancer Discov, 2014. 4(9):
p. 1046-61.
20. Janne, P.A., et al., AZD9291 in EGFR inhibitor-resistant non-small-cell lung
cancer. N Engl J Med, 2015. 372(18): p. 1689-99.
21. Thress, K.S., et al., Acquired EGFR C797S mutation mediates resistance to
AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med, 2015.
21(6): p. 560-2.
22. Piotrowska, Z., et al., Heterogeneity Underlies the Emergence of EGFRT790
Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-
Generation EGFR Inhibitor. Cancer Discov, 2015. 5(7): p. 713-22.
23. Ko, R., et al., Frequency of EGFR T790M mutation and multimutational profiles
of rebiopsy samples from non-small cell lung cancer developing acquired
resistance to EGFR tyrosine kinase inhibitors in Japanese patients. BMC Cancer,
2016. 16(1): p. 864.
24. Li, W., et al., T790M mutation is associated with better efficacy of treatment
beyond progression with EGFR-TKI in advanced NSCLC patients. Lung Cancer,
2014. 84(3): p. 295-300.
25. Jakobsen, J.N. and J.B. Sorensen, Intratumor heterogeneity and chemotherapyinduced
changes in EGFR status in non-small cell lung cancer. Cancer
Chemother Pharmacol, 2012. 69(2): p. 289-99.
26. Gerlinger, M., et al., Intratumor heterogeneity and branched evolution revealed
by multiregion sequencing. N Engl J Med, 2012. 366(10): p. 883-892.
27. Meacham, C.E. and S.J. Morrison, Tumour heterogeneity and cancer cell
plasticity. Nature, 2013. 501(7467): p. 328-37.
28. Shipitsin, M., et al., Molecular definition of breast tumor heterogeneity. Cancer
Cell, 2007. 11(3): p. 259-73.
48
29. Lin, S.C., et al., Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell
Plasticity. Cancer Res, 2016. 76(23): p. 7036-7048.
30. Schepers, A.G., et al., Lineage tracing reveals Lgr5+ stem cell activity in mouse
intestinal adenomas. Science, 2012. 337(6095): p. 730-5.
31. Quintana, E., et al., Phenotypic heterogeneity among tumorigenic melanoma
cells from patients that is reversible and not hierarchically organized. Cancer
Cell, 2010. 18(5): p. 510-23.
32. Sharma, S.V., et al., A chromatin-mediated reversible drug-tolerant state in
cancer cell subpopulations. Cell, 2010. 141(1): p. 69-80.
33. Thomson, S., 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(20): p. 9455-
62.
34. Yauch, R.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(24 Pt 1): p. 8686-98.
35. Witta, S.E., et al., Restoring E-cadherin expression increases sensitivity to
epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res,
2006. 66(2): p. 944-50.
36. Hwang, W., 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(11): p. 3013-3026.
37. Chen, B., et al., The role of epithelial-mesenchymal transition and IGF-1R
expression in prediction of gefitinib activity as the second-line treatment for
advanced nonsmall-cell lung cancer. Cancer Invest, 2013. 31(7): p. 454-60.
38. Fong, H., K.A. Hohenstein, and P.J. Donovan, Regulation of self-renewal and
pluripotency by Sox2 in human embryonic stem cells. Stem Cells, 2008. 26(8):
p. 1931-8.
39. Que, J., et al., Multiple roles for Sox2 in the developing and adult mouse trachea.
Development, 2009. 136(11): p. 1899-907.
40. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4):
p. 663-76.
41. Takahashi, K., et al., Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
42. Tompkins, D.H., et al., Sox2 is required for maintenance and differentiation of
bronchiolar Clara, ciliated, and goblet cells. PLoS One, 2009. 4(12): p. e8248.
43. Azuara, V., et al., Chromatin signatures of pluripotent cell lines. Nat Cell Biol,
49
2006. 8(5): p. 532-8.
44. Bernstein, B.E., et al., A bivalent chromatin structure marks key developmental
genes in embryonic stem cells. Cell, 2006. 125(2): p. 315-26.
45. Clouaire, T., et al., Cfp1 integrates both CpG content and gene activity for
accurate H3K4me3 deposition in embryonic stem cells. Genes Dev, 2012. 26(15):
p. 1714-28.
46. Amador-Arjona, A., et al., SOX2 primes the epigenetic landscape in neural
precursors enabling proper gene activation during hippocampal neurogenesis.
Proc Natl Acad Sci U S A, 2015. 112(15): p. E1936-45.
47. Sholl, L.M., K.B. Long, and J.L. Hornick, Sox2 expression in pulmonary non-small
cell and neuroendocrine carcinomas. Appl Immunohistochem Mol Morphol,
2010. 18(1): p. 55-61.
48. Lu, Y., et al., Evidence that SOX2 overexpression is oncogenic in the lung. PLoS
One, 2010. 5(6): p. e11022.
49. Mu, P., et al., SOX2 promotes lineage plasticity and antiandrogen resistance in
TP53- and RB1-deficient prostate cancer. Science, 2017. 355(6320): p. 84-88.
50. Chou, Y.T., 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(12):
p. 2607-19.
51. Nelson, J.D., O. Denisenko, and K. Bomsztyk, Protocol for the fast chromatin
immunoprecipitation (ChIP) method. Nat Protoc, 2006. 1(1): p. 179-85.
52. Dogan, I., 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): p. 1-6.
53. Rothenberg, S.M., et al., Inhibition of mutant EGFR in lung cancer cells triggers
SOX2-FOXO6-dependent survival pathways. Elife, 2015. 4.