細胞分化 (cell differentiation)，由原本的幹細胞運作程式 (stem cell-program) 轉換成分化運作程式 (differentiation-program)，以驅動細胞進行分化，產生不同功能與型態的細胞類型，且伴隨著細胞增殖的減緩。在這項研究中，我們發現某一群肺癌細胞，透過切換 SOX2 和 SOX9 所驅動的程式，產生癌細胞的塑性，使細胞具備不同的增殖與侵襲能力。SOX2 結合 EPCAM 啟動子 (promoter)，進而誘導 EpCAM–p21Cip1–cyclin A2 訊息路徑，幫助增加癌細胞增殖與屏障能力 (barrier property)。反觀，SOX9 結合 SLUG 啟動子，促使 SLUG 所誘導的癌細胞侵襲與紡錘狀表型 (spindle-like phenotype)。 抑制組織蛋白去乙醯酶 (histone deacetylase; HDAC) 的活性，漸少了 SOX2-positive 細胞族群，轉而增加 SOX9-positive 細胞族群。而 SOX2 的異位表達 (ectopic expression)，抑制 SOX9 表現並增加 SOX9 啟動子上組織蛋白 H3K9 雙甲基化 (histone H3K9 dimethylation; H3K9me2) 的程度。此外，SOX2 和 SOX9 的蛋白質表現，分別與肺癌腫瘤分級，呈現負相關和正相關。綜合上述，我們的研究結果支持了，表觀遺傳調控 (epigenetic regulation) SOX2 與 SOX9 所引導的癌細胞塑性 (cancer cell plasticity) 轉換，並在癌症發展過程中扮演關鍵角色。

Cell differentiation, driven by switching from stem cell- into differentiation-program, generates functionally and morphologically distinct cell types, accompanied with decelerated cell proliferation. In this study, we reported that a group of lung cancer cells generated cancer plasticity by switching between SOX2- and SOX9-mediated program, thus forming differential proliferation and invasion abilities. We found that SOX2 bound EPCAM promoter to induce EpCAM-p21Cip1-cyclin A2 signaling, hence encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound SLUG promoter to encourage SLUG-mediated cancer cell invasion with a spindle-like phenotype. Pharmacological inhibition of HDACs elevated SOX9-positive population from SOX2-postive cells whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on SOX9 promoter. Furthermore, SOX2 and SOX9 expression were negatively and positively correlated with lung tumor grades, respectively. Our findings support the involvement of epigenetic regulation in the SOX2- and SOX9-mediated cancer plasticity, providing critical insights for cancer progression.

REFERENCES

Adam, R.C., Yang, H., Rockowitz, S., Larsen, S.B., Nikolova, M., Oristian, D.S., Polak, L., Kadaja, M., Asare, A., Zheng, D., et al. (2015). Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521, 366-370.
Akiyama, H., Chaboissier, M.C., Martin, J.F., Schedl, A., and de Crombrugghe, B. (2002). The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes & development 16, 2813-2828.
Bass, A.J., Watanabe, H., Mermel, C.H., Yu, S., Perner, S., Verhaak, R.G., Kim, S.Y., Wardwell, L., Tamayo, P., Gat-Viks, I., et al. (2009). SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nature genetics 41, 1238-1242.
Bhattacharjee, A., Richards, W.G., Staunton, J., Li, C., Monti, S., Vasa, P., Ladd, C., Beheshti, J., Bueno, R., Gillette, M., et al. (2001). Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proceedings of the National Academy of Sciences of the United States of America 98, 13790-13795.
Boumahdi, S., Driessens, G., Lapouge, G., Rorive, S., Nassar, D., Le Mercier, M., Delatte, B., Caauwe, A., Lenglez, S., Nkusi, E., et al. (2014). SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma. Nature 511, 246-250.
Boyer, B., and Thiery, J.P. (1993). Epithelium-mesenchyme interconversion as example of epithelial plasticity. Apmis 101, 257-268.
Cano, A., Perez-Moreno, M.A., Rodrigo, I., Locascio, A., Blanco, M.J., del Barrio, M.G., Portillo, F., and Nieto, M.A. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2, 76-83.
Chaffer, C.L., Brennan, J.P., Slavin, J.L., Blick, T., Thompson, E.W., and Williams, E.D. (2006). Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer research 66, 11271-11278.
Chao, Y., Shih, Y.L., Chiu, J.H., Chau, G.Y., Lui, W.Y., Yang, W.K., Lee, S.D., and Huang, T.S. (1998). Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse of human hepatocellular carcinoma. Cancer research 58, 985-990.
Chen, H.M., Yen-Ping Kuo, M., Lin, K.H., Lin, C.Y., and Chiang, C.P. (2003). Expression of cyclin A is related to progression of oral squamous cell carcinoma in Taiwan. Oral oncology 39, 476-482.
Chen, X., Vega, V.B., and Ng, H.H. (2008a). Transcriptional regulatory networks in embryonic stem cells. Cold Spring Harbor symposia on quantitative biology 73, 203-209.
Chen, Y., Shi, L., Zhang, L., Li, R., Liang, J., Yu, W., Sun, L., Yang, X., Wang, Y., Zhang, Y., et al. (2008b). The molecular mechanism governing the oncogenic potential of SOX2 in breast cancer. The Journal of biological chemistry 283, 17969-17978.
Chou, Y.T., Hsieh, C.H., Chiou, S.H., Hsu, C.F., Kao, Y.R., Lee, C.C., Chung, C.H., Wang, Y.H., Hsu, H.S., Pang, S.T., et al. (2012). CITED2 functions as a molecular switch of cytokine-induced proliferation and quiescence. Cell Death Differ 19, 2015-2028.
Chou, Y.T., Lee, C.C., Hsiao, S.H., Lin, S.E., Lin, S.C., Chung, C.H., Chung, C.H., Kao, Y.R., Wang, Y.H., Chen, C.T., et al. (2013). The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung cancer. Stem cells 31, 2607-2619.
Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R., and Wu, C.W. (1997). Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology 17, 353-360.
Cox, J.L., Mallanna, S.K., Luo, X., and Rizzino, A. (2010). Sox2 uses multiple domains to associate with proteins present in Sox2-protein complexes. PloS one 5, e15486.
Doe, C.Q. (2008). Neural stem cells: balancing self-renewal with differentiation. Development 135, 1575-1587.
Garber, M.E., Troyanskaya, O.G., Schluens, K., Petersen, S., Thaesler, Z., Pacyna-Gengelbach, M., van de Rijn, M., Rosen, G.D., Perou, C.M., Whyte, R.I., et al. (2001). Diversity of gene expression in adenocarcinoma of the lung. Proceedings of the National Academy of Sciences of the United States of America 98, 13784-13789.
Giese, A., Bjerkvig, R., Berens, M.E., and Westphal, M. (2003). Cost of migration: invasion of malignant gliomas and implications for treatment. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, 1624-1636.
Giese, A., Loo, M.A., Tran, N., Haskett, D., Coons, S.W., and Berens, M.E. (1996). Dichotomy of astrocytoma migration and proliferation. International journal of cancer Journal international du cancer 67, 275-282.
Gonzalez, B., Denzel, S., Mack, B., Conrad, M., and Gires, O. (2009). EpCAM is involved in maintenance of the murine embryonic stem cell phenotype. Stem cells 27, 1782-1791.
Gordon, G.J., Jensen, R.V., Hsiao, L.L., Gullans, S.R., Blumenstock, J.E., Ramaswamy, S., Richards, W.G., Sugarbaker, D.J., and Bueno, R. (2002). Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma. Cancer research 62, 4963-4967.
Guo, W., Keckesova, Z., Donaher, J.L., Shibue, T., Tischler, V., Reinhardt, F., Itzkovitz, S., Noske, A., Zurrer-Hardi, U., Bell, G., et al. (2012). Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015-1028.
Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A., Saint-Andre, V., Sigova, A.A., Hoke, H.A., and Young, R.A. (2013). Super-enhancers in the control of cell identity and disease. Cell 155, 934-947.
Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Okita, K., and Yamanaka, S. (2009). Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132-1135.
Hotta, A., Cheung, A.Y., Farra, N., Vijayaragavan, K., Seguin, C.A., Draper, J.S., Pasceri, P., Maksakova, I.A., Mager, D.L., Rossant, J., et al. (2009). Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nature methods 6, 370-376.
Huang, H.P., Chen, P.H., Yu, C.Y., Chuang, C.Y., Stone, L., Hsiao, W.C., Li, C.L., Tsai, S.C., Chen, K.Y., Chen, H.F., et al. (2011). Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming. The Journal of biological chemistry 286, 33520-33532.
Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A.E., and Melton, D.A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature biotechnology 26, 795-797.
Jiang, S.S., Fang, W.T., Hou, Y.H., Huang, S.F., Yen, B.L., Chang, J.L., Li, S.M., Liu, H.P., Liu, Y.L., Huang, C.T., et al. (2010). Upregulation of SOX9 in lung adenocarcinoma and its involvement in the regulation of cell growth and tumorigenicity. Clinical cancer research : an official journal of the American Association for Cancer Research 16, 4363-4373.
Kidder, B.L., and Palmer, S. (2012). HDAC1 regulates pluripotency and lineage specific transcriptional networks in embryonic and trophoblast stem cells. Nucleic acids research 40, 2925-2939.
Kimura, O., Kondo, Y., Kogure, T., Kakazu, E., Ninomiya, M., Iwata, T., Morosawa, T., and Shimosegawa, T. (2014). Expression of EpCAM increases in the hepatitis B related and the treatment-resistant hepatocellular carcinoma. Biomed Res Int 2014, 172913.
Landi, M.T., Dracheva, T., Rotunno, M., Figueroa, J.D., Liu, H., Dasgupta, A., Mann, F.E., Fukuoka, J., Hames, M., Bergen, A.W., et al. (2008). Gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival. PloS one 3, e1651.
Lim, S.Y., Sivakumaran, P., Crombie, D.E., Dusting, G.J., Pebay, A., and Dilley, R.J. (2013). Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering. Stem cells translational medicine 2, 715-725.
Liu, S., Cong, Y., Wang, D., Sun, Y., Deng, L., Liu, Y., Martin-Trevino, R., Shang, L., McDermott, S.P., Landis, M.D., et al. (2014). Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem Cell Reports 2, 78-91.
Lu, Y., Futtner, C., Rock, J.R., Xu, X., Whitworth, W., Hogan, B.L., and Onaitis, M.W. (2010). Evidence that SOX2 overexpression is oncogenic in the lung. PloS one 5, e11022.
Ng, V.Y., Ang, S.N., Chan, J.X., and Choo, A.B. (2010). Characterization of epithelial cell adhesion molecule as a surface marker on undifferentiated human embryonic stem cells. Stem cells 28, 29-35.
Ocana, O.H., Corcoles, R., Fabra, A., Moreno-Bueno, G., Acloque, H., Vega, S., Barrallo-Gimeno, A., Cano, A., and Nieto, M.A. (2012). Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Cancer cell 22, 709-724.
Osta, W.A., Chen, Y., Mikhitarian, K., Mitas, M., Salem, M., Hannun, Y.A., Cole, D.J., and Gillanders, W.E. (2004). EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer research 64, 5818-5824.
Que, J., Luo, X., Schwartz, R.J., and Hogan, B.L. (2009). Multiple roles for Sox2 in the developing and adult mouse trachea. Development 136, 1899-1907.
Rockich, B.E., Hrycaj, S.M., Shih, H.P., Nagy, M.S., Ferguson, M.A., Kopp, J.L., Sander, M., Wellik, D.M., and Spence, J.R. (2013). Sox9 plays multiple roles in the lung epithelium during branching morphogenesis. Proceedings of the National Academy of Sciences of the United States of America 110, E4456-4464.
Sakai, D., Suzuki, T., Osumi, N., and Wakamatsu, Y. (2006). Cooperative action of Sox9, Snail2 and PKA signaling in early neural crest development. Development 133, 1323-1333.
Shen, C.I., Lee, H.C., Kao, Y.H., Wu, C.S., Chen, P.H., Lin, S.Z., Lai, P.S., and Su, H.L. (2014). EpCAM induction functionally links to the Wnt-enhanced cell proliferation in human keratinocytes. Cell Transplant 23, 1031-1044.
Shih, J.Y., Tsai, M.F., Chang, T.H., Chang, Y.L., Yuan, A., Yu, C.J., Lin, S.B., Liou, G.Y., Lee, M.L., Chen, J.J., et al. (2005). Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res 11, 8070-8078.
Shipitsin, M., Campbell, L.L., Argani, P., Weremowicz, S., Bloushtain-Qimron, N., Yao, J., Nikolskaya, T., Serebryiskaya, T., Beroukhim, R., Hu, M., et al. (2007). Molecular definition of breast tumor heterogeneity. Cancer cell 11, 259-273.
Shiwarski, D.J., Shao, C., Bill, A., Kim, J., Xiao, D., Bertrand, C.A., Seethala, R.S., Sano, D., Myers, J.N., Ha, P., et al. (2014). To "grow" or "go": TMEM16A expression as a switch between tumor growth and metastasis in SCCHN. Clinical cancer research : an official journal of the American Association for Cancer Research 20, 4673-4688.
Sholl, L.M., Long, K.B., and Hornick, J.L. (2010). Sox2 expression in pulmonary non-small cell and neuroendocrine carcinomas. Applied immunohistochemistry & molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry 18, 55-61.
Su, L.J., Chang, C.W., Wu, Y.C., Chen, K.C., Lin, C.J., Liang, S.C., Lin, C.H., Whang-Peng, J., Hsu, S.L., Chen, C.H., et al. (2007). Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme. BMC genomics 8, 140.
Thiery, J.P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442-454.
Tomida, S., Koshikawa, K., Yatabe, Y., Harano, T., Ogura, N., Mitsudomi, T., Some, M., Yanagisawa, K., Takahashi, T., Osada, H., et al. (2004). Gene expression-based, individualized outcome prediction for surgically treated lung cancer patients. Oncogene 23, 5360-5370.
Tompkins, D.H., Besnard, V., Lange, A.W., Wert, S.E., Keiser, A.R., Smith, A.N., Lang, R., and Whitsett, J.A. (2009). Sox2 is required for maintenance and differentiation of bronchiolar Clara, ciliated, and goblet cells. PloS one 4, e8248.
Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer cell 22, 725-736.
Yamamizu, K., Schlessinger, D., and Ko, M.S. (2014). SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1). Development 141, 4254-4266.