前人研究指出，缺氧扮演一個重要的環境因子對於胚胎發育或是腫瘤發展。更多研究發現，惡劣腫瘤細胞的形成與缺氧環境導致的基因調節有很大的關聯性癌細胞透過何種因子去克服缺氧環境所伴隨而來的養分缺乏以及DNA損害等環境壓力，是目前對於癌症治療急於解答的問題。Oct4在幹細胞中扮演一個相當重要的轉錄因子，尤其在細胞的自我更新 (self-renewal) 能力上。目前在不同的腫瘤細胞中也發現了Oct4表達的情形，但對於Oct4在促進腫瘤之形成所扮演的角色目前仍不清楚。我們的研究中發現，Oct4 的其中一個異構物 Oct4B 可以在肺癌細胞中受到缺氧誘導因子(HIF-2α)的誘導增量。Oct4B 在肺腺癌細胞中的超量表現，會有增加細胞生長 (cell proliferation) 的特性、抵抗活性氧 (ROS) 導致的細胞凋亡反應，以及促進異體移殖(xenograft)的細胞生長。本研究結果指出，Oct4B 具有誘導肺癌細胞由上皮細胞轉化為間質細胞(Epithelial Mesetransition, EMT) 的效果，進一步我們發現Oct4B俱有促進細胞移動(cell migration的能力，我們也發現一個重要的致癌因子Slug，在Oct4B 的表現中被誘導出來。此外上皮生長因子接受器 (epidermal growth factor receptor, EGFR)的活化也在Oct4B 於肺癌細胞的表現中所發現。上述實驗結果首次闡述Oct4B可以在缺氧環境中所誘導表現，且使肺癌細胞適應於壓力環境，同時也增進了肺癌細胞的癌化能力(Oncogenesis)。

Hypoxia, a reduction of normal oxygen levels in cells or tissues, creates a variety of changes in cell metabolism including increased oxidative stress and DNA damage. Though Oct4, a homeobox transcription factor essential for self-renewal of stem cells, is expressed in various cancers, little is known about the functional role of Oct4 in tumorigenesis. In this study, we discovered that hypoxia induces a short isoform of Oct4, termed as Oct4B, in lung cancer through a HIF-2? dependent pathway. Overexpression of Oct4B induced cell proliferation and anchorage-independent growth of lung cancer, indicating the oncogenic potential of Oct4B. In addition, ectopic expression of Oct4B prevented cells from oxidative stress induced apoptosis, suggesting a functional role of Oct4B in anti-apoptosis. Overexpression of Oct4B promoted tumor formation in xenograft mouse model, demonstrating the positive involvement of Oct4B in tumorigenesis. We observed the increased activity of EGFR signaling in both hypoxia-treated or Oct4B-transfected cells. Q-PCR analysis demonstrated that Oct4B enhanced the transcript of TGF-?? a cognate ligand of EGFR. In addition, ectopic expression of Oct4B induced epithelial mesenchymal tans-differentiation and promoted cell migration. Through cDNA microarray and Q-PCR analyses, we identified that Oct4B induces Slug, a key effector in epithelial mesenchymal tans-differentiation.Thus, our results provide a novel mechanism that shows how Oct4B is induced by hypoxia to enable cancer cells to adapt environmental pressures and encourage malignancy in lung cancer

Abstract...................................................5
中文摘要 (Abstract of Chinese).............................7
Chapter1:..................................................8
Introduction...............................................8
1.1 Oct4...................................................8
1.2 Hypoxia................................................9
1.3 Hypoxia and Cancer....................................10
1.4 Hypoxia and EMT.......................................12
1.5 Hypoxia and EGFR......................................13
1.6 Working Hypothesis....................................14
Chapter 2:................................................15
Materials and Methods.....................................15
2.1 DNA plasmid generation................................15
2.2 Cell culture..........................................15
2.3 Lentiviral infection..................................16
2.4 Quantitative Real-time PCR and primer sequence........17
2.5 Luciferase reporter assay.............................17
2.6 Protein extraction and Western blotting...............18
2.7 Hypoxia...............................................21
2.8 Colony formation assay................................21
2.9 WST assay.............................................22
2.10 BrdU incorporation assay.............................22
2.11 Soft agar assay......................................23
2.12 Migration assay......................................24
2.13 Apoptosis cells detection............................25
2.14 Xenograft tumorgenecity assay........................26
2.15 Immunohistochemistry.................................26
Chapter 3: Result.........................................28
3.1 Hypoxia via HIF-2α induces Oct4 expression in lung cancer....................................................28
3.2 Oct4B, but not Oct4A, is induced by hypoxia in lung cancer ...................................................29
3.3 Oct4B induces Slug expression in lung cancer..........29
3.4 Oct4B induces epithelial-mesenchymal transdifferentiation (EMT) and
migration of lung cancer cells............................30
3.5 Oct4B promotes TGF alpha release and enhances EGFR activity..................................................31
3.6 Oct4B promotes cell growth and anti-apoptosis.........31
3.7 Oct4B enhances cell tumorgenecity.....................32
3.8 Model for Oct4B mediated oncogenesis of lung cancer ..33
Chapter 4:Discussion......................................34
Figures...................................................38
Figure 1. Oct4 is induced by hypoxia in lung cancer cells.38
Figure 2. HIF-2α is essential for hypoxia-induced Oct4 expression................................................40
Figure 3. Oct4B is dominantly induced by hypoxia..........42
Figure 4. Oct4-silencing leads to significant morphological change and attenuates Slug expression.....................44
Figure 5. Hypoxia induces Slug by HIF-2α in lung cancer cells. ...................................................46
Figure 6. Oct4B enhances Slug expression, induces epithelial mesenchymal transition, and promotes cell migration.................................................48
Figure 7. Oct4B enhances EGFR activity....................50
Figure 8. Oct4B promotes cell proliferation and anchorage-independent ability ......................................52
Figure 9. Oct4B promotes anti-apoptosis ability...........54
Figure 10. Effect of Oct4B on tumor formation. ...........58
Figure 11. A model of Oct4B mediated oncogenesis of lung cancer ...................................................62
Reference.................................................63

Atlasi, Y., Mowla, S.J., Ziaee, S.A., Gokhale, P.J., and Andrews, P.W. (2008). OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells. Stem Cells 26, 3068-3074.
Batlle, E., Sancho, E., Franci, C., Dominguez, D., Monfar, M., Baulida, J., and Garcia De Herreros, A. (2000). The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2, 84-89.
Ben-Porath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W., Regev, A., and Weinberg, R.A. (2008). An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40, 499-507.
Bertout, J.A., Majmundar, A.J., Gordan, J.D., Lam, J.C., Ditsworth, D., Keith, B., Brown, E.J., Nathanson, K.L., and Simon, M.C. (2009). HIF2alpha inhibition promotes p53 pathway activity, tumor cell death, and radiation responses. Proc Natl Acad Sci U S A 106, 14391-14396.
Blanco, M.J., Moreno-Bueno, G., Sarrio, D., Locascio, A., Cano, A., Palacios, J., and Nieto, M.A. (2002). Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene 21, 3241-3246.
Boiani, M., Eckardt, S., Scholer, H.R., and McLaughlin, K.J. (2002). Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 16, 1209-1219.
Bussmann, L.H., Schubert, A., Vu Manh, T.P., De Andres, L., Desbordes, S.C., Parra, M., Zimmermann, T., Rapino, F., Rodriguez-Ubreva, J., Ballestar, E., et al. (2009). A robust and highly efficient immune cell reprogramming system. Cell Stem Cell 5, 554-566.
Chen, J., Imanaka, N., and Griffin, J.D. (2010). Hypoxia potentiates Notch signaling in breast cancer leading to decreased E-cadherin expression and increased cell migration and invasion. Br J Cancer 102, 351-360.
Chen, Y.C., Hsu, H.S., Chen, Y.W., Tsai, T.H., How, C.K., Wang, C.Y., Hung, S.C., Chang, Y.L., Tsai, M.L., Lee, Y.Y., et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One 3, e2637.
Covello, K.L., Kehler, J., Yu, H., Gordan, J.D., Arsham, A.M., Hu, C.J., Labosky, P.A., Simon, M.C., and Keith, B. (2006). HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev 20, 557-570.
Ema, M., Taya, S., Yokotani, N., Sogawa, K., Matsuda, Y., and Fujii-Kuriyama, Y. (1997). A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A 94, 4273-4278.
Franovic, A., Gunaratnam, L., Smith, K., Robert, I., Patten, D., and Lee, S. (2007). Translational up-regulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer. Proc Natl Acad Sci U S A 104, 13092-13097.
Gordan, J.D., Bertout, J.A., Hu, C.J., Diehl, J.A., and Simon, M.C. (2007). HIF-2alpha promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. Cancer Cell 11, 335-347.
Gunaratnam, L., Morley, M., Franovic, A., de Paulsen, N., Mekhail, K., Parolin, D.A., Nakamura, E., Lorimer, I.A., and Lee, S. (2003). Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells. J Biol Chem 278, 44966-44974.
Harris, A.L. (2002). Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2, 38-47.
Hill, R.P., Marie-Egyptienne, D.T., and Hedley, D.W. (2009). Semin Radiat Oncol 19, 106-111.
Holmquist-Mengelbier, L., Fredlund, E., Lofstedt, T., Noguera, R., Navarro, S., Nilsson, H., Pietras, A., Vallon-Christersson, J., Borg, A., Gradin, K., et al. (2006). Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. Cancer Cell 10, 413-423.
Hu, C.J., Wang, L.Y., Chodosh, L.A., Keith, B., and Simon, M.C. (2003). Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol 23, 9361-9374.
Huang, C.H., Yang, W.H., Chang, S.Y., Tai, S.K., Tzeng, C.H., Kao, J.Y., Wu, K.J., and Yang, M.H. (2009). Regulation of membrane-type 4 matrix metalloproteinase by SLUG contributes to hypoxia-mediated metastasis. Neoplasia 11, 1371-1382.
Keith, B., and Simon, M.C. (2007). Hypoxia-inducible factors, stem cells, and cancer. Cell 129, 465-472.
Lee, J., Kim, H.K., Rho, J.Y., Han, Y.M., and Kim, J. (2006). The human OCT-4 isoforms differ in their ability to confer self-renewal. J Biol Chem 281, 33554-33565.
Lee, S.M. (2006). Is EGFR expression important in non-small cell lung cancer? Thorax 61, 98-99.
Loboda, A., Jozkowicz, A., and Dulak, J. (2010). HIF-1 and HIF-2 transcription factors--similar but not identical. Mol Cells 29, 435-442.
Ma, W., Yan, R.T., Li, X., and Wang, S.Z. (2009). Reprogramming retinal pigment epithelium to differentiate toward retinal neurons with Sox2. Stem Cells 27, 1376-1387.
Nelson, D.A., Tan, T.T., Rabson, A.B., Anderson, D., Degenhardt, K., and White, E. (2004). Hypoxia and defective apoptosis drive genomic instability and tumorigenesis. Genes Dev 18, 2095-2107.
Otrock, Z.K., Hatoum, H.A., Awada, A.H., Ishak, R.S., and Shamseddine, A.I. (2009). Hypoxia-inducible factor in cancer angiogenesis: structure, regulation and clinical perspectives. Crit Rev Oncol Hematol 70, 93-102.
Pan, G.J., Chang, Z.Y., Scholer, H.R., and Pei, D. (2002). Stem cell pluripotency and transcription factor Oct4. Cell Res 12, 321-329.
Pouyssegur, J., Dayan, F., and Mazure, N.M. (2006). Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441, 437-443.
Raval, R.R., Lau, K.W., Tran, M.G., Sowter, H.M., Mandriota, S.J., Li, J.L., Pugh, C.W., Maxwell, P.H., Harris, A.L., and Ratcliffe, P.J. (2005). Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 25, 5675-5686.
Rusch, V., Baselga, J., Cordon-Cardo, C., Orazem, J., Zaman, M., Hoda, S., McIntosh, J., Kurie, J., and Dmitrovsky, E. (1993). Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 53, 2379-2385.
Sahlgren, C., Gustafsson, M.V., Jin, S., Poellinger, L., and Lendahl, U. (2008). Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proc Natl Acad Sci U S A 105, 6392-6397.
Semenza, G.L. (2003). Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3, 721-732.
Sharma, S.V., Bell, D.W., Settleman, J., and Haber, D.A. (2007). Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7, 169-181.
Sung, M.T., Jones, T.D., Beck, S.D., Foster, R.S., and Cheng, L. (2006). OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy. Hum Pathol 37, 662-667.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.
Thomlinson, R.H., and Gray, L.H. (1955). The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 9, 539-549.
Tomasson, M.H. (2009). Cancer stem cells: a guide for skeptics. J Cell Biochem 106, 745-749.
Wang, J.S., and Lin, C.T. (2010). Systemic hypoxia promotes lymphocyte apoptosis induced by oxidative stress during moderate exercise. Eur J Appl Physiol 108, 371-382.
Wang, X., and Schneider, A. (2010). HIF-2alpha-mediated activation of the epidermal growth factor receptor potentiates head and neck cancer cell migration in response to hypoxia. Carcinogenesis 31, 1202-1210.
Wang, X., Zhao, Y., Xiao, Z., Chen, B., Wei, Z., Wang, B., Zhang, J., Han, J., Gao, Y., Li, L., et al. (2009). Alternative translation of OCT4 by an internal ribosome entry site and its novel function in stress response. Stem Cells 27, 1265-1275.
Wiesener, M.S., Jurgensen, J.S., Rosenberger, C., Scholze, C.K., Horstrup, J.H., Warnecke, C., Mandriota, S., Bechmann, I., Frei, U.A., Pugh, C.W., et al. (2003). Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs. FASEB J 17, 271-273.
Yan, W., Fu, Y., Tian, D., Liao, J., Liu, M., Wang, B., Xia, L., Zhu, Q., and Luo, M. (2009). PI3 kinase/Akt signaling mediates epithelial-mesenchymal transition in hypoxic hepatocellular carcinoma cells. Biochem Biophys Res Commun 382, 631-636.
Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., and Yamanaka, S. (2009). Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5, 237-241.
Zavadil, J., Bitzer, M., Liang, D., Yang, Y.C., Massimi, A., Kneitz, S., Piek, E., and Bottinger, E.P. (2001). Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta. Proc Natl Acad Sci U S A 98, 6686-6691