Octamer 4 (Oct4) 是由 Pou5f1 基因轉錄的 POU 轉錄因子，在胚胎幹細胞中維持分化多能性 (plouripotency) 及自我更新 (self-renewal) 的能力。相對於不表現Oct4的體細胞，在不同種類腫瘤觀察到 Oct4 的表現，意味著 Oct4 在腫瘤形成過程中扮演重要角色。在膀胱癌小鼠模式中，Oct4 表現量的增加已證明會促進癌細胞的增生及轉移。雙特異性去磷酸酶6號 (dual-specificity phosphatase 6, DUSP6) 為絲裂原活化蛋白激酶 (mitogen-activated protein kinase, MAPK) 的去磷酸酶 (MAPK phosphatase, MKP) 之一，在胰臟癌中已知會造成細胞外訊息調節激酶2 (extracellular signal-regulated kinase 2, ERK-2) 的去活化，而被視為具有腫瘤抑制的功能。DUSP6 的表現與高復發且低存活率的非小細胞肺癌 (non-small cell lung cancer, NSCLC) 病患有密切的相關性。在未分化的小鼠胚胎幹細胞中利用染色質免疫沉澱分析，發現 DUSP6 可能是 Oct4 下游所調控的基因。在已發表的 cDNA 微陣列資料庫顯示，降低 Oct4 的表現會造成小鼠胚胎幹細胞中 DUSP6 的 mRNA 表現量降低。然而 Oct4 如何調控 DUSP6 表現的機制目前並不清楚。在本研究中，觀察肺癌病患的肺部組織切片染色及肺癌細胞株的結果，發現 Oct4 與 DUSP6 的表現量具有正相關性。在肺癌細胞株過度或降低 Oct4 的表現後，發現 DUSP6 在蛋白質及 mRNA 的表現量也隨之增減，可知 Oct4 與 DUSP6具有正向調控的關係。進一步確認 Oct4 如何在轉錄上調控 DUSP6 基因，我們利用染色質免疫沉澱分析及報導基因分析證明 Oct4是直接結合至 DUSP6 啟動子而增加其活性，以調控DUSP6 基因的表現。在細胞實驗結果也觀察到 Oct4 會增加肺癌細胞的生長速度及轉移能力，並在不同 Oct4 表現量的實驗組中發現 DUSP6 在肺部組織表現的位置與 Oct4 表現的位置相似。綜合以上結果推測 Oct4 促進非小細胞肺癌轉移的機制，可能是藉由調控 DUSP6 基因的表現，啟動一連串的訊息傳遞而影響非小細胞肺癌的轉移。目前 DUSP6 在轉移所扮演的角色尚未明確，需進一步在細胞及動物實驗中將 Oct4 過量表現並降低 DUSP6 的表現量下，觀察肺癌細胞的爬行及侵入能力是否會受到影響，以證實 Oct4 確實可透過調控 DUSP6 而影響肺癌細胞的轉移。

Octamer 4 (Oct4) is a POU domain-containing transcription factor encoded by Pou5f1 and required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. However, Oct4 has been detected in several human tumors, suggesting a potentially critical role in tumorigenesis. Dual-specificity phosphatase 6 (DUSP6) is one of the mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and its inactivation of extracellular signal-regulated kinases 2 (ERK-2) is believed as to function as a tumor suppressor in pancreatic cancer. In contrast, DUSP6 is closely associated with an increased risk of recurrence and decreased overall survival among patients with non-small cell lung cancer. Moreover, chromatin immunoprecipitation (ChIP) assay revealed that DUSP6 may be a potential downstream target of Oct4 in undifferentiated mouse ES cells. Down-regulation of DUSP6 mRNA expression in Oct4-knockdown mouse ES cells was also demonstrated in the public domain database. However, the mechanism underlying the regulation of DUSP6 expression by Oct4 is still unclear. The aim of this study was to study the association of Oct4 and DUSP6 in lung cancer and to elucidate its mechanism of action. My results revealed that there was a positive correlation between the expression of Oct4 and DUSP6 in lung cancer cells. Overexpression of Oct4 enhanced the expression of DUSP6 at both mRNA and protein levels. Chromatin immunoprecipitation (ChIP) and reporter assays showed that Oct4 enhanced the promoter activity of DUSP6 through direct binding to its promoter to regulate DUSP6 gene expression. Oct4 also enhanced the proliferation rate and migratory ability of lung cancer cells in vivo. Taken together, these results suggest that Oct4 regulates DUSP6 expression to initiate signal transduction cascades, leading to promoting the metastasis of non-small lung cancer cells. In the future, I will focus on studying the effects of Oct4-mediated DUSP6 expression on enhancing tumor migration and invasion in vitro and in vivo.

Arkell, R.S., Dickinson, R.J., Squires, M., Hayat, S., Keyse, S.M., and Cook, S.J. (2008). DUSP6/MKP-3 inactivates ERK1/2 but fails to bind and inactivate ERK5. Cell Signal 20, 836-843.

Bloethner, S., Chen, B., Hemminki, K., Muller-Berghaus, J., Ugurel, S., Schadendorf, D., and Kumar, R. (2005). Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines. Carcinogenesis 26, 1224-1232.

Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956.

Brehm, A., Ohbo, K., and Scholer, H. (1997). The carboxy-terminal transactivation domain of Oct4 acquires cell specificity through the POU domain. Mol Cell Biol 17, 154-162.

Chambers, I., and Smith, A. (2004). Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 23, 7150-7160.

Chan, D.W., Liu, V.W., Tsao, G.S., Yao, K.M., Furukawa, T., Chan, K.K., and Ngan, H.Y. (2008). Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells. Carcinogenesis 29, 1742-1750.

Chang, C.C., Shieh, G.S., Wu, P., Lin, C.C., Shiau, A.L., and Wu, C.L. (2008). Oct-3/4 expression reflects tumor progression and regulates motility of bladder cancer cells. Cancer Res 68, 6281-6291.

Chen, H.Y., Yu, S.L., Chen, C.H., Chang, G.C., Chen, C.Y., Yuan, A., Cheng, C.L., Wang, C.H., Terng, H.J., Kao, S.F., et al. (2007). A five-gene signature and clinical outcome in non-small-cell lung cancer. N Engl J Med 356, 11-20.

Chen, J.J., Peck, K., Hong, T.M., Yang, S.C., Sher, Y.P., Shih, J.Y., Wu, R., Cheng, J.L., Roffler, S.R., Wu, C.W., et al. (2001). Global analysis of gene expression in invasion by a lung cancer model. Cancer Res 61, 5223-5230.

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). Oct4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One 3, e2637.

Cheng, L. (2004). Establishing a germ cell origin for metastatic tumors using OCT4 immunohistochemistry. Cancer 101, 2006-2010.

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. Am J Respir Cell Mol Biol 17, 353-360.

Ekerot, M., Stavridis, M.P., Delavaine, L., Mitchell, M.P., Staples, C., Owens, D.M., Keenan, I.D., Dickinson, R.J., Storey, K.G., and Keyse, S.M. (2008). Negative-feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3 gene promoter. Biochem J 412, 287-298.

Furukawa, T., Fujisaki, R., Yoshida, Y., Kanai, N., Sunamura, M., Abe, T., Takeda, K., Matsuno, S., and Horii, A. (2005). Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas. Mod Pathol 18, 1034-1042.

Furukawa, T., Sunamura, M., Motoi, F., Matsuno, S., and Horii, A. (2003). Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer. Am J Pathol 162, 1807-1815.

Gidekel, S., Pizov, G., Bergman, Y., and Pikarsky, E. (2003). Oct-3/4 is a dose-dependent oncogenic fate determinant. Cancer Cell 4, 361-370.

Gollob, J.A., Wilhelm, S., Carter, C., and Kelley, S.L. (2006). Role of Raf kinase in cancer: therapeutic potential of targeting the Raf/MEK/ERK signal transduction pathway. Semin Oncol 33, 392-406.

Gupta, G.P., and Massague, J. (2006). Cancer metastasis: building a framework. Cell 127, 679-695.

Gupta, P., Ho, P.C., Huq, M.M., Ha, S.G., Park, S.W., Khan, A.A., Tsai, N.P., and Wei, L.N. (2008). Retinoic acid-stimulated sequential phosphorylation, PML recruitment, and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression. Proc Natl Acad Sci U S A 105, 11424-11429.

Hakansson, P., Nilsson, B., Andersson, A., Lassen, C., Gullberg, U., and Fioretos, T. (2008). Gene expression analysis of BCR/ABL1-dependent transcriptional response reveals enrichment for genes involved in negative feedback regulation. Genes Chromosomes Cancer 47, 267-275.

Herbst, R.S., Heymach, J.V., and Lippman, S.M. (2008). Lung cancer. N Engl J Med 359, 1367-1380.

Huang, P.Z., Lu, C.L., Li, B.K., Hong, J., Huang, L., Wang, L., Zhang, Y., and Yuan, Y.F. (2010). [OCT4 expression in hepatocellular carcinoma and its clinical significance]. Ai Zheng 29, 111-116.

Jones, T.D., MacLennan, G.T., Bonnin, J.M., Varsegi, M.F., Blair, J.E., and Cheng, L. (2006). Screening for intratubular germ cell neoplasia of the testis using OCT4 immunohistochemistry. Am J Surg Pathol 30, 1427-1431.

Kimura, M., Furukawa, T., Abe, T., Yatsuoka, T., Youssef, E.M., Yokoyama, T., Ouyang, H., Ohnishi, Y., Sunamura, M., Kobari, M., et al. (1998). Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer. Cancer Res 58, 2456-2460.

Klemm, J.D., Rould, M.A., Aurora, R., Herr, W., and Pabo, C.O. (1994). Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77, 21-32.

Kobayashi, S., Boggon, T.J., Dayaram, T., Janne, P.A., Kocher, O., Meyerson, M., Johnson, B.E., Eck, M.J., Tenen, D.G., and Halmos, B. (2005). EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786-792.

Kolch, W. (2005). Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6, 827-837.

Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., et al. (2006). The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431-440.

Monk, M., and Holding, C. (2001). Human embryonic genes re-expressed in cancer cells. Oncogene 20, 8085-8091.

Muda, M., Boschert, U., Dickinson, R., Martinou, J.C., Martinou, I., Camps, M., Schlegel, W., and Arkinstall, S. (1996). MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J Biol Chem 271, 4319-4326.

Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-376.

Pesce, M., and Scholer, H.R. (2001). Oct4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271-278.

Ramnarain, D.B., Park, S., Lee, D.Y., Hatanpaa, K.J., Scoggin, S.O., Otu, H., Libermann, T.A., Raisanen, J.M., Ashfaq, R., Wong, E.T., et al. (2006). Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in glioma cells. Cancer Res 66, 867-874.

Sato, M., Vaughan, M.B., Girard, L., Peyton, M., Lee, W., Shames, D.S., Ramirez, R.D., Sunaga, N., Gazdar, A.F., Shay, J.W., et al. (2006). Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 66, 2116-2128.

Scholer, H.R. (1991). Octamania: the POU factors in murine development. Trends Genet 7, 323-329.

Scholer, H.R., Ruppert, S., Suzuki, N., Chowdhury, K., and Gruss, P. (1990). New type of POU domain in germ line-specific protein Oct4. Nature 344, 435-439.

Sharov, A.A., Masui, S., Sharova, L.V., Piao, Y., Aiba, K., Matoba, R., Xin, L., Niwa, H., and Ko, M.S. (2008). Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data. BMC Genomics 9, 269.

Sun, S., Schiller, J.H., and Gazdar, A.F. (2007). Lung cancer in never smokers--a different disease. Nat Rev Cancer 7, 778-790.

Trono, D. (2003). Virology. Picking the right spot. Science 300, 1670-1671.

Warmka, J.K., Mauro, L.J., and Wattenberg, E.V. (2004). Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras. J Biol Chem 279, 33085-33092.

Xu, S., Furukawa, T., Kanai, N., Sunamura, M., and Horii, A. (2005). Abrogation of DUSP6 by hypermethylation in human pancreatic cancer. J Hum Genet 50, 159-167.

Yano, S., Nokihara, H., Hanibuchi, M., Parajuli, P., Shinohara, T., Kawano, T., and Sone, S. (1997). Model of malignant pleural effusion of human lung adenocarcinoma in SCID mice. Oncol Res 9, 573-579.

Zhang, X., Han, B., Huang, J., Zheng, B., Geng, Q., Aziz, F., and Dong, Q. (2010a). Prognostic Significance of OCT4 Expression in Adenocarcinoma of the Lung. Jpn J Clin Oncol.

Zhang, Z., Kobayashi, S., Borczuk, A.C., Leidner, R.S., Laframboise, T., Levine, A.D., and Halmos, B. (2010b). Dual specificity phosphatase 6 (DUSP6) is an ETS-regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells. Carcinogenesis 31, 577-586.