OCT4-NANOG Promotes Lung Cancer Oncogenesis through Induction of Epithelial-Mesenchymal Transdifferentiations.

簡易檢索 / 詳目顯示

回結果列表

研究生：	陳啟仁 Chen, Chi-Jen
論文名稱：	OCT4-NANOG Promotes Lung Cancer Oncogenesis through Induction of Epithelial-Mesenchymal Transdifferentiations.
指導教授：	吳成文 Wu, Cheng-Wen 劉銀樟 Liu, Yin-Chang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2009
畢業學年度：	97
語文別：	英文
論文頁數：	64
中文關鍵詞：	OCT4 、NANOG 、EMT 、Cancer stem cell
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

OCT4 and NANOG are overexpressed in several cancers and involved in self-renewal signaling of stem cells, and downstream targets of OCT4 and NANOG are more frequently overexpressed in poorly differentiated tumors than in well-differentiated ones. However, little is known about the role of OCT4-NANOG signaling in lung cancer development. We discovered that both OCT4 and NANOG were co-overexpressed in adenocarcinoma. Overexpression of OCT4 and NANOG enhanced anchorage-independent cell growth in lung cancer, and promoted Xenograft lung tumor growth with poorly differentiated phenotype. Interestingly, overexpression of OCT4 and NANOG in lung cancer induced CD133+ cells and sphere formation, suggesting a regulatory role of OCT4-NANOG signaling in cancer stem cell formation. We identified that OCT4-NANOG promoted cell migration and induced epithelial-mesenchymal transition (EMT) through upregulation of SLUG, SNAIL, TWIST and TGF-β RI. As our knowledge, this is the first report demonstrating that OCT4-NANOG signaling promotes oncogenesis through induction of EMT.

前人研究指出，主要調控幹細胞自我更新 (self-renewal)的兩個重要基因
OCT4及NANOG高量表現在多種癌症中。與分化較完全的腫瘤 (well-differentiated tumor)相較之下，OCT4及NANOG所調控的下游(downstream)訊息分子，高量表現在分化不完全的腫瘤組織(poor differentiated tumor)。然而，OCT4及NANOG所調控的訊息路徑，對於肺癌發展所產生的影響則尚未十分清楚。我們發現OCT4及NANOG在肺腺癌組織中 (primary lung adenocarcinoma)呈現共同超量表現的現象。 OCT4及NANOG在肺腺癌細胞中的超量表現，會增加細胞不貼附生長 (anchorage-independent growth)的特性、促進異體移植(Xenograft)的細胞生長、並產生分化未完全的細胞型態 (poor differentiated phenotype)。研究結果顯示，OCT4及NANOG具有誘發肺癌中CD133+ 細胞及增強腫瘤球體 (tumor sphere)形成的作用。換言之，OCT4及NANOG可能經由誘發訊息路徑，共同調節癌幹細胞(cancer stem cells)的產生及增生。此外，我們也發現，藉由活化SLUG、 SNAIL、TWIST及TGF-β RI等因子，OCT4-NANOG具有誘導肺癌細胞由上皮細胞轉化為間質細胞 (Epithelial Mesenchymal transition, EMT) 的效果，進而促進肺癌細胞移動能力(migration ability)。上述實驗結果顯示，OCT4及NANOG經由幹細胞路徑，誘導EMT的發生而提高細胞的癌化能力（Oncogenesis）。

Abstract    5
中文摘要(Abstract of Chinese)    6
Chapter1：    7
Introduction    7
1.1ES signature in cancer progression    7
1.2Cancer stem cells and ES regulatory gene    12
1.3Epithelial-Mesenchymal transition.    16
1.4 A549 cell line    20
Chapter 2：    22
Materials and Methods    22
2.1 DNA plasmid generation    22
2.2 Cell culture, Tranfection reagent, Virus preparation    23
2.4Quantitative PCR and primer sequence    26
2.5 Protein extraction and Western blotting    27
2.6 Colony formation and Anchorage-independent assay    30
2.7Migration assay    32
2.8Immuno-cytochemistry    33
2.9Flow cytometric analysis    34
2.10Sphere formation assay    34
2.11 Patients and tissue samples.    35
2.12Xenograft tumorigenicity assay.    35
Chapter 3：Result    37
3.1 Elevated OCT4 and NANOG expression in human lung cancers.    37
3.2 Ectopic expression of OCT4-NANOG induces spindle phenotype and foci formation in A549 lung cancer cells.    38
3.3 OCT4-NANOG induced CD133+ cells and activated DNMT3B expression.    38
3.4 OCT4-NANOG promoted cell growth and anchorage-independent colony formation.    39
3.5 Ectopic expression of OCT4-NANOG induced EMT    40
3.6 OCT4 induces SLUG transcript in A549 cells.    41
3.7 OCT4-NANOG enhanced tumor growth.    41
Chapter 4 ：    43
Discussion    43
Figures    46
Figure 1. OCT4 and NANOG were up-regulated in primary lung cancer.    47
Figure 2. Ectopic expression of OCT4 and NANOG induced phenotypic change of A549 lung cancer cells.    48
Figure 3. Ectopic overexpression of OCT4 and NANOG in A549 cells.    49
Figure 4. OCT4-NANOG signaling promoted expression of stem cell markers such as CD133 and Dnmt3b.    50
Figure 5. OCT4-NANOG overexpression did not turn on endogenous embryonic stemness circuits.    51
Figure 6. OCT4-NANOG signaling induced sphere formation    52
Figure 7. OCT4-NANOG signaling promoted cell growth.    53
Figure 8. OCT4-NANOG signaling promotes anchorage-independent cell growth.    54
Figure 10. OCT4-NANOG signaling induces expression of key regulators involved in epithelial-mesenchymal transition.    56
Figure 11. OCT4-NANOG signaling induced EMT    57
Figure 12. OCT4-NANOG signaling suppresses E-cadherin but induces Vimentin expression.    58
Figure 13. OCT4 induces SLUG mRNA expression in A549 cells.      59
Figure 14. OCT4-NANOG signaling promoted tumor formation in nude mice.    60
Figure 15. OCT4-NANOG induced cell mitosis and invasion ability.    61

Reference    62

                                

Reference
Al-Hajj, M., M. S. Wicha, et al. (2003). "Prospective identification of tumorigenic breast cancer cells." Proc Natl Acad Sci U S A 100(7): 3983-8.
Batlle, E., E. Sancho, et al. (2000). "The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells." Nat Cell Biol 2(2): 84-9.
Baum, B., J. Settleman, et al. (2008). "Transitions between epithelial and mesenchymal states in development and disease." Semin Cell Dev Biol 19(3): 294-308.
Ben-Porath, I., M. W. Thomson, et al. (2008). "An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors." Nat Genet 40(5): 499-507.
Boiani, M. and H. R. Scholer (2005). "Regulatory networks in embryo-derived pluripotent stem cells." Nat Rev Mol Cell Biol 6(11): 872-84.
Boyer, L. A., T. I. Lee, et al. (2005). "Core transcriptional regulatory circuitry in human embryonic stem cells." Cell 122(6): 947-56.
Chen, Y., L. Shi, et al. (2008). "The molecular mechanism governing the oncogenic potential of SOX2 in breast cancer." J Biol Chem 283(26): 17969-78.
Chiou, S. H., C. L. Kao, et al. (2008). "Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor." PLoS One 3(5): e2090.
Chiou, S. H., C. C. Yu, et al. (2008). "Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma." Clin Cancer Res 14(13): 4085-95.
Dallas, N. A., L. Xia, et al. (2009). "Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition." Cancer Res 69(5): 1951-7.
Derynck, R. and R. J. Akhurst (2007). "Differentiation plasticity regulated by TGF-beta family proteins in development and disease." Nat Cell Biol 9(9): 1000-4.
Dimeo, T. A., K. Anderson, et al. (2009). "A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer." Cancer Res.
Dontu, G., W. M. Abdallah, et al. (2003). "In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells." Genes Dev 17(10): 1253-70.
Eramo, A., F. Lotti, et al. (2008). "Identification and expansion of the tumorigenic lung cancer stem cell population." Cell Death Differ 15(3): 504-14.
Gupta, G. P. and J. Massague (2006). "Cancer metastasis: building a framework." Cell 127(4): 679-95.
Hilbe, W., S. Dirnhofer, et al. (2004). "CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer." J Clin Pathol 57(9): 965-9.
Hu, T., S. Liu, et al. (2008). "Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis." Cancer Res 68(16): 6533-40.
Jeter, C. R., M. Badeaux, et al. (2009). "Functional evidence that the self-renewal gene NANOG regulates human tumor development." Stem Cells 27(5): 993-1005.
Jethwa, P., M. Naqvi, et al. (2008). "Overexpression of Slug is associated with malignant progression of esophageal adenocarcinoma." World J Gastroenterol 14(7): 1044-52.
Karoubi, G., M. Gugger, et al. (2009). "OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention." Interact Cardiovasc Thorac Surg 8(4): 393-7.
Kasai, H., J. T. Allen, et al. (2005). "TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT)." Respir Res 6: 56.
Lapidot, T., C. Sirard, et al. (1994). "A cell initiating human acute myeloid leukaemia after transplantation into SCID mice." Nature 367(6464): 645-8.
Mani, S. A., W. Guo, et al. (2008). "The epithelial-mesenchymal transition generates cells with properties of stem cells." Cell 133(4): 704-15.
McCord, A. M., M. Jamal, et al. (2009). "Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro." Mol Cancer Res 7(4): 489-97.
Mythreye, K. and G. C. Blobe (2009). "The type III TGF-beta receptor regulates epithelial and cancer cell migration through beta-arrestin2-mediated activation of Cdc42." Proc Natl Acad Sci U S A 106(20): 8221-6.
Nakagawa, M., M. Koyanagi, et al. (2008). "Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts." Nat Biotechnol 26(1): 101-6.
Navarro, P., E. Lozano, et al. (1993). "Expression of E- or P-cadherin is not sufficient to modify the morphology and the tumorigenic behavior of murine spindle carcinoma cells. Possible involvement of plakoglobin." J Cell Sci 105 ( Pt 4): 923-34.
Niessen, C. M. and C. J. Gottardi (2008). "Molecular components of the adherens junction." Biochim Biophys Acta 1778(3): 562-71.
Pan, G. J. and D. Q. Pei (2003). "Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog." Cell Res 13(6): 499-502.
Perez-Moreno, M. A., A. Locascio, et al. (2001). "A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions." J Biol Chem 276(29): 27424-31.
Piestun, D., B. S. Kochupurakkal, et al. (2006). "Nanog transforms NIH3T3 cells and targets cell-type restricted genes." Biochem Biophys Res Commun 343(1): 279-85.
Ragge, N. K., B. Lorenz, et al. (2005). "SOX2 anophthalmia syndrome." Am J Med Genet A 135(1): 1-7; discussion 8.
Singh, S. K., C. Hawkins, et al. (2004). "Identification of human brain tumour initiating cells." Nature 432(7015): 396-401.
Siu, M. K., E. S. Wong, et al. (2008). "Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome." Am J Pathol 173(4): 1165-72.
Slack, F. (2009). "let-7 microRNA reduces tumor growth." Cell Cycle 8(12): 1823.
Suva, M. L., N. Riggi, et al. (2009). "Identification of cancer stem cells in Ewing's sarcoma." Cancer Res 69(5): 1776-81.
Takahashi, K., K. Okita, et al. (2007). "Induction of pluripotent stem cells from fibroblast cultures." Nat Protoc 2(12): 3081-9.
Tsuji, T., S. Ibaragi, et al. (2008). "Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth." Cancer Res 68(24): 10377-86.
Viswanathan, S. R., G. Q. Daley, et al. (2008). "Selective blockade of microRNA processing by Lin28." Science 320(5872): 97-100.
Vormoor, J., T. Lapidot, et al. (1994). "SCID mice as an in vivo model of human cord blood hematopoiesis." Blood Cells 20(2-3): 316-20; discussion 320-2.
Wang, Q., W. He, et al. (2009). "Oct3/4 and Sox2 are significantly associated with an unfavorable clinical outcome in human esophageal squamous cell carcinoma." Anticancer Res 29(4): 1233-41.
Wong, D. J., H. Liu, et al. (2008). "Module map of stem cell genes guides creation of epithelial cancer stem cells." Cell Stem Cell 2(4): 333-44.
Wright, M. H., A. M. Calcagno, et al. (2008). "Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics." Breast Cancer Res 10(1): R10.
Xu, J., S. Lamouille, et al. (2009). "TGF-beta-induced epithelial to mesenchymal transition." Cell Res 19(2): 156-72.
Yu, J., M. A. Vodyanik, et al. (2007). "Induced pluripotent stem cell lines derived from human somatic cells." Science 318(5858): 1917-20.
Zhou, Q., H. Chipperfield, et al. (2007). "A gene regulatory network in mouse embryonic stem cells." Proc Natl Acad Sci U S A 104(42): 16438-43.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文