白血病抑制因子促進Gα12介導的鼻咽癌細胞遷移能力

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：42

、訪客IP：13.58.151.231

姓名

邵瑋如(Wei-Ru Shao) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

白血病抑制因子促進Gα12介導的鼻咽癌細胞遷移能力
(Leukemia inhibitory factor promotes Gα12-mediated migration ability of NPC cells)

相關論文

★ 白血病抑制因子調控口腔癌巨噬細胞免疫反應	★ 含EBV病毒產物之外泌小體經由活化纖維母細胞重塑腫瘤微環境
★ 靜磁場於癌細胞的生物效應	★ 白血病抑制因子活化蛋白酶激活受體1進而促進鼻咽癌細胞的遷移

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 (2027-8-1以後開放)

摘要(中)

鼻咽癌是一種具有高度轉移性及侵襲性的頭頸部惡性腫瘤，與其他頭頸癌相比其遠處轉移的發生率最高，是影響鼻咽癌病人預後的關鍵因素。G蛋白偶聯受體(GPCR)的信號增強與多種癌症類型的腫瘤進展有關，Gα12活化small GTPase及其下游信號傳導，調控細胞骨架重組及血管新生促使癌細胞轉移。此外，在實驗室過去的研究中發現白血病抑制因子(LIF)會與鼻咽癌中較高的腫瘤復發率相關，並通過多種機制影響癌細胞遷移，然而Gα12與LIF之間的交互作用仍有待探討。在本研究結果顯示，過表達Gα12會造成鼻咽癌細胞轉化成細長的型態及細胞骨架聚集於細胞偽足的前端，進而促進細胞遷移，此外，qPCR的分析亦顯示Gα12會增加SNAI2表現量，免疫螢光染色結果也觀察到Gα12促使YAP1蛋白入核的表達量增加。LIF的刺激會活化鼻咽癌細胞STAT3，ERK1/2 與 mTOR訊息傳導路徑，而STAT3與 ERK1/2的活化在過表達Gα12的細胞組別中其表現量降低，但Gα12促使mTOR的活化型表達量增加。此外，LIF刺激亦會活化蛋白酶激活受體-1 (PAR1)及其下游信號因子的表達量，而此現象在過表達Gα12的細胞組別中受抑制，使用siRNA 降低 Gα12的表達量則會抑制鼻咽癌細胞的遷移及生長能力，而在LIF的刺激下細胞的生長能力有明顯的提升。綜合以上結果顯示，鼻咽癌細胞Gα12高表達對細胞遷移能力有所提升，在與LIF共同的下游信號途徑STAT3、ERK1/2、mTOR有相互影響的作用，並提高了Gα12介導的細胞遷移能力。

摘要(英)

Nasopharyngeal carcinoma (NPC) is a highly metastatic and aggressive head and neck malignancy. NPC exhibits a highest rate of distant metastasis compared with other types of head and neck cancer. Metastasis is the most important prognostic factor associated with treatment failure in NPC patients. G protein-coupled receptors (GPCR) have been implicated in tumor progression in multiple types of cancer. The activation of Gα12 protein by GPCR triggers small GTPase and its downstream signaling that regulates cytoskeletal reorganization and angiogenesis to promote cancer cell migration ability. Previous studies reveal that higher level of leukemia inhibitor factor (LIF) in NPC tumors is correlated with local recurrence and distal metastasis through multiple mechanisms, however, the interplays between Gα12 and LIF remains largely unexplored. The main purpose of this study is to investigate the interacting network between LIF and Gα12 signaling. We found that overexpression of Gα12 could cause morphological changes and cytoskeleton redistribution in NPC cells, thereby promoting cell mobility. In addition, exogenous expression of Gα12 increased the mRNA expression of SNAI2. Results of immunofluorescence staining demonstrated that Gα12 enhanced nuclear translocation of YAP1 protein in NPC cells. Mechanistically, LIF stimulation activated multiple signaling, including STAT3 (Y705), ERK1/2(T202,Y204) and mTOR(S2448) signaling pathways in NPC cells. Overexpression of Gα12 decreased LIF-mediated activation of STAT3 and ERK1/2, but not mTOR. Further, LIF stimulation induced cleavage of protease-activated receptor-1 (PAR1) and increased expression of its downstream signaling molecules, including mTOR and ERK1/2. Depletion of Gα12 with siRNA inhibited NPC migration and growth ability, which were reversed by LIF stimulation.
Collectively, these data demonstrate that PAR1-Gα12 axis plays a significant role in LIF-mediated NPC migration ability through activation of STAT3, ERK1/2 and mTOR signaling pathways.

關鍵字(中)

★ 鼻咽癌
★ 白血病抑制因子
★ G蛋白偶聯受體
★ 細胞遷移

關鍵字(英)

★ Nasopharyngeal carcinoma
★ LIF
★ GPCR
★ cell migration

論文目次

中文摘要 i
ABSTRACT ii
目錄 iv
第一章、背景介紹 1
一、鼻咽癌 (Nasopharyngeal carcinoma, NPC) 1
二、G蛋白偶聯受體(G protein-coupled receptor, GPCR) 1
三、蛋白酶激活受體1 (Protease-activated receptor-1, PAR-1) 2
四、白血病抑制因子(Leukemia inhibitory factor, LIF) 3
五、Yes-associated Protein 1 (YAP1) 4
第二章、研究動機和目的 5
Figure 1. 實驗架構圖 6
第三章、材料與方法 7
細胞株 7
一、細胞培養與繼代Cell Culture and Passage 7
二、細胞轉染 Transfection 7
三、蛋白質製備與西方墨點法 Protein Preparation and Western Blotting 8
四、RNA萃取及即時聚合酶連鎖反應 RNA Extraction and qPCR 9
五、免疫螢光染色分析Immunofluorescence, IF 9
六、活細胞成像 Live Images 10
七、細胞遷移傷口癒合試驗Wound healing assay 10
八、統計分析方法 11
表一、Western Blot 使用一級抗體列表 12
表二、即時聚合酶連鎖反應所使用之引子 12
表三免疫螢光染色使用一級抗體列表 13
第四章、實驗結果 14
一、Gα12和LIF均在頭頸癌的臨床腫瘤中高表達 14
二、過表達Gα12造成鼻咽癌細胞型態產生變化 14
三、過表達Gα12的NPC細胞遷移功能的變化 15
四、NPC細胞中Gα12的表達量升高對YAP1進入核中表達的影響 15
五、LIF活化的下游信號蛋白p-STAT3在過表達Gα12組別表達量降低 16
六、過表達Gα12活化ERK1/2信號通路 17
七、過表達Gα12的NPC細胞降低了LIF活化PAR1的表達量 17
八、降低NPC細胞中Gα12表達對細胞型態及遷移的影響 18
九、活細胞影像觀察結果 18
第五章、結論與討論 20
圖表 22
Figure 2. Gα12和LIF均在頭頸癌的臨床腫瘤中高表達 22
Figure 3. Gα12過表達導致鼻咽癌細胞形態改變 23
Figure 4. 過表達Gα12的NPC細胞遷移的情形 25
Figure 5. NPC細胞中Gα12表達增加對細胞核中YAP1表達的影響 26
Figure 6. LIF刺激活化的p-STAT3表達量在細胞過表達Gα12時會降低 27
Figure 7. 過表達Gα12與LIF皆活化ERK1/2信號通路 28
Figure 8. 過表達Gα12的NPC細胞降低了PAR1的活化 29
Figure 9. 降低NPC細胞中Gα12表達對細胞型態及遷移的影響 31
Figure 10. 活細胞影像紀錄 33
Figure 11. Gα12促使YAP1進入細胞核中調控細胞功能，Gα12與LIF共同調控ERK1/2、mTOR/p70s6k訊息傳遞路徑。 34
參考文獻 35

參考文獻

1. Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 2018. 68(6): p. 394-424.
2. Chen, Y.-P., et al., Nasopharyngeal carcinoma. 2019. 394(10192): p. 64-80.
3. Mimi, C.Y. and J.-M. Yuan. Epidemiology of nasopharyngeal carcinoma. in Seminars in cancer biology. 2002. Elsevier.
4. Chang, E.T., H.-O.J.C.E. Adami, and P. Biomarkers, The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiology and Prevention Biomarkers, 2006. 15(10): p. 1765-1777.
5. Lo, K.W., K.F. To, and D.P.J.C.c. Huang, Focus on nasopharyngeal carcinoma. Cancer cell, 2004. 5(5): p. 423-428.
6. Tsao, S.W., et al., The role of Epstein–Barr virus in epithelial malignancies. The Journal of pathology, 2015. 235(2): p. 323-333.
7. Chan, S., et al., HLA and nasopharyngeal carcinoma in Chinese—a further study. 1983. 32(2): p. 171-176.
8. Lu, C.C., et al., Genetic susceptibility to nasopharyngeal carcinoma within the HLA‐A locus in Taiwanese. International journal of cancer, 2003. 103(6): p. 745-751.
9. Kongruttanachok, N., et al., Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study. BMC cancer, 2001. 1(1): p. 1-5.
10. Nebert, D.W.J.M.R.F. and M.M.o. Mutagenesis, Role of genetics and drug metabolism in human cancer risk. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1991. 247(2): p. 267-281.
11. Li, Y.Y., et al., Exome and genome sequencing of nasopharynx cancer identifies NF-κB pathway activating mutations. Nature communications, 2017. 8(1): p. 1-10.
12. Teo, P.M., et al., Prognosticators determining survival subsequent to distant metastasis from nasopharyngeal carcinoma. Cancer: Interdisciplinary International Journal of the American Cancer Society, 1996. 77(12): p. 2423-2431.
13. Yeh, S.-A., et al., Treatment outcomes of patients with AJCC stage IVC nasopharyngeal carcinoma: benefits of primary radiotherapy. Japanese journal of clinical oncology, 2006. 36(3): p. 132-136.
14. Hall, A.J.C. and M. Reviews, The cytoskeleton and cancer. Cancer and Metastasis Reviews, 2009. 28(1): p. 5-14.
15. Lee, A., et al., Management of nasopharyngeal carcinoma: current practice and future perspective. 2015. 33(29): p. 3356-3364.
16. Dorsam, R.T. and J.S.J.N.r.c. Gutkind, G-protein-coupled receptors and cancer. 2007. 7(2): p. 79-94.
17. Wootten, D., et al., Mechanisms of signalling and biased agonism in G protein-coupled receptors. Nature reviews Molecular cell biology, 2018. 19(10): p. 638-653.
18. He, J.C., et al., The Gαo/i-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3. Journal of Biological Chemistry, 2005. 280(39): p. 33426-33434.
19. Liu, S.-C., et al., Gα12-mediated pathway promotes invasiveness of nasopharyngeal carcinoma by modulating actin cytoskeleton reorganization. 2009. 69(15): p. 6122-6130.
20. Sah, V.P., et al., The role of Rho in G protein-coupled receptor signal transduction. Annual review of pharmacology and toxicology, 2000. 40: p. 459.
21. Kelly, P., et al., A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion. Journal of Biological Chemistry, 2006. 281(36): p. 26483-26490.
22. Zhang, C., et al., High-resolution crystal structure of human protease-activated receptor 1. Nature, 2012. 492(7429): p. 387-392.
23. Coughlin, S.R.J.N., Thrombin signalling and protease-activated receptors. Nature 2000. 407(6801): p. 258-264.
24. Leger, A.J., L. Covic, and A.J.C. Kuliopulos, Protease-activated receptors in cardiovascular diseases. Circulation, 2006. 114(10): p. 1070-1077.
25. Liu, X., et al., Protease-activated receptor-1 (PAR-1): a promising molecular target for cancer. Oncotarget, 2017. 8(63): p. 107334.
26. Macfarlane, S.R., et al., Proteinase-activated receptors. Pharmacological reviews, 2001. 53(2): p. 245-282.
27. Mo, J.-S., et al., Regulation of the Hippo–YAP pathway by protease-activated receptors (PARs). Genes & development, 2012. 26(19): p. 2138-2143.
28. Saleiban, A., et al., miR‐20b regulates expression of proteinase‐activated receptor‐1 (PAR‐1) thrombin receptor in melanoma cells. Pigment cell & melanoma research, 2014. 27(3): p. 431-441.
29. Wang, Q., et al., Endothelial cell protein C receptor promotes MGC803 gastric cancer cells proliferation and migration by activating ERK1/2. Medical Oncology, 2015. 32(5): p. 1-8.
30. Morris, D.R., et al., Protease-activated receptor-2 is essential for factor viia and xa–induced signaling, migration, and invasion of breast cancer cells. Cancer research, 2006. 66(1): p. 307-314.
31. Zhu, Q., et al., The activation of protease-activated receptor 1 mediates proliferation and invasion of nasopharyngeal carcinoma cells. Oncology reports, 2012. 28(1): p. 255-261.
32. Auvergne, R., et al., PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo. Oncogene, 2016. 35(29): p. 3817-3828.
33. Baker, N.C., et al., Overview of the 2014 food and drug administration cardiovascular and renal drugs advisory committee meeting about vorapaxar. Circulation, 2014. 130(15): p. 1287-1294.
34. Auernhammer, C. and S.J.E.r. Melmed, Leukemia-inhibitory factor—neuroimmune modulator of endocrine function. 2000. 21(3): p. 313-345.
35. Taga, T. and T.J.A.r.o.i. Kishimoto, Gp130 and the interleukin-6 family of cytokines. 1997. 15(1): p. 797-819.
36. Gearing, D.P.J.A.i.i., The leukemia inhibitory factor and its receptor. 1993. 53: p. 31-58.
37. Burdon, T., A. Smith, and P.J.T.i.c.b. Savatier, Signalling, cell cycle and pluripotency in embryonic stem cells. 2002. 12(9): p. 432-438.
38. Nicola, N.A., J.J.J.C. Babon, and g.f. reviews, Leukemia inhibitory factor (LIF). 2015. 26(5): p. 533-544.
39. Liu, S.-C., et al., Leukemia inhibitory factor promotes nasopharyngeal carcinoma progression and radioresistance. 2013. 123(12): p. 5269-5283.
40. Zhang, Y.S., et al., STAT 4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation. 2019. 38(6): p. 1-20.
41. Morris, M.A., C.W. Dawson, and L.S.J.F.o. Young, Role of the Epstein–Barr virus-encoded latent membrane protein-1, LMP1, in the pathogenesis of nasopharyngeal carcinoma. Future oncology, 2009. 5(6): p. 811-825.
42. Zheng, H., et al., Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. Cell Mol Immunol, 2007. 4(3): p. 185-196.
43. Liu, S.-C., et al., Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling. Nature communications, 2018. 9(1): p. 1-16.
44. Hong, W. and K.-L. Guan. The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway. in Seminars in cell & developmental biology. 2012. Elsevier.
45. Kango‐Singh, M. and A.J.D.d.a.o.p.o.t.A.A.o.A. Singh, Regulation of organ size: insights from the Drosophila Hippo signaling pathway. an official publication of the American Association of Anatomists, 2009. 238(7): p. 1627-1637.
46. Lee, M.-J., et al., YAP and TAZ regulate skin wound healing. Journal of Investigative Dermatology, 2014. 134(2): p. 518-525.
47. Lian, I., et al., The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes & development, 2010. 24(11): p. 1106-1118.
48. Zanconato, F., M. Cordenonsi, and S.J.C.c. Piccolo, YAP/TAZ at the roots of cancer. Cancer cell, 2016. 29(6): p. 783-803.
49. Shreberk‐Shaked, M. and M.J.M.o. Oren, New insights into YAP/TAZ nucleo‐cytoplasmic shuttling: new cancer therapeutic opportunities? Molecular oncology, 2019. 13(6): p. 1335-1341.
50. Moroishi, T., C.G. Hansen, and K.-L.J.N.R.C. Guan, The emerging roles of YAP and TAZ in cancer. Nature Reviews Cancer, 2015. 15(2): p. 73-79.
51. Steinhardt, A.A., et al., Expression of Yes-associated protein in common solid tumors. Human pathology, 2008. 39(11): p. 1582-1589.
52. Overholtzer, M., et al., Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proceedings of the National Academy of Sciences, 2006. 103(33): p. 12405-12410.
53. Chen, D., et al., LIFR is a breast cancer metastasis suppressor upstream of the Hippo-YAP pathway and a prognostic marker. Nature medicine, 2012. 18(10): p. 1511-1517.
54. Zucchini, C., et al., ROCK2 deprivation leads to the inhibition of tumor growth and metastatic potential in osteosarcoma cells through the modulation of YAP activity. Journal of Experimental & Clinical Cancer Research, 2019. 38(1): p. 1-14.
55. Wei, H., et al., Verteporfin suppresses cell survival, angiogenesis and vasculogenic mimicry of pancreatic ductal adenocarcinoma via disrupting the YAP‐TEAD complex. Cancer science, 2017. 108(3): p. 478-487.
56. Moya, I.M., et al., Peritumoral activation of the Hippo pathway effectors YAP and TAZ suppresses liver cancer in mice. Science, 2019. 366(6468): p. 1029-1034.
57. Chan, S.W., et al., A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells. 2008. 68(8): p. 2592-2598.
58. Gupta, G.P. and J.J.C. Massagué, Cancer metastasis: building a framework. Cell, 2006. 127(4): p. 679-695.
59. O’Hayre, M., M.S. Degese, and J.S.J.C.o.i.c.b. Gutkind, Novel insights into G protein and G protein-coupled receptor signaling in cancer. Current opinion in cell biology, 2014. 27: p. 126-135.

指導教授

劉淑貞(Shu-Chen Liu)

審核日期

2022-9-20

推文