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研究生:黃彥銘
研究生(外文):Yen-MingHuang
論文名稱:研究內皮唾酸蛋白在腫瘤相關的纖維母細胞調節血管新生的功能
論文名稱(外文):Study of Endosialin in Cancer Associated Fibroblast-mediated Angiogenesis
指導教授:施桂月
指導教授(外文):Guey-Yueh Shi
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:53
中文關鍵詞:內皮唾酸蛋白
外文關鍵詞:endosialin
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中文摘要
內皮唾酸蛋白(endosialin),又稱為CD248或是第一型腫瘤內皮標誌蛋白,是一個穿膜醣蛋白。它由六個結構區域組成,包括一個C型類凝集功能區,一個sushi功能區,一個由三個類內皮生長因子重複區塊組成的功能區,一個類黏蛋白功能區,一個穿膜區以及膜內片段。研究顯示內皮唾酸蛋白會高量表現在大部分腫瘤的基質區域以及血管周圍。除此之外,藉由腹腔腸部原位癌移植的動物實驗模式,內皮唾酸蛋白的基因剔除小鼠較野生型小鼠有較嚴重的腫瘤生成以及轉移,而且這些長在內皮唾酸蛋白基因剔除小鼠的腫瘤都有較多的未成熟小血管及較少的成熟大血管,顯示內皮唾酸蛋白似乎藉由透過血管新生作用以調控腫瘤生成過程。然而,在腫瘤血管新生時內皮唾酸蛋白的來源以及功能仍是未知。腫瘤相關的纖維母細胞 (Cancer-associated fibroblasts, CAFs) 是腫瘤基質區域的主要細胞族群。我們分離了口腔癌病人的腫瘤相關的纖維母細胞以及其周圍正常組織的齒齦纖維母細胞,發現腫瘤相關的纖維母細胞有較高量的內皮唾酸蛋白的表現,顯示它可能是一個腫瘤相關的纖維母細胞的標誌蛋白。除此之外,我們也在腫瘤相關的纖維母細胞的細胞培養液中發現了內皮唾酸蛋白片段的存在,此細胞培養液可以顯著的促進人類臍帶靜脈內皮細胞的生長以及其管狀結構的形成,而加入專一性針對內皮唾酸蛋白的抗體則可以阻斷這樣的現象。另外,我們使用酵母菌蛋白表現系統純化出了內皮唾酸蛋白的類內皮生長因子重複區塊的蛋白,此重組蛋白亦可以顯著的促進人類臍帶靜脈內皮細胞的生長以及其管狀結構形成。而此蛋白在動物實驗中也具有促進血管新生的功能。綜合以上結果,我們發現內皮唾酸蛋白的片段會被腫瘤相關的纖維母細胞所釋放出來,而且其類內皮生長因子重複區塊具有血管新生因子的功能。
Endosialin (also known as CD248 or tumor endothelial marker 1) is a transmembrane glycoprotein. It is composed of six domains, including a lectin-like domain, a sushi domain, a three epidermal growth factor (EGF)-like repeats, a mucin-like domain, a transmembrane domain, and a cytoplasmic tail. Previous studies showed that endosialin was highly expressed in fibroblasts-rich stroma and vasculature of carcinomas. Furthermore, reduced tumor growth and metastasis were observed in endosialin knockout mice but not in wild type mice in an abdominal orthotopic xenograft model. Meanwhile, the tumors from endosialin knockout mice contained more small and immature vessels but less large and mature vessels, suggesting that endosialin may play a role in tumor progression by promoting angiogenesis. However, the source and function of endosialin in angiogenesis remain unknown. Cancer-associated fibroblasts (CAFs) are the major cell population in tumor stroma. We collected CAFs and human gingival fibroblasts (HGFs) from oral cancer patients. We found that the expression level of endosialin in CAFs was higher than HGFs, indicating that endosialin may be a marker of CAFs. Meanwhile, endosialin fragments were also identified in conditioned medium of CAFs. CAFs-conditioned medium could promote human umbilical vein endothelial cells (HUVECs) proliferation and tube formation in vitro whereas addition of endosialin specific antibody could effectively attenuate these effects. In addition, recombinant EGF-like repeats of endosialin (ESD3) protein functioned as an angiogenic factor in vitro and in vivo. ESD3 could activate angiogenesis-associated signaling pathways such as extracellular signal-regulated kinase and Akt in HUVECs. It also promoted cell proliferation and tube formation of HUVECs and induced angiogenesis in a murine Matrigel plug assay. Based on these observations, soluble endosialin fragments may be released from CAFs and the EGF domain of endosialin may be an angiogenic factor in CAFs-mediated angiogenesis.
Content Page
Introduction
1.Cancers 1
2.Identification of endosialin 2
3.Expression pattern of endosialin 3
4.Function of endosialin 4
5.Cancer-associated fibroblasts (CAFs) 5
6.Angiogenesis 6
7.Specific aim 8
Materials and Methods
1.Cell culture 9
2.Protein quantification 10
3.SDS-PAGE (Sodium Dodecyl Sulfate-Polyacryamide Gel Electrophoresis) 10
4.Coomassie blue staining 11
5.Silver staining 12
6.Western blotting 12
7.Effect of HGF and CAF-conditioned medium on HUVECs proliferation and Western blotting analysis of HGF
and CAF-conditioned medium 13
8.Effect of endosialin antibody on CAF-conditioned
medium-promoted HUVECs proliferation 14
9.Effect of endosialin antibody on CAF-conditioned
medium-induced tube formation of HUVECs 15
10.Expression and purification of recombinant endosialin EGF-like repeats (rESD3) protein by a pichia pastoris expression system 16
11.Effects of rESD3 protein on proliferation of HUVECs 17
12.Effects of rESD3 protein on tube formation of HUVECs 17
13.Signal activation of HUVECs by treatment with rESD3 protein 18
14.In vivo Matrigel plug assay 18
Results
1.Endosialin is highly expressed in CAFs 19
2.CAF-conditioned medium induces HUVECs proliferation 19
3.Endosialin fragments are identified in conditioned
medium from CAFs 19
4.Addition of endosialin antibody attenuates CAF-conditioned medium promoted HUVECs proliferation 20
5.Endosialin antibody attenuates CAF-conditioned
medium mediated tube formation of HUVECs 20
6.Expression and purification of rESD3 protein 21
7.Effect of rESD3 protein on proliferation of HUVECs 21
8.Effect of rESD3 protein on tube formation of HUVECs 21
9.Effect of rESD3 protein on angiogenic-related signal activation of HUVECs 22
10.rESD3 protein can induce angiogenesis 22
Discussion 23
Figure legends
Figure 1. The expression level of endosialin in CAFs is higher than in HGFs 28
Figure 2. Conditioned medium from CAFs has higher
activity in promoting endothelial cell proliferation
than that medium from HGFs 29
Figure 3. Endosialin fragments are identified in conditioned media from CAFs and HGFs 30
Figure 4. Endosialin antibody attenuates the growth
of HUVECs promoted by CAF-conditioned medium 31
Figure 5. Endosialin antibody attenuates the in vitro tube formation of HUVECs mediated by CAF-conditioned medium 32
Figure 6. Expression and purification of rESD3 protein 33
Figure 7. rESD3 protein promotes HUVECs proliferation
in a dose dependent manner 34
Figure 8. rESD3 protein promotes tube formation of HUVECs 35
Figure 9. rESD3 protein activates angiogenic-related signals of HUVECs 36
Figure 10. rESD3 protein induces angiogenesis in
in vivo Matrigel plug assay 37
Figure 11. The model we proposed in this report 38
Reference 39
Reagents, Drugs, Chemicals and Instruments 43
Abbreviation 46
Appendixes
Appendix 1. Structure of endosialin and its homologues thrombomodulin and C1qRp 48
Appendix 2. cDNA sequence and protein sequence of
endosialin 49
Resume 53

1Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008. CA Cancer J Clin. 2008; 58: 71-96.
2Bickers DR, Lowy DR. Carcinogenesis: a fifty-year historical perspective. J Invest Dermatol. 1989; 92: 121S-31S.
3Preston-Martin S, Pike MC, Ross RK, Jones PA, Henderson BE. Increased cell division as a cause of human cancer. Cancer Res. 1990; 50: 7415-21.
4Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 100: 57-70.
5Hahn WC, Weinberg RA. Rules for making human tumor cells. N Engl J Med. 2002; 347: 1593-603.
6Hahn WC, Weinberg RA. Modelling the molecular circuitry of cancer. Nat Rev Cancer. 2002; 2: 331-41.
7Comoglio PM, Trusolino L. Invasive growth: from development to metastasis. J Clin Invest. 2002; 109: 857-62.
8Bhowmick NA, Moses HL. Tumor-stroma interactions. Curr Opin Genet Dev. 2005; 15: 97-101.
9Pandya NM, Dhalla NS, Santani DD. Angiogenesis--a new target for future therapy. Vascul Pharmacol. 2006; 44: 265-74.
10Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008; 358: 2039-49.
11Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004; 25: 581-611.
12Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006; 6: 392-401.
13Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K, Sahai E. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 2007; 9: 1392-400.
14Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res. 2005; 97: 512-23.
15Le XF, Pruefer F, Bast RC, Jr. HER2-targeting antibodies modulate the cyclin-dependent kinase inhibitor p27Kip1 via multiple signaling pathways. Cell Cycle. 2005; 4: 87-95.
16Overall CM, Lopez-Otin C. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer. 2002; 2: 657-72.
17Mueller MM, Fusenig NE. Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer. 2004; 4: 839-49.
18Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA, Old LJ. Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proc Natl Acad Sci U S A. 1992; 89: 10832-6.
19Christian S, Ahorn H, Koehler A, Eisenhaber F, Rodi HP, Garin-Chesa P, Park JE, Rettig WJ, Lenter MC. Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium. J Biol Chem. 2001; 276: 7408-14.
20St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B, Kinzler KW. Genes expressed in human tumor endothelium. Science. 2000; 289: 1197-202.
21Greenlee MC, Sullivan SA, Bohlson SS. CD93 and related family members: their role in innate immunity. Curr Drug Targets. 2008; 9: 130-8.
22MacFadyen JR, Haworth O, Roberston D, Hardie D, Webster MT, Morris HR, Panico M, Sutton-Smith M, Dell A, van der Geer P, Wienke D, Buckley CD, Isacke CM. Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium. FEBS Lett. 2005; 579: 2569-75.
23Christian S, Winkler R, Helfrich I, Boos AM, Besemfelder E, Schadendorf D, Augustin HG. Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. Am J Pathol. 2008; 172: 486-94.
24Rouleau C, Curiel M, Weber W, Smale R, Kurtzberg L, Mascarello J, Berger C, Wallar G, Bagley R, Honma N, Hasegawa K, Ishida I, Kataoka S, Thurberg BL, Mehraein K, Horten B, Miller G, Teicher BA. Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clin Cancer Res. 2008; 14: 7223-36.
25Simonavicius N, Robertson D, Bax DA, Jones C, Huijbers IJ, Isacke CM. Endosialin (CD248) is a marker of tumor-associated pericytes in high-grade glioma. Mod Pathol. 2008; 21: 308-15.
26Carson-Walter EB, Winans BN, Whiteman MC, Liu Y, Jarvela S, Haapasalo H, Tyler BM, Huso DL, Johnson MD, Walter KA. Characterization of TEM1/endosialin in human and murine brain tumors. BMC Cancer. 2009; 9: 417.
27Tomkowicz B, Rybinski K, Foley B, Ebel W, Kline B, Routhier E, Sass P, Nicolaides NC, Grasso L, Zhou Y. Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proc Natl Acad Sci U S A. 2007; 104: 17965-70.
28Tomkowicz B, Rybinski K, Sebeck D, Sass P, Nicolaides NC, Grasso L, Zhou Y. Endosialin/TEM-1/CD248 regulates pericyte proliferation through PDGF receptor signaling. Cancer Biol Ther. 2010; 9. 11731.
29Becker R, Lenter MC, Vollkommer T, Boos AM, Pfaff D, Augustin HG, Christian S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K. FASEB J. 2008; 22: 3059-67.
30Nanda A, Karim B, Peng Z, Liu G, Qiu W, Gan C, Vogelstein B, St Croix B, Kinzler KW, Huso DL. Tumor endothelial marker 1 (Tem1) functions in the growth and progression of abdominal tumors. Proc Natl Acad Sci U S A. 2006; 103: 3351-6.
31Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest. 1985; 76: 2178-81.
32Bohlson SS, Silva R, Fonseca MI, Tenner AJ. CD93 is rapidly shed from the surface of human myeloid cells and the soluble form is detected in human plasma. J Immunol. 2005; 175: 1239-47.
33Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C, Huang HC, Chang YJ, Chen PS, Wu HL. Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation. 2005; 111: 1627-36.
34De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer. 2008; 123: 2229-38.
35Elenbaas B, Weinberg RA. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res. 2001; 264: 169-84.
36Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature. 2004; 432: 332-7.
37Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005; 121: 335-48.
38Crawford Y, Kasman I, Yu L, Zhong C, Wu X, Modrusan Z, Kaminker J, Ferrara N. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell. 2009; 15: 21-34.
39Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer. 2008; 8: 579-91.
40Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer. 2008; 8: 592-603.
41Eskens FA, Verweij J. The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; a review. Eur J Cancer. 2006; 42: 3127-39.
42Schmeidler-Sapiro KT, Ratnoff OD, Gordon EM. Mitogenic effects of coagulation factor XII and factor XIIa on HepG2 cells. Proc Natl Acad Sci U S A. 1991; 88: 4382-5.
43Hamada H, Ishii H, Sakyo K, Horie S, Nishiki K, Kazama M. The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood. 1995; 86: 225-33.
44Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest. 1992; 67: 519-28.
45Takano S, Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood. 1990; 76: 2024-9.
46Lohi O, Urban S, Freeman M. Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids. Curr Biol. 2004; 14: 236-41.
47Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010; 141: 52-67.

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