LOXL2在胆管癌侵袭和转移中作用的初步研究
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摘要
胆管癌是常见的胆道恶性肿瘤之一,其发病数有呈逐年上升的趋势,而影响其治疗和病人远期生存率的主要因素是肿瘤的局部浸润和转移。肿瘤的侵袭和转移是多基因参与、多步骤完成的复杂过程,其中肿瘤细胞粘附、运动能力的改变,与细胞外基质间的相互作用是肿瘤发生侵袭转移的基础,也是肿瘤浸润转移的关键环节。探讨影响这些关键环节的调控因素将有助于我们更深入的理解胆管癌侵袭和转移的机制。
赖氨酰氧化酶样蛋白- 2(Lysyl Oxidase Like- 2 Protein , LOXL2)是赖氨酰氧化酶(lysyl oxidase, LOX)家族的成员之一,基因定位于8p21.2–p21.3。Kirschmann等最初报道了在肿瘤发生、发展中有LOXL2的参与,在具有侵袭和转移潜能的乳腺癌来源的细胞系中LOXL2蛋白呈高表达,且LOXL2蛋白的表达上调与乳腺肿瘤的侵袭、转移潜能具有相关性。后来的研究结果也证实LOX2的高表达与肿瘤侵袭呈正相关,甚至在MDCK(一种低转移潜能的乳腺癌细胞系)细胞系中LOXL2过表达可诱导完整的上皮-间叶样表型转化(Epithelial-Mesenchymal transition, EMT)。在LOXL2-shRNA表达的恶性肿瘤中采用RNAi技术沉默LOXL2基因表达后能促使肿瘤生长速度减慢,同时伴随着肿瘤细胞凋亡的增加,这些结果使LOXL2基因的重要生物学特性得到证明。
能否将LOXL2作为一种新的判断胆管癌或者是肿瘤预后的一个分子标志?深入研究LOXL2在胆管癌侵袭转移的作用,有助于我们更深刻的理解肿瘤侵袭转移的发生、发展机制,从而为干预肿瘤侵袭转移提供新的靶点。
本课题正是基于LOXL2可能是判断胆管癌预后的一个新的分子标志,它可能调控着肿瘤侵袭、转移关键环节的设想,我们以临床胆管癌组织标本和胆管癌QBC939细胞为研究对象,研究其在胆管癌浸润和转移中的作用及其可能的分子机制,也同时为深入研究肿瘤侵袭和转移的调控机制寻求新的思路和更多的理论依据。
主要研究内容与方法
1.胆管癌组织LOXL2的表达及其与临床恶性表型间关系。应用免疫组织化学法检测LOXL2在临床胆管癌组织标本中的表达情况,并分析其与胆管癌临床、病理参数的关系。
2.沉默LOXL2基因表达后对肿瘤转移相关蛋白表达的影响。运用RNAi技术,沉默胆管癌QBC939细胞中LOXL2的表达,观察胆管癌细胞中肿瘤转移相关蛋白CD44,MMP-9的表达变化情况。胆管癌QBC939细胞中CD44,MMP-9蛋白的表达用免疫组织化学的方法进行检测。
结果与讨论
胆管癌组织中LOXL2的过表达与肿瘤有无发生侵袭、转移差异有统计学意义,但与性别、年龄、肿瘤部位、肿瘤的分化程度差异无关。此研究结果表明,LOXL2的过度表达可能与胆管癌的侵袭、转移相关,其过度表达或许可以作为胆管癌预后的一个分子标志。
RNAi沉默胆管癌QBC939细胞LOXL2基因表达后,胆管癌QBC939细胞中CD44、MMP-9蛋白的表达较之空质粒阴性对照组明显下降。提示LOXL2可能在转录水平通过以下环节调控肿瘤的侵袭、转移:①LOXL2高表达促进CD44的表达,可相应增强胆管癌细胞与细胞外基质的粘附能力,胆管癌细胞粘附能力的提高促使了癌细胞对细胞外基质和基底膜的粘附,进而促进肿瘤细胞侵袭、转移的发生;②LOXL2的高表达可以通过细胞内某些重要的信号转导通路,增强MMP-9的表达而促进细胞外基质的降解,从而促进肿瘤的转移。
结论
1.LOXL2的过度表达可能与胆管癌的侵袭、转移相关,可以作为判断胆管癌恶性程度的一个分子标志。
2.LOXL2可能在肿瘤细胞粘附、降解细胞外基质等重要环节介导了胆管癌的侵袭和转移。
Metastasis is the main factor to infect the surgivcal manners selection and long term survival rate of patients with cholangiocaicinoma. Tumor cell adhension, moving and degradation of extracellular matrix is the most important step in tumor metastasis. Investing the controlling of the malig-phenotype initiating is the key point in revealing the mechanism of tumor meatstasis.
Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase (LOX) protein family that consists of five members defined by highly conserved COOH-terminal sequences. Recent investigations have illustrated that the biological role of LOX extends beyond the oxidation of structuralproteins of the ECM. Indeed, several reports describe its influence on cell proliferation, intracellular signal responses, and cell migration, which reveal that it can act as an antagonist or a protagonist of malignant processes. Previous study data have shown that most tumor cells (squamous cell carcinoma in head and neck, breast carcinoma, colonic carcinoma, etc) express elevated levels of LOXL2 but not in normal tissue and the expression level of LOXL2 is directly proportional to invasion and metastasis ability of maligmant cells. Taken together, these results raise the possibility of using LOXL2 expression as an additional predictive/prognostic marker for carcinoma progression. It is speculated that LOXL2 plays the role of common regulator in malignant tumour metastasis.
To test this hypothesis, we evaluated the expression pattern of LOXL2 in cholangiocarcinoma and the role of LOXL2 in the metastasis of cholangiocarcinoma. Immunohistochemistry method was used to quantify LOXL2 expression, as well as tumor metastasis associated CD44、MMP-9 in cholangiocarcinoma cells line(QBC939). The expression degree of CD44、MMP-9 in QBC939 cells was evaluated following LOXL2 expression down-regulated by Vector-mediated RNAi.
The positive expression rate was observed in surgical resection cholangiocarcinoma tissues as 70.83 %(34/48) , there was no correlation between the expression of LOXL2 and such factors as sex, age of patients with cholangiocarcinoma and differentiation grade , but a significant correlation with its metastasis was found (P<0.05). After pGenesil-shLOXL2 plasmid was constructed and transfected into QBC939 cell, we examined the lower expression of LOXL2 protein by immunocytochemistry and the LOXL2 protein significantly decreased the expression of CD44、MMP-9 protein in QBC939 cells.
The results demonstrate that LOXL2 overexpression is a critical molecular marker of cholangiocarcinoma and is strongly associated with invasion and metastasis of this tumor. Maybe LOXL2 overexpression is an important factor in keeping high metastatic phentotype of cholangiocarcinoma cells in vitro.LOXL2 controll cholangiocarcinoma invasion and metastasis by mediating tumor cells adnension, degradation of extracellular matrix.
赖氨酰氧化酶样蛋白- 2(Lysyl Oxidase Like- 2 Protein , LOXL2)是赖氨酰氧化酶(lysyl oxidase, LOX)家族的成员之一,基因定位于8p21.2–p21.3。Kirschmann等最初报道了在肿瘤发生、发展中有LOXL2的参与,在具有侵袭和转移潜能的乳腺癌来源的细胞系中LOXL2蛋白呈高表达,且LOXL2蛋白的表达上调与乳腺肿瘤的侵袭、转移潜能具有相关性。后来的研究结果也证实LOX2的高表达与肿瘤侵袭呈正相关,甚至在MDCK(一种低转移潜能的乳腺癌细胞系)细胞系中LOXL2过表达可诱导完整的上皮-间叶样表型转化(Epithelial-Mesenchymal transition, EMT)。在LOXL2-shRNA表达的恶性肿瘤中采用RNAi技术沉默LOXL2基因表达后能促使肿瘤生长速度减慢,同时伴随着肿瘤细胞凋亡的增加,这些结果使LOXL2基因的重要生物学特性得到证明。
能否将LOXL2作为一种新的判断胆管癌或者是肿瘤预后的一个分子标志?深入研究LOXL2在胆管癌侵袭转移的作用,有助于我们更深刻的理解肿瘤侵袭转移的发生、发展机制,从而为干预肿瘤侵袭转移提供新的靶点。
本课题正是基于LOXL2可能是判断胆管癌预后的一个新的分子标志,它可能调控着肿瘤侵袭、转移关键环节的设想,我们以临床胆管癌组织标本和胆管癌QBC939细胞为研究对象,研究其在胆管癌浸润和转移中的作用及其可能的分子机制,也同时为深入研究肿瘤侵袭和转移的调控机制寻求新的思路和更多的理论依据。
主要研究内容与方法
1.胆管癌组织LOXL2的表达及其与临床恶性表型间关系。应用免疫组织化学法检测LOXL2在临床胆管癌组织标本中的表达情况,并分析其与胆管癌临床、病理参数的关系。
2.沉默LOXL2基因表达后对肿瘤转移相关蛋白表达的影响。运用RNAi技术,沉默胆管癌QBC939细胞中LOXL2的表达,观察胆管癌细胞中肿瘤转移相关蛋白CD44,MMP-9的表达变化情况。胆管癌QBC939细胞中CD44,MMP-9蛋白的表达用免疫组织化学的方法进行检测。
结果与讨论
胆管癌组织中LOXL2的过表达与肿瘤有无发生侵袭、转移差异有统计学意义,但与性别、年龄、肿瘤部位、肿瘤的分化程度差异无关。此研究结果表明,LOXL2的过度表达可能与胆管癌的侵袭、转移相关,其过度表达或许可以作为胆管癌预后的一个分子标志。
RNAi沉默胆管癌QBC939细胞LOXL2基因表达后,胆管癌QBC939细胞中CD44、MMP-9蛋白的表达较之空质粒阴性对照组明显下降。提示LOXL2可能在转录水平通过以下环节调控肿瘤的侵袭、转移:①LOXL2高表达促进CD44的表达,可相应增强胆管癌细胞与细胞外基质的粘附能力,胆管癌细胞粘附能力的提高促使了癌细胞对细胞外基质和基底膜的粘附,进而促进肿瘤细胞侵袭、转移的发生;②LOXL2的高表达可以通过细胞内某些重要的信号转导通路,增强MMP-9的表达而促进细胞外基质的降解,从而促进肿瘤的转移。
结论
1.LOXL2的过度表达可能与胆管癌的侵袭、转移相关,可以作为判断胆管癌恶性程度的一个分子标志。
2.LOXL2可能在肿瘤细胞粘附、降解细胞外基质等重要环节介导了胆管癌的侵袭和转移。
Metastasis is the main factor to infect the surgivcal manners selection and long term survival rate of patients with cholangiocaicinoma. Tumor cell adhension, moving and degradation of extracellular matrix is the most important step in tumor metastasis. Investing the controlling of the malig-phenotype initiating is the key point in revealing the mechanism of tumor meatstasis.
Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase (LOX) protein family that consists of five members defined by highly conserved COOH-terminal sequences. Recent investigations have illustrated that the biological role of LOX extends beyond the oxidation of structuralproteins of the ECM. Indeed, several reports describe its influence on cell proliferation, intracellular signal responses, and cell migration, which reveal that it can act as an antagonist or a protagonist of malignant processes. Previous study data have shown that most tumor cells (squamous cell carcinoma in head and neck, breast carcinoma, colonic carcinoma, etc) express elevated levels of LOXL2 but not in normal tissue and the expression level of LOXL2 is directly proportional to invasion and metastasis ability of maligmant cells. Taken together, these results raise the possibility of using LOXL2 expression as an additional predictive/prognostic marker for carcinoma progression. It is speculated that LOXL2 plays the role of common regulator in malignant tumour metastasis.
To test this hypothesis, we evaluated the expression pattern of LOXL2 in cholangiocarcinoma and the role of LOXL2 in the metastasis of cholangiocarcinoma. Immunohistochemistry method was used to quantify LOXL2 expression, as well as tumor metastasis associated CD44、MMP-9 in cholangiocarcinoma cells line(QBC939). The expression degree of CD44、MMP-9 in QBC939 cells was evaluated following LOXL2 expression down-regulated by Vector-mediated RNAi.
The positive expression rate was observed in surgical resection cholangiocarcinoma tissues as 70.83 %(34/48) , there was no correlation between the expression of LOXL2 and such factors as sex, age of patients with cholangiocarcinoma and differentiation grade , but a significant correlation with its metastasis was found (P<0.05). After pGenesil-shLOXL2 plasmid was constructed and transfected into QBC939 cell, we examined the lower expression of LOXL2 protein by immunocytochemistry and the LOXL2 protein significantly decreased the expression of CD44、MMP-9 protein in QBC939 cells.
The results demonstrate that LOXL2 overexpression is a critical molecular marker of cholangiocarcinoma and is strongly associated with invasion and metastasis of this tumor. Maybe LOXL2 overexpression is an important factor in keeping high metastatic phentotype of cholangiocarcinoma cells in vitro.LOXL2 controll cholangiocarcinoma invasion and metastasis by mediating tumor cells adnension, degradation of extracellular matrix.
引文
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1. Bar-OrA, Nuttall RK, DuddyM, et al. A nalyses of allmatrixmetallop ro teinase members in leukocytes emphasize monocytes as major inflammato ry mediato rs in multip le sclero sis [ J ].Brain,2003,126:273822749.
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3. Strongin A Y, Collier I, Bannikov G, et al. M echanism of cellsurface activation of 722kDa type IV co llagenase [ J ]. J Bio lChem, 1995,270 (10):15331215338.
4. Goh ji K, Fujimo to N, FujiiA,et al. P rogno stic significance ofcirculating matrix metallop ro teinase22 to tissue inh ibito r ofmetallop ro teinase22 ratio in recurrence of uro thelial cancer aftercomp lete resection [J ]. Cancer Res, 1996,56 (14): 319623198.
5. Kraiem Z, Ko rem S. M atrix metallop ro teinases and thethyro id [J ]. Thyro id, 2000,10 (12):106121069.
6. Sternlicht MD, Werb Z. How matrix metalloproreinases regulate cell behavior [J]. Annu Rev Cell Biol, 2001, 17:463-516
7. Pepper Ms. Pole of the matrix metalloproreinase and plasminogen activator-plasmin systems in angiogenesis [J]. Arterioscler Thromb Vase Biol, 2001, 21(7):1104-1117
8. Sato H, Takino T, Okada Y, et al. A matrix metalloproreinase expressed on the surface of invasive tumor cells [J]. Nature, 1994, 370(6484):61-65
9. Dhanaraj V, Ye QZ, Johnson LL, et al. X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproreinase superfamily [J]. Structure, 1996, 4(4):375-386
10.袁发焕,王海燕,李惊子. ECM, MMPs/TIMPs及其调节研究进展[J].国外医学生理病理学与临床分册,2000, 20(2):93-96
11. Kadono Y, Okada Y, Namiki M. Transformation of epithelial Madin-Darby canine kidney cells with p60-src induced expression of membrinetype 1 matrix metalloproreinase and invasiveness [J]. Cancer Res, 1998, 58(10):2240-2244
12. Huo N, Ichikawa Y, Kamiyama M, et al. MMP-7 (matrilysin) accelerated growth of human umbilical vein endothelial cells [J]. Cancer Lett, 2002, 177(1):95-100
13. Boulay A, Masson R, Chenard MP, et al. High cancer cell death in syngeneic tumorsdeveloped in host mice deficient for the stromelysin-3 matrix metalloproreinase [J]. Canc Res, 2001, 61(5):2189-2193
14. Mitsiades N, Yu WH, Poulaki V, et al. Matrix metalloproreinase-7 mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity [J]. Cancer Res,2001, 61(2):577-581
15. Liotta L. Isolation of a protein that stimulates blood vessel growth [J]. Nature, 1985, 318(6041):14
16. Fang J, Shing Y, Wiederschain D, etal. Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model [J]. Proc Natl Acad Sci USA, 2000, 97(8):3884-3889
17. Ivaska J, Heino J. Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix [J]. Cell Mol Life Sci, 2000, 57(1):16-24
18. Deryugina EI, Bourdon MA, Jungwirth K, etal. Functional activation of integrin alpha V beta 3 in tumor cells expressing membranetype 1 matrix metalloproteinase [J]. Int J Cancer, 2000, 86(1):15-23
19. Korem S. M atrix metallop ro teinase and thyro id [J]. Thyro id,2000, 10 (12) : 106121069.
20. Sato H,Seik iM. Regulato ry mechanism of 92 kDa type IV collagenase gene exp ression wh ich is associated with invasiveness of tumo r cells [J]. Oncogene,1993, 8 (2):395-405.
21. Tierney G M, Griffin NR, Stuart RC. A pilot study of the safety and effects of the matrix metalloproteinase inhibitor marimastat in gastric cancer [J]. Eur J Cancer,1999,35(4) ;563～8.
22. Jones L, Ghaneh P, Humphreys M. The matrix metalloproteinases and their inhibitors in the treatment of pancreatic cancer [J ] .Ann N YAcad Sci,1999,880 :288 - 307 ;288～307.
23. Gato C, Rieppim, Borsotti P. a novel inhibitor of matrix metalloproteinases with antiangiogenic activity [J]. ClinCancer Res ,1999 ,5(11) ;3603～7.
24. Lokes, Okeshwar BL. MMP inhibition in prostate cancer [J]. Ann N YAcad Sci. 1999, 878:271 - 89.
25. WATSON SA, MORRIS TM, COLLINS HM. Inhibition of tumour growth bymarimastat in a human xenograft model of gastric cancer: relationship with levels of circulating CEA [J]. Br J Cancer,1999,81(1) ;19～23.
26. Tanka H, Nishida K, Sugita K. Antitumor efficacy of hypothemycin, a new Ras - signaling inhibitor [J]. Jpn J Cancer Res,1999,90 (10) ;1139～45.
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1. Bar-OrA, Nuttall RK, DuddyM, et al. A nalyses of allmatrixmetallop ro teinase members in leukocytes emphasize monocytes as major inflammato ry mediato rs in multip le sclero sis [ J ].Brain,2003,126:273822749.
2. Westermarck J, KahariVM. Regulation of matrix metallop roteinase exp ression in tumor invasion [J ]. FA SEB J, 1999, 13(8):7812792.
3. Strongin A Y, Collier I, Bannikov G, et al. M echanism of cellsurface activation of 722kDa type IV co llagenase [ J ]. J Bio lChem, 1995,270 (10):15331215338.
4. Goh ji K, Fujimo to N, FujiiA,et al. P rogno stic significance ofcirculating matrix metallop ro teinase22 to tissue inh ibito r ofmetallop ro teinase22 ratio in recurrence of uro thelial cancer aftercomp lete resection [J ]. Cancer Res, 1996,56 (14): 319623198.
5. Kraiem Z, Ko rem S. M atrix metallop ro teinases and thethyro id [J ]. Thyro id, 2000,10 (12):106121069.
6. Sternlicht MD, Werb Z. How matrix metalloproreinases regulate cell behavior [J]. Annu Rev Cell Biol, 2001, 17:463-516
7. Pepper Ms. Pole of the matrix metalloproreinase and plasminogen activator-plasmin systems in angiogenesis [J]. Arterioscler Thromb Vase Biol, 2001, 21(7):1104-1117
8. Sato H, Takino T, Okada Y, et al. A matrix metalloproreinase expressed on the surface of invasive tumor cells [J]. Nature, 1994, 370(6484):61-65
9. Dhanaraj V, Ye QZ, Johnson LL, et al. X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproreinase superfamily [J]. Structure, 1996, 4(4):375-386
10.袁发焕,王海燕,李惊子. ECM, MMPs/TIMPs及其调节研究进展[J].国外医学生理病理学与临床分册,2000, 20(2):93-96
11. Kadono Y, Okada Y, Namiki M. Transformation of epithelial Madin-Darby canine kidney cells with p60-src induced expression of membrinetype 1 matrix metalloproreinase and invasiveness [J]. Cancer Res, 1998, 58(10):2240-2244
12. Huo N, Ichikawa Y, Kamiyama M, et al. MMP-7 (matrilysin) accelerated growth of human umbilical vein endothelial cells [J]. Cancer Lett, 2002, 177(1):95-100
13. Boulay A, Masson R, Chenard MP, et al. High cancer cell death in syngeneic tumorsdeveloped in host mice deficient for the stromelysin-3 matrix metalloproreinase [J]. Canc Res, 2001, 61(5):2189-2193
14. Mitsiades N, Yu WH, Poulaki V, et al. Matrix metalloproreinase-7 mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity [J]. Cancer Res,2001, 61(2):577-581
15. Liotta L. Isolation of a protein that stimulates blood vessel growth [J]. Nature, 1985, 318(6041):14
16. Fang J, Shing Y, Wiederschain D, etal. Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model [J]. Proc Natl Acad Sci USA, 2000, 97(8):3884-3889
17. Ivaska J, Heino J. Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix [J]. Cell Mol Life Sci, 2000, 57(1):16-24
18. Deryugina EI, Bourdon MA, Jungwirth K, etal. Functional activation of integrin alpha V beta 3 in tumor cells expressing membranetype 1 matrix metalloproteinase [J]. Int J Cancer, 2000, 86(1):15-23
19. Korem S. M atrix metallop ro teinase and thyro id [J]. Thyro id,2000, 10 (12) : 106121069.
20. Sato H,Seik iM. Regulato ry mechanism of 92 kDa type IV collagenase gene exp ression wh ich is associated with invasiveness of tumo r cells [J]. Oncogene,1993, 8 (2):395-405.
21. Tierney G M, Griffin NR, Stuart RC. A pilot study of the safety and effects of the matrix metalloproteinase inhibitor marimastat in gastric cancer [J]. Eur J Cancer,1999,35(4) ;563～8.
22. Jones L, Ghaneh P, Humphreys M. The matrix metalloproteinases and their inhibitors in the treatment of pancreatic cancer [J ] .Ann N YAcad Sci,1999,880 :288 - 307 ;288～307.
23. Gato C, Rieppim, Borsotti P. a novel inhibitor of matrix metalloproteinases with antiangiogenic activity [J]. ClinCancer Res ,1999 ,5(11) ;3603～7.
24. Lokes, Okeshwar BL. MMP inhibition in prostate cancer [J]. Ann N YAcad Sci. 1999, 878:271 - 89.
25. WATSON SA, MORRIS TM, COLLINS HM. Inhibition of tumour growth bymarimastat in a human xenograft model of gastric cancer: relationship with levels of circulating CEA [J]. Br J Cancer,1999,81(1) ;19～23.
26. Tanka H, Nishida K, Sugita K. Antitumor efficacy of hypothemycin, a new Ras - signaling inhibitor [J]. Jpn J Cancer Res,1999,90 (10) ;1139～45.