CCR7-SLC/ELC生物学轴在乳腺癌亲嗜性淋巴结转移中的作用及机制探讨
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摘要
目的：探讨CCR7-SLC/ELC生物学轴在乳腺癌淋巴结转移中的作用及机制。方法：1.采用免疫组织化学方法分别检测48例乳腺癌石蜡组织,23例腋窝转移淋巴结,15例癌旁组织及同期15例纤维腺瘤中CCR7,SLC,ELC蛋白表达情况,了解CCR7-SLC/ELC生物学轴是否参与乳腺癌淋巴结转移。2.用Boyden趋化小室测定外源性趋化因子SLC,ELC对乳腺癌细胞株MCF-7趋化及侵袭活性的影响；用Westen-Blot法检测所取新鲜乳腺癌转移淋巴结标本中SLC,ELC的表达情况。取转移淋巴结组织匀浆上清液代替外源趋化因子,测定其对MCF-7趋化及侵袭活性,同时测定使用SLC,ELC单克隆抗体后趋化及侵袭活性的改变,探讨CCR7-SLC/ELC轴促进淋巴结转移的可能机制。结果：①乳腺癌组织中CCR7阳性表达率为62.5%(30/48),转移淋巴结中CCR7的表达率为87.0%(20/23),两组比较差异有统计学意义,P<0.05。15例癌旁组织及纤维腺瘤中的CCR7表达率分别为33.3%(5/15),20.0%(3/15),俩组比较差异无统计学意义。但无论是癌旁或纤维腺瘤与乳腺癌组织及转移淋巴结比较均有统计学意义,P<0.05。在对乳腺癌组织中CCR7表达与临床病理特征比较中发现,有淋巴结转移的CCR7表达率为66.7%(20/30),无淋巴结转移的为33.3%(6/18),差异有统计学意义,P<0.01。肿瘤不同分期及雌孕激素不同表达组间CCR7的表达无明显差异。②乳腺癌组织中SLC的表达率为66.7%(32/48)；转移淋巴结,癌旁及纤维腺瘤中的表达率分别为91.3%(21/23),46.7%(7/15),40.0%(6/15)。其中乳腺癌组织与转移淋巴结中SLC的表达差异有统计学意义P<0.025,而与癌旁及纤维腺瘤比较无明显统计学意义P>0.05。临床病理特征比较中发现不同分期及淋巴结有无转移组间差异有统计学意义P<0.05。③乳腺癌组织中ELC的表达率为70.8%(34/48),转移淋巴结,癌旁及纤维腺瘤中的ELC表达率分别为95.7%(22/23),66.7%(10/15),53.3%(8/15)。其中癌组织中ELC的表达与转移淋巴结比较有统计学意义,P<0.025,与癌旁及纤维腺瘤比较无统计学意义。临床病理特征比较中发现不同分期及淋巴结有无转移组间差异有统计学意义P<0.05。④不论是外源性SLC,ELC还是转移淋巴结的蛋白上清液均能增加MCF-7趋化及侵袭活性.SLC,ELC单克隆抗体的封闭能明显抑制其活性。结论：CCR7-SLC/ELC轴与乳腺癌癌亲嗜性淋巴结转移中有关,同时SLC,ELC的表达能提高肿瘤的侵袭性及恶性程度,这或许是该轴促进乳腺癌淋巴结转移的机制。
Abstract:. Objective:To detect the expression o f chemokine receptor CCR7 and chemokine SLC,ELC protein in breast carcinoma and discuss their relationship with lymph node metastasis. Method:1.The expression of chemokine receptor CCR7 and chemokine SLC,ELC protein in 48 cases breast carcinoma,23 cases metastatic lymph node,15 cases normal tissue beside carcinoma,15 cases fibroadenoma were detected by immunohistochemicaltechnique.2. The chemotaxis and invasion assays were performed on breast cancer cells-MCF-7 in presence of exogenous SLC and ELC;Detect the expression of SLC and ELC in novel lymph node with breast cancer metastasis by Westen-Blot.Second replace the exogenous SLC and ELC with the clear supernatant liquid of novel lymph node with breast cancer metastasis to study the chemotaxis and invasion of breast cancer cells-MCF-7 facilitated by endogenous SLC and ELC,and when SLC and ELC was blocked Result:①There were 30 cases of positive expression of CCR7 protein in 48 cases of breast carcinoma tissue62.5%(30/48),20 cases in 23 cases of metastatic lymph node 87.0%(20/23),5 cases in 15 cases of normal tissue beside carcinoma33.3%(5/15),3 cases in 15 cases of fibroadenoma20.0%(3/15). The positive expression of CCR7 protein in metastatic lymph node was significant than that in other teams.P<0.05. The positive expression of CCR7 protein in breast carcinoma with lymph node metastasis was significant than that without lymph node metastas.P<0.01. The positive rate in clinical stageⅠⅡ,Ⅲhas not significant difference P>0.05.The positive expression of CCR7 was correlated with lymph node.②There were 32 cases of positive expression of SLC protein in 48 cases of breast carcinoma tissue66.7%(32/48),21cases in 23 cases of metastatic lymph node91.3%(21/23),7 cases in 15 cases of normal tissue beside carcinoma46.7%(7/15),6 cases in 15 cases of fibroadenoma40.0%(6/15). The positive expression of SLC protein in metastatic lymph node was significant than that in other teams.P<0.025. There was not significant difference between carcinoma, normal tissue beside carcinoma and fibroadenoma.The positive expression of SLC protein in breast carcinoma with lymph node metastasis was significant than that without lymph node metastas.P<0.05. The positive rate in clinical stageⅠⅡ,Ⅲhas significant difference P<0.05.The positive expression of SLC was correlated with lymph node and promote invasion.③The expression of ELC has similary result with SLC protein.④Both exotgenous and endogenous SLC and ELC can facilitate the chemotaxis and invasion of the breast cancer cells-MCF-7,both chemotaxis and invasion could be blocked by neutralazing anti-SLC,ELC antibody. Conclusion:CCR7/ SLC,ELC axis may contribute to lymph node metastasis and promote invasion in breast cancer.
Abstract:. Objective:To detect the expression o f chemokine receptor CCR7 and chemokine SLC,ELC protein in breast carcinoma and discuss their relationship with lymph node metastasis. Method:1.The expression of chemokine receptor CCR7 and chemokine SLC,ELC protein in 48 cases breast carcinoma,23 cases metastatic lymph node,15 cases normal tissue beside carcinoma,15 cases fibroadenoma were detected by immunohistochemicaltechnique.2. The chemotaxis and invasion assays were performed on breast cancer cells-MCF-7 in presence of exogenous SLC and ELC;Detect the expression of SLC and ELC in novel lymph node with breast cancer metastasis by Westen-Blot.Second replace the exogenous SLC and ELC with the clear supernatant liquid of novel lymph node with breast cancer metastasis to study the chemotaxis and invasion of breast cancer cells-MCF-7 facilitated by endogenous SLC and ELC,and when SLC and ELC was blocked Result:①There were 30 cases of positive expression of CCR7 protein in 48 cases of breast carcinoma tissue62.5%(30/48),20 cases in 23 cases of metastatic lymph node 87.0%(20/23),5 cases in 15 cases of normal tissue beside carcinoma33.3%(5/15),3 cases in 15 cases of fibroadenoma20.0%(3/15). The positive expression of CCR7 protein in metastatic lymph node was significant than that in other teams.P<0.05. The positive expression of CCR7 protein in breast carcinoma with lymph node metastasis was significant than that without lymph node metastas.P<0.01. The positive rate in clinical stageⅠⅡ,Ⅲhas not significant difference P>0.05.The positive expression of CCR7 was correlated with lymph node.②There were 32 cases of positive expression of SLC protein in 48 cases of breast carcinoma tissue66.7%(32/48),21cases in 23 cases of metastatic lymph node91.3%(21/23),7 cases in 15 cases of normal tissue beside carcinoma46.7%(7/15),6 cases in 15 cases of fibroadenoma40.0%(6/15). The positive expression of SLC protein in metastatic lymph node was significant than that in other teams.P<0.025. There was not significant difference between carcinoma, normal tissue beside carcinoma and fibroadenoma.The positive expression of SLC protein in breast carcinoma with lymph node metastasis was significant than that without lymph node metastas.P<0.05. The positive rate in clinical stageⅠⅡ,Ⅲhas significant difference P<0.05.The positive expression of SLC was correlated with lymph node and promote invasion.③The expression of ELC has similary result with SLC protein.④Both exotgenous and endogenous SLC and ELC can facilitate the chemotaxis and invasion of the breast cancer cells-MCF-7,both chemotaxis and invasion could be blocked by neutralazing anti-SLC,ELC antibody. Conclusion:CCR7/ SLC,ELC axis may contribute to lymph node metastasis and promote invasion in breast cancer.
引文
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13. Barberos,et al. Stromal cell-derived factor 1 alpha stimulates human glioblastoma cell growgh through the activation of both extracellular signal regulated kinases 1/2 and akt[J]. Cancer Res,2003;63(4): 196-199.
14. Violet F. The chemoine network:A target in cancer biology[J]. Advanced drug delivery reviews,2006;58(4):962-974.
15. T.Jcuril. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J].Nat.Med,2004;4(4):942-945.
16. Y.koxama.The enhanced expressions of CXCR4 and CCR7 MRNA in breast cancer tissue do not always correlate with cancer metastasis[J]. Breast disease,2007;61(1):78-85.
17. Iwao Takanami.Overexpression of CCR7 MRNA in no small cell lung cancer:correlation with lymph node metastasis [J].cancer, 2003;105(6):186-189.
18. L.pitkin et al. Expression of CC chemokine receptor in tonsillar cancer predicts cerbical nodal metastasis and systemic relapse[J]. British journal of oral and maxillofacial surger,2007;45(5):1-7.
19. Gustavo A,Heresi et al. Expression of the chemokine receptor CCR7 in prostate cancer presenting with generalize lymph adenopathy [J].Seminars and original investigations,2005;23(4):261-267.
1. Rot, A; von Andrian, UH. Chemokines in innate and adaptive host defense:basic chemokinese grammar for immune cells. Annu Rev Immunol.2004;22:891-928
2. Coussens, LM; Werb, Z. Inflammation and cancer. Nature.2002; 420:860-7.
3. Robinson, SC; Coussens, LM. Soluble mediators of inflammation during tumor development. Adv Cancer Res.2005;93:159-87.
4. Aggarwal, BB; Shishodia, S; Sandur, SK; Pandey, MK; Sethi, G. Inflammation and cancer:how hot is the link? Biochem Pharmacol. 2006;72:1605-21.
5. Szlosarek, P; Charles, KA; Balkwill, FR. Tumour necrosis factor-alpha as a tumour promoter. Eur J Cancer.2006; 42:745-50.
6. Wang, D; Wang, H; Brown, J; Daikoku, T; Ning, W; Shi, Q, et al. CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J Exp Med.2006;203:941-51.
7. Cha, YI; Dubois, RN. NSAIDs and Cancer Prevention:Targets Downstream of COX-2. Annu Rev Med.2006
8. Murakami, T., Cardones, A. R., Hwang, S. T. Chemokine receptors and melanoma metastasis J. Dermatol. Sci.2004;36,71-78
9. Tanaka, T., Bai, Z., Srinoulprasert, Y., Yang, B. G., Hayasaka, H., Miyasaka, M. Chemokines in tumor progression and metastasis Cancer Sci2005.; 96,317-322
10. Balkwill, F. Cancer and the chemokine network Nat. Rev. Cancer 2004;4,540-550
11. Chen, G. S., Yu, H. S., Lan, C. C., Chow, K. C., Lin, T. Y., Kok, L. F., Lu, M. P., Liu, C. H., Wu, M. T. CXC chemokine receptor CXCR4 expression enhances tumorigenesis and angiogenesis of basal cell carcinoma Br. J. Dermatol 2006. in press
12. Li YM, Pan Y, Wei Y et al. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 2004; 6:459-469.
13. Hall JM, Korach KS. Stromal cell-derived factor 1, a novel target for estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Molecular Endocr 2005; 17:792-803.
14. Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses Annu. Rev. Immuno 2004
15. Chavey, C; Bibeau, F; Gourgou-Bourgade, S; Burlinchon, S; Boissiere, F; Laune, D, et al. Oestrogen receptor negative breast cancers exhibit high cytokine content. Breast Cancer Res.2007;9:R15
16. Helbig G, et al. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. Biol Chem,2003,278:21631-8
17. Schioppa, T; Uranchimeg, B; Saccani, A; Biswas, SK; Doni, A; Rapisarda, A, et al. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med.2003;198:1391-402.
18. Staller, P; Sulitkova, J; Lisztwan, J; Moch, H; Oakeley, EJ; Krek, W. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature.2003;425:307-11
2. A.keener c.et al. A possible role for CXCR4 and its ligand the CXC chemokine cell-derived factor-l,in the development of bone marrow metastasis[J]. Cancer, 2004;55(3):115-102.
3. Sehgal A. et al. CXCR4 a chemokine receptor is overexpressed in and required for proliferation of glioblastoma tumour cells[J]. surg oncol, 1998;43(4):69-99.
4. Albert zlotnik. Chemokine in neoplastic progression [J].Seminars in cancer biology,2004; 14(4):181-185.
5. Li yan,G.mark Anderson. Theropeutic potential of cytokine and chemokine antagonistis in cancer therapy[J].Cancer,2005;34 (4):24-28.
6. Anja Muller,Bernhard Homey et al. Involement of chemokine receptor in breast cancer metastasis[J].Nature,2001;410(3):50-56.
7. J.Blay.CCR7 receptor expression correlate with node involvement and survival in primary breast carcinoma[J]. Breast cancer, 2006;131(2):134-139.
8. Bunzo Nakata et al. Chemokine receptor CCR7 expression correlates with lymph node metastasis in pancreatic cancer[J]. Clinical translation research,2008;74(3):69-75.
9. Hideaki Tsuzuki et al. Oral and oropharyngeal squamous cell carcinomas expressing CCR7 have poor prognose[J].Auris nasus larunx,2006;33(5):37-42.
10. B.Schmauber,.S.Endrich et al. The chemokine receptor CCR7 is expressed on epithelium of non-inflamed gastric mucosa, helicobacter pylori gastritis,gastic carcinoma and its precursor lesions and up regulated by H.pylori[J].Clinical Experimental immunology, 2008;139(4):323-327.
11. J panse,K Friedrichs et al. Chemokine CXCL13 is overexpressed in the tumour tissue and in the peripheral blood of breast cancer patients[J].British journal of cancer,2008;99(5):930-938.
12. F.Toulza.Breast tumor cell soluble factors induce monocytes to produce angiogenicbut not angiostatic CXC chemokines [J].cancer,2005;115(4):429-436.
13. Barberos,et al. Stromal cell-derived factor 1 alpha stimulates human glioblastoma cell growgh through the activation of both extracellular signal regulated kinases 1/2 and akt[J]. Cancer Res,2003;63(4): 196-199.
14. Violet F. The chemoine network:A target in cancer biology[J]. Advanced drug delivery reviews,2006;58(4):962-974.
15. T.Jcuril. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J].Nat.Med,2004;4(4):942-945.
16. Y.koxama.The enhanced expressions of CXCR4 and CCR7 MRNA in breast cancer tissue do not always correlate with cancer metastasis[J]. Breast disease,2007;61(1):78-85.
17. Iwao Takanami.Overexpression of CCR7 MRNA in no small cell lung cancer:correlation with lymph node metastasis [J].cancer, 2003;105(6):186-189.
18. L.pitkin et al. Expression of CC chemokine receptor in tonsillar cancer predicts cerbical nodal metastasis and systemic relapse[J]. British journal of oral and maxillofacial surger,2007;45(5):1-7.
19. Gustavo A,Heresi et al. Expression of the chemokine receptor CCR7 in prostate cancer presenting with generalize lymph adenopathy [J].Seminars and original investigations,2005;23(4):261-267.
1. Rot, A; von Andrian, UH. Chemokines in innate and adaptive host defense:basic chemokinese grammar for immune cells. Annu Rev Immunol.2004;22:891-928
2. Coussens, LM; Werb, Z. Inflammation and cancer. Nature.2002; 420:860-7.
3. Robinson, SC; Coussens, LM. Soluble mediators of inflammation during tumor development. Adv Cancer Res.2005;93:159-87.
4. Aggarwal, BB; Shishodia, S; Sandur, SK; Pandey, MK; Sethi, G. Inflammation and cancer:how hot is the link? Biochem Pharmacol. 2006;72:1605-21.
5. Szlosarek, P; Charles, KA; Balkwill, FR. Tumour necrosis factor-alpha as a tumour promoter. Eur J Cancer.2006; 42:745-50.
6. Wang, D; Wang, H; Brown, J; Daikoku, T; Ning, W; Shi, Q, et al. CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J Exp Med.2006;203:941-51.
7. Cha, YI; Dubois, RN. NSAIDs and Cancer Prevention:Targets Downstream of COX-2. Annu Rev Med.2006
8. Murakami, T., Cardones, A. R., Hwang, S. T. Chemokine receptors and melanoma metastasis J. Dermatol. Sci.2004;36,71-78
9. Tanaka, T., Bai, Z., Srinoulprasert, Y., Yang, B. G., Hayasaka, H., Miyasaka, M. Chemokines in tumor progression and metastasis Cancer Sci2005.; 96,317-322
10. Balkwill, F. Cancer and the chemokine network Nat. Rev. Cancer 2004;4,540-550
11. Chen, G. S., Yu, H. S., Lan, C. C., Chow, K. C., Lin, T. Y., Kok, L. F., Lu, M. P., Liu, C. H., Wu, M. T. CXC chemokine receptor CXCR4 expression enhances tumorigenesis and angiogenesis of basal cell carcinoma Br. J. Dermatol 2006. in press
12. Li YM, Pan Y, Wei Y et al. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 2004; 6:459-469.
13. Hall JM, Korach KS. Stromal cell-derived factor 1, a novel target for estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Molecular Endocr 2005; 17:792-803.
14. Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses Annu. Rev. Immuno 2004
15. Chavey, C; Bibeau, F; Gourgou-Bourgade, S; Burlinchon, S; Boissiere, F; Laune, D, et al. Oestrogen receptor negative breast cancers exhibit high cytokine content. Breast Cancer Res.2007;9:R15
16. Helbig G, et al. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. Biol Chem,2003,278:21631-8
17. Schioppa, T; Uranchimeg, B; Saccani, A; Biswas, SK; Doni, A; Rapisarda, A, et al. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med.2003;198:1391-402.
18. Staller, P; Sulitkova, J; Lisztwan, J; Moch, H; Oakeley, EJ; Krek, W. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature.2003;425:307-11