【作者】 周转；

【导师】 杨治华；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 博士

【摘要】 食管癌的死亡率在我国恶性肿瘤中居第四位,五年生存率低于10%。抑制食管癌淋巴结转移和邻近组织侵袭是有效治疗食管癌、提高食管癌生存率的关键。我们采用两种策略研究食管癌侵袭转移相关功能基因:即采用基因芯片分析高侵袭转移潜能亚细胞系与亲本细胞系差异表达基因和功能性单体库筛选分离功能抗原基因。采用基因芯片技术分析时,我们首先采用体外Matrigel侵袭克隆筛选建立高侵袭高转移食管癌亚细胞系EC9706-P4,应用全基因组芯片比较EC9706-P4与其亲本细胞系EC9706的基因表达谱,获得124个差异表达基因,其中48.3%(60/124)是与侵袭转移相关的基因。通过表达谱分析和生物信息学分析,表明TM4SF3可能是候选的食管癌侵袭转移相关的功能基因。Western blotting表明TM4SF3在57.1%的食管癌组织和高侵袭食管癌细胞系中显著上调表达。RNA干扰和外源性基因表达研究表明TM4SF3可显著促进食管癌细胞侵袭。在EC9706细胞过表达TM4SF3促进体内移植瘤基质侵袭、自发肺转移,并缩短小鼠生存期。进一步研究其分子机制表明TM4SF3显著上调ADAM12m,再上调ILK的表达。RNAi抑制ADAM12m表达能显著抑制TM4SF3介导的侵袭。这些研究结果表明TM4SF3是一个食管癌表达上调的具有促进癌细胞侵袭转移的功能性基因。因此该基因有可能作为靶向食管癌侵袭转移治疗的分子靶标。有关TM4SF3基因侵袭转移功能及分子机制的研究国内外还尚未见到报道。在克隆TM4SF3基因时我们还首次发现了一个功能性的SNP改变,即TM4SF3 397bp位点的SNP(G→C),我们的实验结果证明这一SNP改变能够消除TM4SF3对ADAM12m的表达调节,显著影响TM4SF3促肿瘤侵袭转移潜能。在研究基因芯片中差异表达的基因时,我们也同时发现ADAM12显著上调,因此我们对该基因在食管癌转移发挥的功能做了深入的研究。采用119例食管癌组织芯片和98例原发肿瘤和转移性淋巴结配对免疫组化研究表明ADAM12m在肿瘤组织显著过表达,并表现为伴随侵袭、淋巴结转移呈梯度上调,预后的相关性分析表明过表达的ADAM12m是一个独立于食管癌淋巴结转移或者局部侵袭之外的有重要意义的预后分子标志物。RNA干扰或者外源性表达ADAM12m表明其在体内外均显著促进食管癌侵袭和自发性肺转移,并缩短实验小鼠的生存期。分子机理研究表明过表达的ADAM12m参与稳定点状粘附,并通过促进FAK磷酸化和ILK表达参与肿瘤侵袭转移。ADAM12m介导的FAK磷酸化依赖于Integrinβ3,并且通过促进Integrinβ3-FAK相互作用以促进FAK磷酸化。而且ADAM12m过表达与FAK磷酸化上调和ILK表达上调在临床组织标本具有高频度的相关性。因此,ADAM12m过表达及其相关信号通路的活化是食管癌进展中的高频事件,是促进食管癌侵袭和转移的重要功能性基因,因此也可能是治疗食管癌侵袭转移的有价值的靶标。这些关于ADAM12m在肿瘤侵袭转移中的功能及其分子机制的研究,国内外尚未见到报道。功能性单抗库筛选功能性抗原基因的研究中,采用转移性食管癌原发瘤中分离的有核细胞免疫Balb/c小鼠,制备含有1260个杂交瘤克隆株的大容量食管癌功能性单抗库。活细胞免疫荧光筛选获得了545株识别食管癌细胞膜蛋白的单抗。通过免疫组化筛选获得了485株与正常人食管组织不反应或者较弱反应的相对特异的单抗。通过迁移、侵袭、粘附、增殖等功能筛选获得了70株抑制迁移,7株抑制侵袭,5株抑制增殖,32株抑制与肺内皮粘附,48株与淋巴内皮粘附相关的单抗。选取其中20株进行抗原特征分析,并对其中8株单抗纯化抗体制备抗体亲和柱亲和纯化相应的功能性抗原蛋白。采用MALDI-TOF和LCQ-TOF鉴定了其中3个抗体识别的功能性抗原及抗原相关序列,包括hDUS2(单抗12B8),1FAKI(单抗3D11),GRLF1(单抗4B9)。生物信息学分析这3个抗原基因具有粘附、运动和增殖等相关功能,表明功能性单抗库技术能有效筛选鉴定肿瘤相关抗原基因。通过这种方式筛选获得的功能性抗体及其靶抗原具有潜在的应用价值,表明功能性单抗库也是研究食管癌侵袭转移的有效策略。更多还原

【Abstract】 Esophageal cancer is the fourth most common malignancy in China and its 5-years survival rate is less than 10%. Esophageal cancer is characterized by rapid clinical progression and poor prognosis due to adjacent tissue invasion and distant organs metastasis at a very early stage. Therefore, disrupting invasion and metastasis is essential to develop effective treatments and longer the survival time of esophageal cancer. To investigate genes involved in esophageal carcinoma invasion and metastasis, we applied two different strategies, as by microarray comparison high invasive and metastasis potential subcell line with parent cell line or by building functional mAb libray fowolling functional mAb screen and antigen identification.For microarray analyses, an in vitro metastasis model via matrigel invasion clonal selection approach was employed, and a highly invasive sub-line EC9706-P4 was established. Then the expression profile of the sub-line and the parental cell line EC9706 was analyzed by gene microarray analysis. By this screen, TM4SF3 (transmembrane 4 superfamily 3) was identified as a candidate promoter for esophageal carcinoma cell invasion and metastasis. Western blotting revealed that TM4SF3 was overexpressed in 57.1% (8/14) of esophageal carcinomas and esophageal carcinoma cell lines with high-invasive potential. RNAi knockdown and ectopic expression indicated it promoted cell migration and invasion. Upregulating TM4SF3 expression in EC9706 cells promoted xenograft tumor invading into surrounding tissues, enhanced lung metastasis, and shortened the lifespan of mice in a spontaneous metastasis model. Further studies demonstrated that ADAM12m was upregulated by TM4SF3 overexpression in vitro and in vivo. Abrogating up-expression of ADAM12m by siRNA significantly suppressed TM4SF3-mediated invasion. Furthermore, a SNP of 397bp (G→C) in TM4SF3 abrogated ADAM12m upregulated by TM4SF3, and decreased its promoting invasion and metastasis potential.By gene microarray analysis, we also identified ADAM12m, a membrane-anchored form of a disintegrin and metalloprotease, was over expressed in EC9706-P4 cells. Immunohistochemical analysis on 119 cases tissue microarray and 98 cases paired primary cancer tissues and lymph nodes with tumor metastasis showed ADAM12m was overexpressed in tumor, and gradient upregulated in tumor invasion and lymph node metastasis. The overexpressed ADAM12m was a valuable prognostic factor independent of lymph node metastasis or local invasion. RNAi-mediated knockdown and ectopic expression of ADAM12m showed that ADAM12m promoted esophageal carcinoma metastasis in vitro and in vivo. Subsequent mechanistic studies showed that ADAM12m overexpression participated in focal adhesion and conferred metastatic properties to cancer cells by promoting FAK phosphorylation and ILK expression. The ADAM12 mediated FAK phosphorylation is integrinβ3 dependent and due to the enhancement of integrinβ3-FAK interaction. Furthermore, the correlation of ADAM12m overexpression with up-regulated FAK phosphorylation and ILK expression was frequently observed in clinical samples. Thus, ADAM12m overexpression and the related pathway activation, should be frequent events in esophageal carcinoma progression, and contributes to the cancer invasion and metastasis. Therefore, AMAM12 may be a valuable target for the treatment of esophageal carcinoma invasion and metastasis.To screen functional mAbs associated with esophageal carcinoma invasion and metastasis, an esophageal carcinoma functional monoclonal antibodies library containing 1,260 clones, was prepared by immunized Balb/c mouse with nucleated cells separated from metastatic esophageal carcinoma primary tumor. From this library, 545 membrane reactive clones were got by viable cell immunofluorescence assay. Then 485 clones, which did not or weakly reacted with normal esophageal tissue were selected out by immunohistochemitry. By migration, invasion, adhesion and proliferation assays, 70 clones showed the ability of migration inhibition, 7 clones showed the ability of invasion inhibition, 5 clones showed the ability of proliferation inhibition, 32 clones could inhibit the adhesion of esophageal tumor cells with HLEC and 48 clones could inhibit or promote the adhesion of esophageal tumor cells with LYEC. Then 20 of these clones were picked out for further assayed and at last 8 clones were cultured to produce antibody for antibody affinity assay. Three antigens purified by antibody affinity assay were identified by MALDI- TOF and LCQ-TOF, and were homology to hDUS2 (12B8 mAb), 1FAK1 (3D11 mAb) and GRLF1 (4B9 mAb). These functional mAb or antigens recognized by them might have potential application for development of therapies targeting esophageal carcinoma invasion and metastasis. Moreover, these mAb might be valuable for investigate the mechanism of esophageal carcinoma invasion and metastasis.更多还原