【作者】 陆意；

【导师】 王明海； 姚航平；

【作者基本信息】 浙江大学 ， 肿瘤学， 2009， 博士

【摘要】 背景：受体酪氨酸激酶RON在上皮细胞的生长、分化、增殖等过程中发挥着一定的作用。RON在乳腺，结肠，肺，甲状腺，皮肤，膀胱和胰腺肿瘤等组织中呈高度表达，而在正常上皮组织中呈低表达或不表达。RON的异常表达常伴随变异体产生。变异体RON△160的过度表达是结肠癌细胞的一个特征。RON含有多个功能性的结构域，如胞外的SEMA结构域、PSI基团、IPT单位和胞内的邻膜区、激酶区和C-末端磷酸化位点等。不同的结构域在RON介导的生物学活性中发挥着不同的作用。缺失或截断都会导致RON受体磷酸化活性改变。变异体RON△160缺失胞外段第一个IPT单位，使它具有致瘤性。C-末端位于RON第20外显子，在调节RON活性中发挥两个作用。一方面，其包含一个多功能结合位点—Y¹³⁵³VQL-PAT-Y¹³⁶⁰序列。该位点能招募下游的信号分子，如PI-3K，Smad，Grb2，STAT3等。当RON与特异性配体结合后，该位点的酪氨酸残基发生磷酸化、传导信号。但是包含两个突变位点Y¹³⁵³和Y¹³⁶⁰的突变体RON^M1254T显示这个多功能位点对致瘤活性不是必需的。另一方面，体外实验显示整个C-末端对RON激酶活性有自我抑制作用，主要通过是C-末端Tyr1353／1360残基与激酶区Tyr1238／1239残基之间的结合起作用。研究发现C-末端的Tyr1353／1360残基发生突变或被Phe替代后，C-末端与激酶区的结合更加紧密，进一步抑制RON活性。因此，C末端在RON介导的生物学活性中的作用有待于进一步研究。目的：本课题旨在研究RON蛋白在正常人消化道上皮组织及其癌变细胞中的表达状态，同时着重探讨RON-C末端肽段在RON及其变异体RON160所介导的信号传导及致瘤活性中的作用。材料与方法：采用单克隆抗体杂交瘤技术，制备并鉴定数个具有生物学活性的特异性抗RON单克隆抗体。通过免疫组织化学的方法，用抗RON单抗（2F2），检测了RON在正常人消化道及癌症组织中的表达。所用的组织芯片类型包括：正常成人、胚胎及多种消化道肿瘤组织。利用PCR方法构建了C末端缺失的两个RON变异体——RON-CF和RON△160-CF。通过细胞表达、蛋白化学分析、生物学功能及动物体内实验等实验方法，研究了C末端在RON及RON△160在促进细胞侵袭性生长中的一些作用及机理。结果：1、成功制备了特异性抗RON单克隆抗体Zt／g4、2F2和2C6，初步鉴定了它们在免疫组化、ELISA、免疫沉淀、免疫荧光等方面的性状。其中Zt／g4和2F2主要识别RON胞外段，两者都具有激活RON磷酸化的活性。2C6主要识别胞内段，没有明显抑制或激活RON的生物学活性；2、组织芯片的免疫组化结果显示，RON在消化系统的胚胎和成人组织中几乎不表达或呈低水平表达，两者的表达和分布没有明显差异；在食道癌、胃癌、胰腺癌、结肠癌组织中，RON蛋白呈异常表达，其中50％以上的结直肠癌组织中RON为过度表达：3、C末端缺失的RON蛋白呈现明显的生化及生物学功能的异常，主要表现为：1) C末端缺失后导致RON受体无法发生二聚体化，蛋白磷酸化的作用消失；2)在RON介导的信号通路中，C末端缺失后RON／RON△160的自体磷酸化功能丧失、下游信号蛋白（Erk1／2、AKT）的磷酸化也抑制，RON△160介导的细胞浆内β-catenin的积聚的功能减弱；3)C末端去除后导致RON△160介导的细胞增殖、形态改变、迁移和动物体内致瘤效应等活性明显丧失。结论：RON蛋白在正常人胚胎和成人消化道上皮组织中呈微量表达状态，而在部分消化道肿瘤组织，特别是在大肠癌中呈过度表达，提示RON的过表达可能和大肠癌的发生和进展有一定的病理联系。RON蛋白C末端是调节RON生物学功能的重要结构组成部分，通过影响RON受体的磷酸化，调节胞内信号蛋白途经的激活，从而调节细胞的生物学功能。更多还原

【Abstract】 BackgroundThe RON receptor tyrosine kinase, a member of the MET protooncogene family,plays an important role in epithelial cell growth, differentiation, and transformation.RON is minimally expressed in normal epithelial tissues. However, overexpression isobserved in various epithelial tumors, indluding those from breast, colon, lung, thyroid,skin, bladder, and pancreas.. In primary epithelial cancers, RON expression is oftenaccompanied with generation of splicing or truncated variants。Over-expresion of RON△160 is an example in colon cancer. The RON protein contains multiple functionalstructures, including the extracellular ligand binding domain, sema domain, PSI motif,and IPT units, The intracellular sequences contain juxtamembrane domain, kinasedomain, and C-terminal docking site. Thses domains play significant roles in regulationof RON-mediated biological activities. Any deletion or truncation affects RONphosphorylation, leading to increased or reduced tyrosine kinase activities. RON△160,generated by alternative deletion of the first IPT unit, is a typical example. It has celltransforming activities and caused tumor growth in animal study. The RON C-terminus contains about 55 amino acids coded by exon 20. Using RON^1254T mutant it has beenshown that C-teminus is not required for RON-mediated tumor growth in vivo.However, two functions of the C-teminus have been identified. The first one is themultifunctional docking site in the C-terminus. The site contains a unique sequence（Y1353VQL-XXX-Y1360MNL-）. Ligand stimulation results in Y1353 and Y1360,which act as the docking site recruiting downstream signaling molecules. The idnetifedmolecules include PI-3K, Smad, Grb2, STAT3 and others. The second function is itsauto-inhibitory activity acting on the tyrosine kinase domain. There is a report showingthat deletion of the C-terminal tail enhances RON kinase activity. This is modeld byinteraction of the C-terminus with the kinase catalytic domain. Clearly, C-terminus isciritcally important in regulating RON mediated biological activities.AimThe project is to determine RON expression in human gastrointestinal tissuesderived from embryonic, normal adult and cancerous samples. The roles of C-terminusin regulation of RON or RON△160-mediated signaling transduction and tumor growthwere also studied.Method and materials:Three monoclonal antibodies signated as Zt/g4, 2F2 and 2C6 were produced fromBalb/c mice through standard hybridoma technology. They were characterized andevaluated in immunohistochemical stainig, ELISA, Wetern bloting, immunoprecipitation,and immune fluorescent analysis. Antibody 2F2 was also used in tissuemicroarrays by immunohistochemical staining to study RON expression in variousgastrointestic tissues samples. These samples were derived from normal embryonic,adult, and tumor tissues. In addition, two RON variants, RON-cf and RON△160-cf,free of the C-terminal tail were produced by PCR techniques and used as the model tostudy their significance in regulation of RON or RON160 activities. Through a serious of in vitro and in vivo experiments, including protein expression, phosphorylation,signaling transduction, cellular functions, and tumor growth in mice, the effect of theC-terminus in regulating RON or RON160-mediated tumorigenic activites and itsunderlying mechanisms were studied.Results1. The monoclonal antibodies （Zt/g4, 2F2 and 2C6） are produceded and identifiedin immunohistochemistry, ELISA, immunoprecipitation and Immuno-fluorescentassaies. Zt/g4 and 2F2 recognizes the extracellular binding sites of RON, and 2C6recognizes the intracellular binding site. Zt/g4 stimulates the phosphorylation of RON,2F2 obviously inhibits RON△160 induced tumor formation, and 2C6 has a neutralactivity. 2. Immunohistochemistry of tissue microarrays by 2F2 has revealed that theexpression patterns were relatively similar between embryonic and adult tissues, butwere altered significantly in cancerous samples. Overexpression of RON was found inmore than 50% of CRC cases, particularly oncogenic RON variants, RON△160.3. Thedeletion of C-terminus results in alteration of RON biochemical and biological acticities:1) Deletion of the C-terminal tail impairs the dimerization of RON receptor, whichresults in the inactive state of RON. 2 ) In analyzing RON-mediated signaling events, wefind that deletion of the C-terminal tail significantly inhibits RON or RON△160auto-phosphorylation and subsequent activation of downstream signaling componentssuch as Erk1/2 and AKT. Deletion of the C-terminal tail was also affect RON△160-mediated cytoplasmicβ-catenin accumulation. 3 ) Truncation of the C-terminal tailsignificantly iinpaires RON or RON△160-mediated cell proliferation, morphologicalchanges, migration, and tumorigenic growth.ConclusionIn samples of normal human embryonic and adult gastrointestinal epithelial tissues, RON expression is minimal or at relatively low level. Increased expression of RON ingastrointestinal cancer samples including colon cancer was observed. These resultsindicate that RON may be ivolved in pathogengesis and carcinogenesis of certain typesof tumors. The C-ternimus is an important structural component that regulates RON orRON160-mediated biological activity. By controlling RON phosphorylation,downstream signaling cascades, or RON△160-mediated cytoplasmicβ-cateninaccumulation, the C-terminus play a vital role not only in maintaining the structuralintegrities, but also in regulating RON-mediated tumorigenic activities.更多还原