The Role Of C-terminus In RON/RON△160 Mediated Signal Transduction And Tumor Growth

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.