Identification&Functional Study Of MC3-AG Associated With Colorectal Cancer

BackgroundColorectal cancer (CRC) is a leading cause of cancer related morbidity andmortality worldwide. Traditional CRC biomarkers such as carcinoembryonicantigen (CEA) and CA199are not suitable for screening and early diagnosis dueto low sensitivities. Genomic and proteomic researches have been advancingrapidly in recent years, facilitating the discovery of a range of promising CRCbiomarkers. However, few has been further validated and applied to the clinicalpractice after translational study. Hence, identification of novel biomarkers forCRC and translation the study data into patient care may open the door to a moreaccurate and target specific personalized medicine with improved patientsurvival.Using a homogenate preparation of colon cancer tissues from metastaticlymph nodes, Fan et al. developed a series of CRC-specific monoclonalantibodies through hybridoma technique in1980s. Among those antibodies, MC3is a specific and sensitive antibody that reacts with an unknown antigenpresent in up to90%cancer tissues. MC3-Ag is an ideal candidate biomarker forCRC for its expression is correlated to TNM (tumor-node-metastasis) stagingand its overexpression is observed in metastatic CRC. Tremendous works hasbeen done in our laboratory concerning MC3-Ag on its expression profile inCRC, its diagnostic and predictive value as a novel biomarker and also thetargeted therapy, with a lot of papers published. However, MC3has not yetgained recognition and acceptance, primarily because the target antigen of theMC3antibody had never been identified.In the present study, proteomics approaches were applied to successfullyisolate and identify the MC3Ab immunoreactive protein MC3-Ag. Thefollowing study went on to validate its diagnostic and prognostic value.Functional studies revealed its specific regulation of multiple malignantbehaviors of CRC including proliferation, anti-apoptosis and metastasis. Finally,the interaction protein with MC3-Ag was identified and the downstreammolecular events and pathways were further explored.Objectives1. To isolate and identify MC3-Ag specific to CRC2. To study the impact of MC3-Ag/Txl-2on multiple malignant behaviors ofCRC3. To examine the differential functions of MC3-Ag/Txl-2alternative splicedvariants in CRC4. To explore the underlying mechanisms mediated the function ofMC3-Ag/Txl-2in CRC Methods1Proteomics approaches including immunoprecipitation, two-dimensional gelelectrophoresis (2-DE) and matrix-assisted laser desorption/ionization time offlight mass spectrometry (MALDI-TOF-MS) were applied to isolate and identifyMC3-Ag. Paired immunohistochemical study, immunofluorescence andconfocal microscopy and Western blot analysis were done for further validation.Tissue arrays were applied to examine the MC3-Ag expression profiles innormal tissues and multiple tumor tissues.2Immunohistochemical study was done in243cases of clinical samples tostudy the association of MC3-Ag/Txl-2expression with clinicopathologicalparameters and further validate its prognostic value. Txl-2siRNA vector wasconstructed and stably transfected into the highly invasive colon cancer cell lineSW620. MTT assay, colony formation assay, cell cycle analysis by flowcytometry (FCM) were done to examine the impact of Txl-2on cell proliferation;Annexin V/PI double staining and FCM were done to examine the serumdeprivation-induced and chemodrug-induced apoptosis. Wound healing assay,adhesion assay, Transwell invasion assay and soft agar assay were done toexamine the function of Txl-2on tumor cell invasive and metastatic potential.Further in vivo assays including nude mice tumorigenesis assay and tail veinassay of metastasis were carried out to verify the results from in vitro studies.3RT-PCR was done to examine the mRNA of three Txl-2alternative splicedvariants and total mRNA level from18cases of colon cancer tissues, adjacenttissues and normal tissues. Isoform-specific cDNA expression vectors wereconstructed and stably transfected into colon cancer cells. The differentialfunctions of each individual Txl-2isoform in CRC were investigated.Site-directed mutation of catalytic active sites was manipulated to determine the functional domain and critical sequences.4Yeast two-hybrid system and coimmunoprecipitation were utilized toidentify the Txl-2interaction protein. Immunofluorescence and confocalmicroscopy were done to observe the colocalization of Txl-2and Ran.Interventions by siRNA oligos, antibody and pathway inhibitors were done toinvestigate the therapeutic effect and determine the downstream events andspecific signal pathway.Results1. MC3-Ag was identified as Thioredoxin like-2(Txl-2) by proteomic studyImmunoprecipitation of MC3-Ag using SW480cell lysates or fresh CRCtissue homogenates showed that MC3Ab consistently identified two proteinbands around30kDa. Then the immunoprecipitates were subjected to2-DEfollowed by silver staining or Western blot respectively. The results showed thatMC3antibody consistently detected two immunoreactive spots at the basic pHrange near pH5with Mr of32and28kDa, respectively. Then the two spotswere subjected to in-gel trypsin digestion and MALDI-TOF-MS analysis. ThePMFs were subjected to database searches by ProFound and the two MC3immunoreactive spots were identified as two isoforms of Txl-2(Swiss-Prot:Q86XW9-2and Q86XW9-3). The following paired immunohistochemical study,immunofluorescence and confocal microscopy and Western blot analysis usingboth MC3Ab and anti Txl-2Ab further validated the that Txl-2is the antigenfor MC3Ab. Additionally, expression profiles of MC3-Ag in normal tissues andmultiple tumor tissues were examined by tissue arrays.2. Knockdown of Txl-2significantly suppressed colon cell proliferation,invasion and metastasis, and sensitized tumor cells to apoptosis. Immunohistochemical study in243cases of CRC tissues revealed thatMC3-Ag/Txl-2expression was correlated with TNM staging and its expressionwas significantly upregulated in metastatic CRC. The following survivalanalysis indicated that MC3-Ag/Txl-2could serve as an independent prognosisbiomarker for CRC patients. Txl-2expression was significantly suppressed bystable transfection of siRNA vectors in highly invasive SW620cells.Knockdown of Txl-2expression significantly suppressed cell proliferation withreduced ability of colony formation and cell cycle G0/G1arrest. Inhibition ofTxl-2expression significantly increased the cell apoptotic ratio induced byserum-deprivation or chemodrugs. Knockdown of Txl-2also significantlyinhibited cell migration, adhesion, invasion and anchorage-independent growth.In vivo assay including nude mice tumor formation assay and tail veinmetastasis assay further validated the in vitro results.3. Three isoforms of Txl-2exerted divergent impacts on multiple malignantbehaviors of colon cancer cells.mRNA level of each Txl-2isoform and total Txl-2mRNA level wereexamined in18cases of clinical samples. Total Txl-2mRNA was elevated incolon cancer tissues compared to adjacent tissues and normal mucosa. Among3isoforms, mRNA of Txl-2b and Txl-2c were significantly upregulated in coloncancer tissues while Txl-2a was rarely expressed in all samples (2/18cases).EGFP recombinant expression vectors of each Txl-2isoform were constructedand stably transfected into LoVo cells. Both in vitro and in vivo studiesindicated that3isoforms of Txl-2exerted divergent impacts on multiplemalignant behaviors of colon cancer cells. Txl-2b contributes to thephysiopathology of colon cancer progression and tumorigenesis with its role onfavoring different aspects of colon cancer progression; Txl-2a overexpression produced a less significant modulation of function whereas Txl-2c exhibited arelatively inhibitive effect. Site-directed mutation in catalytic active sitesrevealed that Trx domain is critical for Txl-2mediated invasion and metastasis.4. Txl-2b interacted with the small GTPase Ran and promoted tumor cellmetastasis through PI3K pathwayYeast two-hybrid system verification and co-immunoprecipitation wereutilized to identify the small GTPase family member Ran as a Txl-2b interactingprotein, but not for Txl-2a and Txl-2c. Immunofluorescence and confocalmicroscopy revealed that Txl-2was colocalized with Ran. Transfection ofsiRNA-Ran oligos could partially reverse the Txl-2b mediated tumor metastasis.Further study revealed that PI3K activation participated in Txl-2-Ran-MMPmediated tumor cell invasion and metastasis. Besides, Txl-2was also found tointeract with microtubules and influence colon cancer cell metastasis throughcell shape remodeling.【Conclusion】In the present study, we successfully identified Txl-2as the antigen of themonoclonal antibody MC3associated with CRC. Txl-2exerts multiple impactson tumor cell malignant phenotypes with isoform-specific modulation. Txl-2bdirectly interacts with Ran and PI3K pathway participates in Txl-2b-Ran-MMPmediated tumor invasion and metastasis. The current study provides a novelbiomarker and target molecule for diagnosis and treatment of CRC and opensthe door to a novel understanding of cancer specific alternative splicing and itsfunctioning mechanisms.