The Study Of The Expression And Function Of Cell Adhesion Molecule2in Human Nasopharyngeal Carcinoma

Nasopharyngeal cancer is an common malignant tumor in people living in the centralregion of southern China’s Guangdong province. Moreover, the incidence is rising year byyear in other regions, including Native people in Southeast Asia, the local Arabs of NorthAfrica and parts of the Middle East. Recently, Epstein-Barr virus, genetic predisposition,dietary and environmental factors are all believed to be involved in NPC development.These factors alone or in combination, will lead to changes in multiple genes andepigenetic, in which inactivation or loss of tumor suppressor genes have a closerelationship with nasopharyngeal carcinoma, but a complete understanding of thepathogenesis of NPC in the context of global gene expression, transcriptional pathwaysand biomarker assessment remains to be elucidated. Pathologically, we found thatnasopharyngeal carcinoma is a poorly differentiated cancer with highly metastatic, and itsoccurrence site very subtle, and therefore cause great difficulties for diagnosis andtreatment, and resulting in the majority of patients have been diagnosed in the late andoften accompanied with metastasis. Presently, the major method of treatment isradiotherapy, and the five-year survival rate after treatment is always below60%.Therefore, it is urgent for us to identify molecular mechanisms of pathogenesis of NPC, tofind valuable biomarker for early diagnosis and valid treatment.Herein, cell adhesion molecule2(CADM2) is an immunoglobulin (Ig)-like celladhesion molecule that belongs to the CADMs family. CADMs family consists of fourconserved members. All CADMs proteins display the same domain organization with threeimmunoglobulin-like domains, a single trans-membrane region, and a short cytosolic tailwith a protein4.1interaction sequences and a PDZ type II motif domain. It is assumed thatall members of the family regulates cell adhesion by its Ig-like domains, and theintracellular protein4.1motif and PDZ II motif binding to them respective ligands,participate in the formation of cell structures, cell movement, intracellular signalconductivity etc. So the characteristics of these domains are very important for us to studythe function of CADMs. The studies of this family mainly focused on CADM1. Recentstudies suggest that promoter methylation and loss of expression of the CADM1gene wasfound in cancers from NSCLC, pancreatic cancers, hepatocellular Carcinomas, prostatecancers, the esophagus, stomach, pancreas, and uterine cervix, as well as meningioma. Inaddition, clinico-pathological analyses have revealed that the inactivation of CADM1 occurs more frequently in tumors in advanced stages. CADM1has also been shown toexert an anti-adhesive and anti-proliferative effect on many human tumor cells. Besides, ithas been reported that CADM1/TSLC1/IGSF4also plays critical roles in epidermaladhesion, stem cell quiescence and wound repair in different tissues, and loss ofCADM1/TSLC1/IGSF4expression might be favorable for cancer cells to escape theimmunological surveillance conducted by the host system. Recently, a study by Fukuharahas shown that the expression of TSLL1(also known as CADM3) and TSLL2(also knownas CADM4) was silenced or markedly reduced in many human glioma cell lines or someprostate cancer cell lines, suggesting that loss of expression of these genes might beinvolved in some human cancers. Properties and functions of CADM2largely remain to beelucidated. A few studies have reported that CADM2was silenced by promotermethylation in human prostate cancer, human renal cell carcinoma ovarian cancer,lymphoma and melanoma. However, the function of CADM2and its cascade in eachbiological situation would be required for further discussion. Until now，there is never anystudy that has shown a relationship between CADM2and NPC.In this study, we first examined CADM2expression in nasopharyngeal cancer celllines by semi-quantitative RT-PCR; we cloned human CADM2genes into the inducibleexpression of lent-viral vector; selected NPC cells expressing low level of CADM2totransfected with CADM2lentivirus; then, the effect of CADM2on the NPC cells wasanalyzed by doubling time assay, EdU incorporation assay. Furthermore, we also testedwhether CADM2could suppress the growth of NPC cells in nude mice in vivo; In order totesting the in-situ expression of clinical specimens, we produced specific monoclonalantibodies anti CADM2;We sought to determine whether tumor cell growth inhibition byCADM2was associated with a greater degree of cell-cycle dysregulation as determined byDNA flow cytometry analysis, and we determined the altered expression of relativeregulatory proteins by western blot and IF. It would be valuable to know which domains ofCADM2are significant for its ability to conduct tumor suppress, a series of HA-taggedCADM2variants were constructed and transfected into NPC cells. Finally, by using IHCand semi-RT-PCR, we tested the expression of CADM2, p-Akt, p27KIP1, p21WAF1/CIP1inNPC tissues, analyzed the relationship between them, and the development of the NPC.The results showed that CADM2was frequently down-regulated in NPC cell lines bysemi-RT-PCR. Re-expression of CADM2in NPC cells can extend the doubling time ofnasopharyngeal carcinoma cells, and inhibited the clone formation ability, anchorage-independent growth in vitro and tumorigenesis of NPC cell in vivo. For testingthe expression of CADM2in clinical tissue specimens in situ, we successfully achieved thespecific antibody against CADM2. Furthermore, Cell cycles analysis indicated thatCADM2inhibited NPC cell growth in vitro by arresting cells in G0/G1phase withdown-regulation of CDK2, cyclinE and up-regulation of their inhibitors. Surprisingly, wealso show that the extracellular region of CADM2is essential for it to exert its inhibitoryeffect. In addition, CADM2acts as a signaling molecule leading to progressive inactivationof Akt. Finally, we found that CADM2was downregulated in NPC tissues, and negativelycorrelated with p-Akt, a positive correlation with p27KIP1, p21WAF1/CIP1, also promotesp27KIP1, p21WAF1/CIP1localization in the nucleus, and closely related to the development ofNPC.In conclusion, the results indicate that CADM2is downregulated or silenced in NPC.It contributes to tumor inhibition through its effects on the PI3K/Akt/Skp2/p27KIP1、p21WAF1/CIP1axis. This finding may facilitate the determination of diagnostic andtherapeutic targets for NPC as well as provide insights about the molecular pathogenesis ofNPC.