XAGE Gene Expression And Function In Tumor Growth And Metastasis

Cancer is a major disease with serious threat to human health.Thus to explore new diagnostic or therapeutic target is the crucial task of biological research.As we known,invasion and metastasis are important characters of malignant tumors which followed by a series of changes of gene expression.In previous study,we found the expression level of XAGE-1b(a member of CTAs) was higher in high metastatic cell line ACC-M cells than in low metastatic cell line ACC-2 cells.Deceased invasion ratio in vitro and in vivo was seen in ACC-M cells with knockdown of the XAGE-1b expression.These data suggest that XAGE-1b might play an important role in the metastasis process of ACC.In other studies,XAGE-1 has been reported with high expression in Ewing's sarcoma,melanoma and lung adenocarcinoma,which is regulated by methylation and can stimulate cellular and antibody response in patients with prostate or lung cancer. This paper focused on the expression pattern of XAGE-1 in oral tumors and different transcriptional regulation mechanism as well as its role in the proliferation/metastasis of ACC and the potential immunotherapy implication.The study has 5 parts.In the first part,Real-time PCR and IHC were applied to assay the expression pattern of XAGE-1 in oral tumors.Data showed that the expression level of 4 variants of XAGE-1 was in a greater difference.The expression of lb is highest which associated in some degree with that of 1d.For the first time, XAGE-1 was shown express positively in ACC tumor and in lymph nodes.In the second part,different fragments on the upstream promoter area of XAGE-1a or lb were cloned and fused with luciferase.Data showed that the transcriptional activity of fragment from lb was much higher than that from la.The 181 bp sequence between 1a and 1b might be the main cause of such great difference.Data also showed that the transcriptional activity of 1b in SPC-A1 and ACC-M cell lines was higher than that in 293T and ACC-2 cell lines respectively.At the same time,subcellular localization studies showed that 1a,1b,1c were located in the nuclear while 1d was located in whole cell in 293T,COS7 or ACC-2 cells.In order to study the role that XAGE play in the proliferation and metastasis processes of ACC cells,in the third part,stable clone overexpressing XAGE-1b were established in ACC-2 cells,which were assayed in vitro or grown as xenografts in nude mice.A significant increase in cell proliferation and invasion was observed of XAGE-1b-expressing ACC-2 cells.Synchronously,knockdown of XAGE-1b by RNA interference suppressed ACC-M cells' growth in vitro and in vivo.In the fourth part, the mechanism for XAGE-1b to promote cell growth and metastasis were studied. Transient XAGE-1b transfection reduced G0-G1 phase cell populations and increased G2-M phase populations.XAGE-1b also can confer ACC-2 cells with the ability of resistance to the apoptosis induced by 1250u/ml TNF-a or serum deprivation.Further, Mercury signaling pathway system was used to analysis the transcriptional regulation of XAGE-1b on several important signaling molecules.The results showed that expression of XAGE-1b partly activated the transcription of RB,CRE,C-Myc,HSE, E2F,GRE,NF-κB elements and depressed that of P53,AP1.Among them,the RB(P＜0.01) and C-Myc(P＜0.05) activation were remarkable.The activation effect was according with the concentration of XAGE-1b expressing plasmid.In order to further clarify the immunological background of ACC-2 cells and help with the application of XAGE-1 on the immunotherapy,HLA typing was conducted with ACC-2 cell line and the result was HLA-A24/68,B72/72,DR1/1.At the same time we established a HLA-DR typing method based on melting curve analysis.In short,we studied the expression pattern of XAGE-1 in oral tumor including ACC tumor and found XAGE-1b was the dominant transcript.XAGE-1d shared a different subcellular localization from other 3 transcript with typical nuclear distribution.XAGE-1b can promote the proliferation and metastasis of ACC cells in vitro and in vivo.The possible mechanism may associate with the regulation function on cell cycle,cell apoptosis and important signal transduction pathway.Such results suggest that XAGE-1b plays an important role in oncogenesis of ACC,and acts as an important diagnostic and prognostic marker of ACC.It also provided new clues for the study of transcriptional regulation function of CTAs.