Molecular Mechanism Of EB Virus LMP1 Regulates CXCR4 Activity By EGFR Mediated Tyrosylprotein Sulfotransferasel TPST-1

Nasopharyngeal carcinoma (NPC) is a malignancy characterized by high metastatic potential and most patients have poor prognosis because of early-stage and rapid metastasis to other sites. The latent membrane protein (LMP), an important oncoprotein encoded by Epstein-Barr virus (EBV), is closely related with carcinogenesis and metastasis of NPC.Chemokines affect the adhesion, invasion and metastasis of tumor cell. The mechanism of how chemokines and their receptors are involved in tumor metastasis has not been fully understood yet. CXCR4, the SDF-1 chemokine receptor, is the overexpression in tumors, and the expression and activation of CXCR4 can induce tumor cell metastasis to the sites with high expression of SDF-1 (such as bone and lymph node).It is related with growth and metastasis of diverse tumor.In recent years, the sulfated tyrosine (tyrosine sulfation), an important post-translational modification (PTM) form, have attracted much attention because of mediation of chemokine receptor activity. The primary role of tyrosine sulfation is to induce the secretion of secretory proteins or membrane proteins interaction with other proteins, especially interaction with its ligand. The tyrosine 21 of Chemokine receptor CXCR4 is considered as its main sulfation sites. In vivo, tyrosylprotein sulfotransferase (TPST) is responsible for catalysis of tyrosine sulfation and two subunits of TPST are encoded by genes of TPST-1 and TPST-2, both have highly conserved sulfotransferase region. TPST-1 located in the Golgi catalyzed tyrosine sulfation and participated in tyrosine sulfation cycle.Our previous study has found that EBV encoded LMP1 induced the EGFR expression through NF-κB signal transduction pathways, and increased the phosphorylation of EGFR in NPC cells. After being phosphorylated, new transcription factor EGFR was translocated into nucleus to transactivate the key regulators of the cell cycle including cyclin D1 and cyclin E. Bioinformatic analysis revealed that TPST-1 gene (GenBank AF038009) contains EGFR binding sites, which were located in 5'UTR region, that is TGTTT (located-28-24). Therefore, we thought that EGFR might exert CXCR4 sulfation by modulating TPST-lto affect the binding of CXCR4 and its ligand. Therefore, we observed that LMP1 regulates the activity of CXCR4 by EGFR and is correlated with the metastatic Potential of NPC cell.1. CXCR4 sulfation induces the metastasis of NPCPost-translational modifications of the amino termini of CCchemokine receptors, in particular tyrosine sulfation, play a critical role in the ability of these receptors to associate with their natural ligands. Using high metastatic 5-8F and non-metastatic 6-1 OB NPC cells as a study model, we found that high metastatic 5-8F cells is high sulfation level. The data indicated that tyrosine sulfation is associated with cellular metastasis.Chemotaxis assays and Matrigel invasion assays were used for analysis of Chemotactic activity and invasion capacity in sodium chlorate inhibited high metastatic 5-8F cells and in non-metastatic 6-10B cells transfected with WT-CXCR4 or MUT-CXCR4 expressing plasmids. We found that the Chemotactic activity and invasion capacity of high metastatic 5-8F cells was reduced after inhibiting its sulfation, and Non-metastatic 6-10B cells transfected with WT-CXCR4 expression plasmids can enhance cellular Chemotactic activity and invasion capacity. These results suggested that tyrosine sulfation of proteins, especially tyrosine sulfation of CXCR4, is correlated with the metastatic potential of NPC cells, and No.21 tyrosine sulfation of CXCR4 plays an important role in this metastatic process.By RT-PCR and Western Blot, We found that mRNA and protein levels of TPST-1 is higher in high CXCR4 sulfation level 5-8F cells than in low CXCR4 sulfation level 6-10B cells. Further study indicated that TPST-1 siRNA inhibited significantly CXCR4 sulfation level in 5-8F cells. It has been found that the cell surface receptor EGFR and FGFR can locate in the nucleus, and the new study found that another cell surface receptor CXCR4 also can enter the nucleus. Therefore, we firstly detected CXCR4 localization in highly metastatic 5-8F cells and non-metastatic 6-10B cells. By the Western Blot analysis and immunofluorescent analysis with a laser scanning confocal microscope, we found that CXCR4 protein appears in 5-8F nucleus but not in 6-10B nucleus from both quantitative and qualitative levels. After treated with specific sulfation inhibitors or TPST-1siRNA, stimulated 5-8F cells with SDF-la 30nm for 30min. We found that the nuclear translocation of CXCR4 decreased. It implied that the CXCR4 sulfation is closely related to CXCR4 nuclear localization. 2. LMP1 induces the metastasis of NPC by regulating tyrosine sulfation, functional activity and nuclear translocation of CXCR4.We have found that CXCR4 sulfation is higher in 5-8F cells than that in 6-10B cells, meanwhile, our previous studies have found that LMP1 expression is higher in 5-8F cells than 6-10B cells. In addition, it is well known that LMP1 plays an important role in invasion and metastasis of NPC cells. All these results implied that LMP1 infulenced metastasis of NPC via activation of CXCR4 sulfation.Firstly, by flow cytometry we further confirmed that LMP1 expression is higher in high sulfation 5-8F cells than in 6-10B cells.Using tetracycline-rehulated LMP1 expression in a NPC cell line, we found that LMP1 induced CXCR4 expression in a dose-dependent manner. We also demonstrated that LMP1 increasd CXCR4 sulfation in a dose-dependent manner through immunoprecipitation(IP) and Western Blot. Similarly, we treated the Tet-on LMP1 HNE2 cells with DOX in different doses for 24h, then with SDF-la in different dose. By chemotactic experiments conducted, we found that LMP1 can up-regulate chemotactic activity of CXCR4 in a dose-dependent manner. These results further clarified that LMP1 influenced CXCR4 function activity by upregulating CXCR4 sulfation.We have confirmed that LMP1 enhanced function activity of CXCR4 by upregulating CXCR4 sulfation, then we will further explore whether LMP1 induced nuclear translocation of CXCR4. By Western Blot analysis and immunofluorescent analysis with a laser scanning confocal microscope at qualitative and quantitative levels, we found that LMP1 could regulate nuclear translocation of CXCR4 in a dose-dependent manner in Tet-on LMP1 HNE2. Then we used Tet-on LMP1 HNE2 cells transfected with WT-CXCR4 or MUT-CXCR4 expression plasmid for 24h, respectively, then induced by DOX (0,6ug/ml) by the qualitative level through immunofluorescent confocal technology, we found that LMP1 induced CXCR4 protein nuclear accumulation in cells which transfected with WT-CXCR4. These results indicated that LMP1 affects the nuclear translocation of CXCR4 by upregulating CXCR4 sulfation.Finally, HNE2-PSG5 and HNE2-LMP1 cells were transfected with WT-CXCR4 and MUT-CXCR4 expression plasmid for 24h, then we found LMP1 only induced capacity of invasion and metastasis of cells transfected with WT-CXCR4 plasmid but not affected capacity of invasion and metastasis of the cells transfected with MUT-CXCR4 plasmid by Matrigel invasion assay. Futher demonstrated that LMP1 induced invasion and metastasis of NPC cells by inducing No.21 tyrosine sulfation of CXCR4.3. EBV encoded LMP1 mediated CXCR4 sulfation through EGFROur preliminary work confirmed that LMP1 increased the expression of transcription factor EGFR, and we found that there is a EGFR binding sites in TPST-1 promoter domain by the genetic information Therefore, we speculated that LMP1 may mediate TPST-1 by activating EGFR signal transduction.By using Real-time-PCR and Western Blotting in Tet-on LMP1 HNE2 cells, we found that LMP1 upregulated TPST-1 mRNA and protein levels in a dose-dependent manner. After introducting EGFR siRNA into Tet-on LMP1 HNE2 cells for 24h to block EGFR the expression, we found that TPST-1 expression decreased, and CXCR4 sulfation was inhibited. These results primary demonstrated that LMP1 induced CXCR4 sulfation by up-mediating TPST-1 in EGFR dependent manner.Data showed that EGFR could bind to TPST-1 promoter in vivo under the control of LMP1 using chromatin immuniprecipitation (ChIP) assay. Reporter gene analysis showed that LMP1 increased TPST-1 promoter activity, however, the activity of TPST-1 promoter decreased by mutating off the binding site of EGFR and TPST-1. The results further indicated that LMP1 induced TPST-1 in EGFR dependent. we used NPC cell line as the basic experimental model to investigate sulfation, subcellular localization and function of CXCR4 by LMP1 from comparing difference CXCR4 sulfation level in different metastatic potential of NPC cells. This study used Western Blot, chemotaxis, invasion experiments, chromatin immunoprecipitation-PCR, site directed mutagenesis, reporter gene analysis, and other experimental methods of molecular biology to clarify the molecular mechanism that LMP1 regulates CXCR4 at post-translational modification by combining with blocking strategies, and gained the following important Innovative findings:1. It is first to discover that CXCR4 sulfation could induce the metastasis of NPC cells. high metastatic cells is high CXCR4 sulfation level. Special inhibitor-sodium cholorate could inhibit the Chemotactic activity and invasion capacity of highly metastatic cells, while which is enhanded by transfecting wild-type CXCR4 expression plasmid. high metastatic cells is high TPST-1 expression, and TPST-1siRNA can effectively block CXCR4 sulfation. Simultaneously, it is first to show that CXCR4 sulfation could induce the nuclear translocation of CXCR4 in NPC cells. Using specific sulfation inhibitors and TPST-1siRNA can effectively block CXCR4 protein nuclear translocation.2. It is first to find that EB virus LMP1 could up-regulate CXCR4 sulfation level, nuclear translocation and functional activity in a dose-dependent manner. We also found that LMP1 induced the nuclear translocation of CXCR4 by up-regulating CXCR4 sulfation dependent, and LMP1 could induce metastasis of NPC by regulating CXCR4 sulfation.3. It is first to indicate that LMP1 could up-regulate the TPST-1 mRNA and protein levels in a dose-dependent effect. Using EGFRsiRNA can decrease TPST-1 expression. LMP1 could enhanced the binding transcription factor EGFR and TPST-1 promoter, and increased TPST-1 promoter activity, but, mutating EGFR in TPST-1 promoter binding site can be down regulation of TPST-1 promoter activity by LMP1.This paper takes EBV encoded LMP1 mediated TPST-1 by activating transcription factor EGFR as an entrancing point to illustrate the metastatic mechanism and biological significance of LMP1-EGFR-TPST-1-CXCR4 signal transduction pathway aspect of PTM of CXCR4 sulfaton in NPC cells. The study will be give intersect between the EB virus and chemokine receptor signal transduction, provide a new study model, extend a new field for studying EBV molecular mechanism of carcinogenesis, provide a new experimental evidence for targeting LMP1 and CXCR4 anti-cancer signal transduction therapy. This study will be helpful for valid therapy and prevention of metastasis to illustrate the mechanism of NPC metastasis at the molecular level, and thus provide a new theoretical basis for prevention and treatment.