EB病毒LMP1通过转录因子EGFR调节酪氨酰蛋白硫酸转移酶TPST-1介导趋化因子受体CXCR4活性的分子机制
|
摘要
鼻咽癌是以高转移性为突出特点的恶性肿瘤,大部分患者由于早期发生转移而预后不良。EB病毒(Epstein-Barr Virus, EBV)编码的主要致瘤蛋白即潜伏膜蛋白1 (latent membrane protein, LMP1)与鼻咽癌的发生和转移密切相关。
趋化因子影响肿瘤细胞的黏附、侵袭和转移,趋化因子及其受体参与肿瘤转移的机制尚未完全阐明。趋化因子SDF-1 (CXCL12)受体CXCR4表达和活化可促进肿瘤细胞向高表达SDF-1的部位(如骨和淋巴结)转移,与多种肿瘤的生长和转移相关。
最近几年,酪氨酸的硫酸化(tyrosine sulfation)作为一种重要的翻译后修饰(post-translational modification, PTM)的方式,因其对趋化因子受体活性调节而受到重视。酪氨酸的硫酸化的主要作用是促进分泌型蛋白的分泌或膜蛋白与其它蛋白特别是配体之间的相互作用。趋化因子受体CXCR4第21位的酪氨酸是其主要的硫酸化位点。体内负责催化酪氨酸硫酸化的酶为酪氨酰蛋白硫酸转移酶(tyrosylprotein sulfotransferase, TPST),编码该酶两种亚基的基因即TPST-1和TPST-2,都具有高度保守的硫酸转移酶区域,定位于高尔基体中的TPST-1可对酪氨酸进行硫酸化,参与酪氨酸的硫酸化循环。
我们前期工作发现,LMP1在鼻咽癌细胞中通过NF-κB信号通路上调EGFR的表达并使其发生磷酸化；作为一个新型的转录因子,磷酸化的EGFR转位入核可反式活化与细胞增殖有关的基因如cyclinD1和cyclinE。同时,我们通过生物信息学发现TPST-1基因(GenBank AF038009)5'UTR内存在一个可能的EGFR结合位点,即TGTTT(位于-28—-24位之间)。这一发现使我们有理由推测EGFR可能通过该酶催化CXCR4硫酸化从而影响其与配体的结合。因此,我们提出LMP1可能通过EGFR调节CXCR4的活性促进鼻咽癌细胞的转移。
1. CXCR4硫酸化促进鼻咽癌细胞的转移
酪氨酸的硫酸化作为一种重要的翻译后修饰的方式,其在趋化因子受体活性调节中发挥重要的作用。利用高转移的5-8F和不转移的6-10B鼻咽癌细胞为基本模型,研究发现高转移的5-8F细胞呈高硫酸化状态。提示,硫酸化可能与细胞的转移能力有关。
进一步利用硫酸化特异性抑制剂氯酸钠(sodium chlorate)抑制高转移的5-8F细胞蛋白硫酸化活性,通过趋化实验(chemotaxis)和Matrigel侵袭实验发现抑制5-8F细胞硫酸化可以抑制细胞的趋化活性和侵袭转移能力。同时利用不转移鼻咽癌6-10B细胞,转染野生型WT-CXCR4和突变型MUT-CXCR4表达质粒后,进行趋化实验和Matrigel侵袭实验,发现转染WT-CXCR4表达质粒后细胞的趋化活性和侵袭转移能力明显增强,而转染MUT-CXCR4表达质粒后并没影响细胞的趋化活性和侵袭转移能力。这些结果提示,细胞蛋白的硫酸化,特别是CXCR4的硫酸化与细胞的转移相关,而CXCR4第21位酪氨酸硫酸化在促进鼻咽癌细胞转移的过程中起到非常重要的作用。
我们检测高转移5-8F细胞和不转移6-10B细胞的TPST-1的表达发现,高转移5-8F细胞TPST-1的mRNA水平和蛋白水平均高于6-10B细胞。进一步抑制5-8F的TPST-1可以抑制其CXCR4的硫酸化水平,而在6-10B细胞中转染TPST-1表达质粒可以上调CXCR4的硫酸化水平。近年来,在人们发现细胞表面的膜受体EGFR和FGFR能够移位入核之后,新近有研究发现细胞膜表面受体CXCR4同样能够入核。我们首先检测高转移和不转移的5-8F细胞和6-10B细胞的CXCR4是否存在核定位,采用亚细胞组分结合Western Blot及激光共聚焦技术,从定性和定量水平均发现5-8F细胞核内存在CXCR4蛋白的表达。进一步利用特异性硫酸化抑制剂和TPST-1 siRNA处理5-8F细胞后,SDF-1α30nm刺激细胞30min,发现其细胞核内的CXCR4蛋白明显减少。提示,CXCR4的核移位与CXCR4的硫酸化密切相关。
2.EB病毒LMP1通过调节CXCR4功能活性促进鼻咽癌细胞转移
上一个部分我们在5-8F和6-10B细胞中研究发现CXCR4的硫酸化与鼻咽癌细胞的转移密切相关。我们实验室前期研究发现LMP1在高转移5-8F细胞的表达高于不转移6-10B细胞,结合LMP1在鼻咽癌细胞侵袭和转移过程中发挥重要作用,提示我们：在鼻咽癌细胞中,LMP1可能是通过对CXCR4硫酸化的调控,进而影响鼻咽癌的转移。
首先应用流式细胞术进一步确证高硫酸化的5-8F细胞高表达LMP1后,采用可调控LMP1表达的细胞系,本研究发现在鼻咽癌细胞系中LMP1可诱导CXCR4的表达,并呈剂量诱导效应。进一步采用免疫共沉淀将CXCR4沉淀分离后,Western Blot检测酪氨酸硫酸化水平即CXCR4硫酸化变化。结果发现,随DOX剂量的增加,CXCR4的硫酸化也随之增加。提示,LMP1能够上调CXCR4的硫酸化。同样我们应用Tet-on LMP1 HNE2细胞,用不同DOX剂量诱导24h后,在不同浓度的SDF-1α的作用下,进行趋化实验,结果发现LMP1能够上调CXCR4的趋化活性,说明LMP1通过上调CXCR4硫酸化进而影响其功能活性。
业已证实LMP1调控CXCR4的硫酸化及其功能活性,那么我们将探讨LMP1是否诱导CXCR4核移位。采用亚细胞组分结合Western Blot及激光共聚焦技术,我们从定性和定量水平均发现Tet-on LMP1HNE2细胞核内部分存在CXCR4的蛋白表达,且随LMP1表达的增加而增加。进一步我们在Tet-on LMP1 HNE2细胞中转染WT-CXCR4和MUT-CXCR4表达质粒24h后,分别在DOX (0,6ug/ml)剂量诱导下,采用亚细胞组分结合激光共聚焦技术,我们从定性水平发现LMP1促进了WT-CXCR4组细胞CXCR4蛋白的核积聚。这些结果表明LMP1通过调控CXCR4的硫酸化进而影响CXCR4的核移位。
最后利用HNE2-PSG5和HNE2-LMP1细胞,分别转染WT-CXCR4和MUT-CXCR4表达质粒24h后,Matrigel侵袭实验发现LMP1促进转染WT-CXCR4质粒组细胞的侵袭转移能力,并没有影响MUT-CXCR4质粒组细胞侵袭转移能力。进一步说明LMP1通过诱导CXCR4第21位酪氨酸硫酸化功能活性进而促进鼻咽癌细胞的转移。
3.EB病毒LMP1通过EGFR介导CXCR4硫酸化
我们的前期工作证实LMP1上调转录因子EGFR的表达,而且我们通过基因信息学发现酪氨酰蛋白硫酸转移酶TPST-1的启动子区存在EGFR的结合位点。因此我们推测LMP1有可能是通过EGFR介导TPST-1上调CXCR4的硫酸化。
采用鼻咽癌Tet-on LMP1 HNE2细胞,应用Real-time-PCR和Western Blot技术发现LMP1上调TPST-1的mRNA和蛋白水平,EGFR siRNA处理Tet-on LMP1 HNE2细胞24h阻断EGFR表达后,TPST-1表达下调,初步说明LMP1诱导的TPST-1是EGFR所依赖的。
采用ChIP发现,LMP1可以促进EGFR与TPST-1的结合,采用报告基因分析发现,LMP1增加TPST-1启动子活性,而突变EGFR与TPST-1的结合位点,我们发现LMP1增加的TPST-1启动子活性显著下调。进一步说明LMP1诱导的TPST-1和CXCR4的硫酸化是EGFR所依赖的。
小结：我们以鼻咽癌细胞系为基本实验模型,从比较具有不同转移潜能的鼻咽癌细胞系之间CXCR4硫酸化状态的差异入手,进行LMP1对CXCR4硫酸化及亚细胞定位和功能的研究,本研究主要采用阻断策略结合Western Blotting、趋化实验、侵袭实验、ChIP、体外定点突变等分子生物学实验方法,从蛋白硫酸化修饰的角度来阐明LMP1调节CXCR4的分子机制,取得如下重要的创新性发现：
1.首次发现CXCR4的硫酸化可以促进鼻咽癌细胞的转移。抑制高转移5-8F细胞的硫酸化可以抑制其CXCR4的趋化活性和细胞的侵袭转移能力,且转染野生型的CXCR4表达质粒可以促进不转移的6-10B细胞CXCR4的趋化活性和细胞侵袭转移能力。高转移鼻咽癌细胞高表达硫酸化转移酶TPST-1,且TPST-1siRNA可以有效的阻断CXCR4的硫酸化。首次发现鼻咽癌5-8F细胞核内存在CXCR4蛋白的表达。利用特异性硫酸化抑制剂和TPST-1siRNA抑制CXCR4的硫酸化可有效阻断5-8F细胞CXCR4蛋白核移位。
2.首次发现EB病毒LMP1上调CXCR4的硫酸化水平、功能活性和促进CXCR4蛋白的核移位,并呈一定剂量效应。且发现LMP1调控CXCR4核移位是通过其上调CXCR4的硫酸化实现的。同时发现LMP1上调CXCR4的硫酸化与鼻咽癌细胞转移相关。
3.首次发现LMP1可以上调TPST-1的mRNA和蛋白表达水平,并呈剂量依赖效应。EGFR siRNA可以抑制LMP1上调的TPST-1表达。在LMP1调控下,转录因子EGFR与TPST-1的启动子结合增强。并发现LMP1增加TPST-1启动子活性,突变EGFR在TPST-1启动子上的结合位点可以下调LMP1调控的TPST-1的启动子活性。
本论文以EBV的致瘤蛋白LMP1通过转录因子EGFR对酪氨酸硫酸化转移酶TPST-1的调控为切入点,从CXCR4硫酸化翻译后修饰的角度来阐明LMP1-EGFR-TPST-1-CXCR4信号转导通路在鼻咽癌转移中的分子机制及其生物学意义。通过本研究,将EB病毒与趋化因子及其受体通过信号转导联系起来,提出了新的工作模式。为EB病毒致瘤分子机理研究开拓一个新的视野,为靶向LMP1和CXCR4抗信号转导治疗肿瘤提供了新的实验依据,这将有助于从分子水平揭示鼻咽癌转移发生机制,为鼻咽癌的防治提供新的理论依据。
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 NPC
Post-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 EGFR
Our 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.
趋化因子影响肿瘤细胞的黏附、侵袭和转移,趋化因子及其受体参与肿瘤转移的机制尚未完全阐明。趋化因子SDF-1 (CXCL12)受体CXCR4表达和活化可促进肿瘤细胞向高表达SDF-1的部位(如骨和淋巴结)转移,与多种肿瘤的生长和转移相关。
最近几年,酪氨酸的硫酸化(tyrosine sulfation)作为一种重要的翻译后修饰(post-translational modification, PTM)的方式,因其对趋化因子受体活性调节而受到重视。酪氨酸的硫酸化的主要作用是促进分泌型蛋白的分泌或膜蛋白与其它蛋白特别是配体之间的相互作用。趋化因子受体CXCR4第21位的酪氨酸是其主要的硫酸化位点。体内负责催化酪氨酸硫酸化的酶为酪氨酰蛋白硫酸转移酶(tyrosylprotein sulfotransferase, TPST),编码该酶两种亚基的基因即TPST-1和TPST-2,都具有高度保守的硫酸转移酶区域,定位于高尔基体中的TPST-1可对酪氨酸进行硫酸化,参与酪氨酸的硫酸化循环。
我们前期工作发现,LMP1在鼻咽癌细胞中通过NF-κB信号通路上调EGFR的表达并使其发生磷酸化；作为一个新型的转录因子,磷酸化的EGFR转位入核可反式活化与细胞增殖有关的基因如cyclinD1和cyclinE。同时,我们通过生物信息学发现TPST-1基因(GenBank AF038009)5'UTR内存在一个可能的EGFR结合位点,即TGTTT(位于-28—-24位之间)。这一发现使我们有理由推测EGFR可能通过该酶催化CXCR4硫酸化从而影响其与配体的结合。因此,我们提出LMP1可能通过EGFR调节CXCR4的活性促进鼻咽癌细胞的转移。
1. CXCR4硫酸化促进鼻咽癌细胞的转移
酪氨酸的硫酸化作为一种重要的翻译后修饰的方式,其在趋化因子受体活性调节中发挥重要的作用。利用高转移的5-8F和不转移的6-10B鼻咽癌细胞为基本模型,研究发现高转移的5-8F细胞呈高硫酸化状态。提示,硫酸化可能与细胞的转移能力有关。
进一步利用硫酸化特异性抑制剂氯酸钠(sodium chlorate)抑制高转移的5-8F细胞蛋白硫酸化活性,通过趋化实验(chemotaxis)和Matrigel侵袭实验发现抑制5-8F细胞硫酸化可以抑制细胞的趋化活性和侵袭转移能力。同时利用不转移鼻咽癌6-10B细胞,转染野生型WT-CXCR4和突变型MUT-CXCR4表达质粒后,进行趋化实验和Matrigel侵袭实验,发现转染WT-CXCR4表达质粒后细胞的趋化活性和侵袭转移能力明显增强,而转染MUT-CXCR4表达质粒后并没影响细胞的趋化活性和侵袭转移能力。这些结果提示,细胞蛋白的硫酸化,特别是CXCR4的硫酸化与细胞的转移相关,而CXCR4第21位酪氨酸硫酸化在促进鼻咽癌细胞转移的过程中起到非常重要的作用。
我们检测高转移5-8F细胞和不转移6-10B细胞的TPST-1的表达发现,高转移5-8F细胞TPST-1的mRNA水平和蛋白水平均高于6-10B细胞。进一步抑制5-8F的TPST-1可以抑制其CXCR4的硫酸化水平,而在6-10B细胞中转染TPST-1表达质粒可以上调CXCR4的硫酸化水平。近年来,在人们发现细胞表面的膜受体EGFR和FGFR能够移位入核之后,新近有研究发现细胞膜表面受体CXCR4同样能够入核。我们首先检测高转移和不转移的5-8F细胞和6-10B细胞的CXCR4是否存在核定位,采用亚细胞组分结合Western Blot及激光共聚焦技术,从定性和定量水平均发现5-8F细胞核内存在CXCR4蛋白的表达。进一步利用特异性硫酸化抑制剂和TPST-1 siRNA处理5-8F细胞后,SDF-1α30nm刺激细胞30min,发现其细胞核内的CXCR4蛋白明显减少。提示,CXCR4的核移位与CXCR4的硫酸化密切相关。
2.EB病毒LMP1通过调节CXCR4功能活性促进鼻咽癌细胞转移
上一个部分我们在5-8F和6-10B细胞中研究发现CXCR4的硫酸化与鼻咽癌细胞的转移密切相关。我们实验室前期研究发现LMP1在高转移5-8F细胞的表达高于不转移6-10B细胞,结合LMP1在鼻咽癌细胞侵袭和转移过程中发挥重要作用,提示我们：在鼻咽癌细胞中,LMP1可能是通过对CXCR4硫酸化的调控,进而影响鼻咽癌的转移。
首先应用流式细胞术进一步确证高硫酸化的5-8F细胞高表达LMP1后,采用可调控LMP1表达的细胞系,本研究发现在鼻咽癌细胞系中LMP1可诱导CXCR4的表达,并呈剂量诱导效应。进一步采用免疫共沉淀将CXCR4沉淀分离后,Western Blot检测酪氨酸硫酸化水平即CXCR4硫酸化变化。结果发现,随DOX剂量的增加,CXCR4的硫酸化也随之增加。提示,LMP1能够上调CXCR4的硫酸化。同样我们应用Tet-on LMP1 HNE2细胞,用不同DOX剂量诱导24h后,在不同浓度的SDF-1α的作用下,进行趋化实验,结果发现LMP1能够上调CXCR4的趋化活性,说明LMP1通过上调CXCR4硫酸化进而影响其功能活性。
业已证实LMP1调控CXCR4的硫酸化及其功能活性,那么我们将探讨LMP1是否诱导CXCR4核移位。采用亚细胞组分结合Western Blot及激光共聚焦技术,我们从定性和定量水平均发现Tet-on LMP1HNE2细胞核内部分存在CXCR4的蛋白表达,且随LMP1表达的增加而增加。进一步我们在Tet-on LMP1 HNE2细胞中转染WT-CXCR4和MUT-CXCR4表达质粒24h后,分别在DOX (0,6ug/ml)剂量诱导下,采用亚细胞组分结合激光共聚焦技术,我们从定性水平发现LMP1促进了WT-CXCR4组细胞CXCR4蛋白的核积聚。这些结果表明LMP1通过调控CXCR4的硫酸化进而影响CXCR4的核移位。
最后利用HNE2-PSG5和HNE2-LMP1细胞,分别转染WT-CXCR4和MUT-CXCR4表达质粒24h后,Matrigel侵袭实验发现LMP1促进转染WT-CXCR4质粒组细胞的侵袭转移能力,并没有影响MUT-CXCR4质粒组细胞侵袭转移能力。进一步说明LMP1通过诱导CXCR4第21位酪氨酸硫酸化功能活性进而促进鼻咽癌细胞的转移。
3.EB病毒LMP1通过EGFR介导CXCR4硫酸化
我们的前期工作证实LMP1上调转录因子EGFR的表达,而且我们通过基因信息学发现酪氨酰蛋白硫酸转移酶TPST-1的启动子区存在EGFR的结合位点。因此我们推测LMP1有可能是通过EGFR介导TPST-1上调CXCR4的硫酸化。
采用鼻咽癌Tet-on LMP1 HNE2细胞,应用Real-time-PCR和Western Blot技术发现LMP1上调TPST-1的mRNA和蛋白水平,EGFR siRNA处理Tet-on LMP1 HNE2细胞24h阻断EGFR表达后,TPST-1表达下调,初步说明LMP1诱导的TPST-1是EGFR所依赖的。
采用ChIP发现,LMP1可以促进EGFR与TPST-1的结合,采用报告基因分析发现,LMP1增加TPST-1启动子活性,而突变EGFR与TPST-1的结合位点,我们发现LMP1增加的TPST-1启动子活性显著下调。进一步说明LMP1诱导的TPST-1和CXCR4的硫酸化是EGFR所依赖的。
小结：我们以鼻咽癌细胞系为基本实验模型,从比较具有不同转移潜能的鼻咽癌细胞系之间CXCR4硫酸化状态的差异入手,进行LMP1对CXCR4硫酸化及亚细胞定位和功能的研究,本研究主要采用阻断策略结合Western Blotting、趋化实验、侵袭实验、ChIP、体外定点突变等分子生物学实验方法,从蛋白硫酸化修饰的角度来阐明LMP1调节CXCR4的分子机制,取得如下重要的创新性发现：
1.首次发现CXCR4的硫酸化可以促进鼻咽癌细胞的转移。抑制高转移5-8F细胞的硫酸化可以抑制其CXCR4的趋化活性和细胞的侵袭转移能力,且转染野生型的CXCR4表达质粒可以促进不转移的6-10B细胞CXCR4的趋化活性和细胞侵袭转移能力。高转移鼻咽癌细胞高表达硫酸化转移酶TPST-1,且TPST-1siRNA可以有效的阻断CXCR4的硫酸化。首次发现鼻咽癌5-8F细胞核内存在CXCR4蛋白的表达。利用特异性硫酸化抑制剂和TPST-1siRNA抑制CXCR4的硫酸化可有效阻断5-8F细胞CXCR4蛋白核移位。
2.首次发现EB病毒LMP1上调CXCR4的硫酸化水平、功能活性和促进CXCR4蛋白的核移位,并呈一定剂量效应。且发现LMP1调控CXCR4核移位是通过其上调CXCR4的硫酸化实现的。同时发现LMP1上调CXCR4的硫酸化与鼻咽癌细胞转移相关。
3.首次发现LMP1可以上调TPST-1的mRNA和蛋白表达水平,并呈剂量依赖效应。EGFR siRNA可以抑制LMP1上调的TPST-1表达。在LMP1调控下,转录因子EGFR与TPST-1的启动子结合增强。并发现LMP1增加TPST-1启动子活性,突变EGFR在TPST-1启动子上的结合位点可以下调LMP1调控的TPST-1的启动子活性。
本论文以EBV的致瘤蛋白LMP1通过转录因子EGFR对酪氨酸硫酸化转移酶TPST-1的调控为切入点,从CXCR4硫酸化翻译后修饰的角度来阐明LMP1-EGFR-TPST-1-CXCR4信号转导通路在鼻咽癌转移中的分子机制及其生物学意义。通过本研究,将EB病毒与趋化因子及其受体通过信号转导联系起来,提出了新的工作模式。为EB病毒致瘤分子机理研究开拓一个新的视野,为靶向LMP1和CXCR4抗信号转导治疗肿瘤提供了新的实验依据,这将有助于从分子水平揭示鼻咽癌转移发生机制,为鼻咽癌的防治提供新的理论依据。
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 NPC
Post-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 EGFR
Our 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.
引文
[1]A. Ben-Baruch, The multifaceted roles of chemokines in malignancy, Cancer Metastasis Rev, 2006,25(3),357-71.
[2]H. Kulbe, N. R. Levinson, F. Balkwill et al., The chemokine network in cancer-much more than directing cell movement, Int J Dev Biol,2004,48(5-6),489-96.
[3]A. Orimo, P. B. Gupta, D. C. Sgroi et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion, Cell,2005,121(3),335-48.
[4]M. Cotton, and A. Claing, G protein-coupled receptors stimulation and the control of cell migration, Cell Signal,2009,21(7),1045-53.
[5]F. Balkwill, The significance of cancer cell expression of the chemokine receptor CXCR4, Semin Cancer Biol,2004,14(3),171-9.
[6]A. Krohn, Y. H. Song, F. Muehlberg et al., CXCR4 receptor positive spheroid forming cells are responsible for tumor invasion in vitro, Cancer Lett,2009,280(1),65-71.
[7]J. I. Lee, B. H. Jin, M. A. Kim et al., Prognostic significance of CXCR-4 expression in oral squamous cell carcinoma, Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2009,107(5), 678-84.
[8]H. J. Lee, S. W. Kim, H. Y. Kim et al., Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer, Int J Oncol,2009,34(2),473-80.
[9]K. Sasaki, S. Natsugoe, S. Ishigami et al., Expression of CXCL12 and its receptor CXCR4 in esophageal squamous cell carcinoma, Oncol Rep,2009,21(1),65-71,
[10]Y. Lee, S. J. Kim, H. D. Park et al., PAUF functions in the metastasis of human pancreatic cancer cells and upregulates CXCR4 expression, Oncogene,2009.
[11]A. Muller, B. Homey, H. Soto et al., Involvement of chemokine receptors in breast cancer metastasis, Nature,2001,410(6824),50-6.
[12]H. Tsutsumi, T. Tanaka, N. Ohashi et al., Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents, Biopolymers,2007,88(2),279-89.
[13]A. Zlotnik, Chemokines and cancer, Int J Cancer,2006,119(9),2026-9.
[14]J. A. Burger, and T. J. Kipps, CXCR4:a key receptor in the crosstalk between tumor cells and their microenvironment, Blood,2006,107(5),1761-7.
[15]M. Farzan, T. Mirzabekov, P. Kolchinsky et al., Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry, Cell,1999,96(5),667-76.
[16]J. Gutierrez, L. Kremer, A. Zaballos et al., Analysis of post-translational CCR8 modifications and their influence on receptor activity, J Biol Chem,2004,279(15),14726-33.
[17]M. Farzan, G. J. Babcock, N. Vasilieva et al., The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry, J Biol Chem,2002,277(33),29484-9.
[18]C. Seibert, M. Cadene, A. Sanfiz et al., Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence, Proc Natl Acad Sci U S A,2002,99(17),11031-6.
[19]J. R. Bundgaard, J. W. Sen, A. H. Johnsen et al., Analysis of tyrosine-O-sulfation, Methods Mol Biol,2008,446,47-66.
[20]J. Liu, S. Louie, W. Hsu et al., Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease, Am J Respir Cell Mol Biol,2008,38(6),738-43.
[21]C. Seibert, and T. P. Sakmar, Toward a framework for sulfoproteomics:Synthesis and characterization of sulfotyrosine-containing peptides, Biopolymers,2008,90(3),459-77.
[22]P. A. Baeuerle, and W. B. Huttner, Tyrosine sulfation of yolk proteins 1,2, and 3 in Drosophila melanogaster, J Biol Chem,1985,260(10),6434-9.
[23]M. J. Stone, S. Chuang, X. Hou et al., Tyrosine sulfation:an increasingly recognised post-translational modification of secreted proteins, N Biotechnol,2009,25(5),299-317.
[24]J. W. Kehoe, and C. R. Bertozzi, Tyrosine sulfation:a modulator of extracellular protein-protein interactions, Chem Biol,2000,7(3), R57-61.
[25]R. A. Colvin, G. S. Campanella, L. A. Manice et al., CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis, Mol Cell Biol,2006,26(15),5838-49.
[26]F. Monigatti, B. Hekking, and H. Steen, Protein sulfation analysis-A primer, Biochim Biophys Acta,2006,1764(12),1904-13.
[27]Y. Ouyang, W. S. Lane, and K. L. Moore, Tyrosylprotein sulfotransferase:purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins, Proc Natl Acad Sci U S A,1998,95(6), 2896-901.
[28]R. Beisswanger, D. Corbeil, C. Vannier et al., Existence of distinct tyrosylprotein sulfotransferase genes:molecular characterization of tyrosylprotein sulfotransferase-2, Proc Natl Acad Sci U S A,1998,95(19),11134-9.
[29]S. Goettsch, R. A. Badea, J. W. Mueller et al., Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment, J Mol Biol,2006,361(3), 436-49.
[30]S. Hemmerich, D. Verdugo, and V. L. Rath, Strategies for drug discovery by targeting sulfation pathways, Drug Discov Today,2004,9(22),967-75.
[31]S. H. Lee, and M. Hannink, Molecular mechanisms that regulate transcription factor localization suggest new targets for drug development, Adv Drug Deliv Rev,2003,55(6), 717-31.
[32]Y. Tao, X. Song, X. Deng et al., Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1, Exp Cell Res,2005,303(2),240-51.
[33]S. U. Woo, J. W. Bae, C. H. Kim et al., A significant correlation between nuclear CXCR4 expression and axillary lymph node metastasis in hormonal receptor negative breast cancer, Ann Surg Oncol,2008,15(1),281-5.
[34]J. Hu, X. Deng, X. Bian et al., The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma, Clin Cancer Res, 2005,11(13),4658-65.
[35]S. E. Ong, B. Blagoev, I. Kratchmarova et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol Cell Proteomics,2002,1(5),376-86.
[36]E. K. Frow, J. Reckless, and D. J. Grainger, Tools for anti-inflammatory drug design:in vitro models of leukocyte migration, Med Res Rev,2004,24(3),276-98.
[37]L. H. Wang, Q. Liu, B. Xu et al., Identification of nuclear localization sequence of CXCR4 in renal cell carcinoma by constructing expression plasmids of different deletants, Plasmid, 2010,63(1),68-72.
[38]W. H.-m. SONG Li-bing, ZHENG Mu-sheng, et al., Study on tumor heterogeneity of Nasopharyngeal Carcinoma cell line (SUNE 1) [J], Chinese Journal of Cancer,1998,17(5), 324-327.
[39]Y. J. i. SONG Li-bing, WANG Hui-min, et al., Molecular mechanisms of tumorgenesis and metastasis in nasopharyngeal carcinoma cell sublines [J]. Chinese Journal of Cancer, 2002,21(2),158-162.().
[40]A. Albini, Y. Iwamoto, H. K. Kleinman et al., A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res,1987,47(12),3239-45.
[41]L. B. Song, J. Li, W. T. Liao et al., The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells, J Clin Invest,2009,119(12),3626-36.
[42]R. Horuk, Chemokine receptors, Cytokine Growth Factor Rev,2001,12(4),313-35.
[43]A. P. Vicari, and C. Caux, Chemokines in cancer, Cytokine Growth Factor Rev,2002,13(2), 143-54.
[44]M. Przybylski, A. Kozlowska, P. P. Pietkiewicz et al., Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B, Biomed Pharmacother,2009.
[45]L. Kubarek, A. Kozlowska, M. Przybylski et al., Down-regulation of CXCR4 expression by tamoxifen is associated with DNA methyltransferase 3B up-regulation in MCF-7 breast cancer cells, Biomed Pharmacother,2009,63(8),586-91.
[46]A. Zlotnik, Involvement of chemokine receptors in organ-specific metastasis, Contrib Microbiol,2006,13,191-9.
[47]A. M. Oliveira, D. A. Maria, M. Metzger et al., Thalidomide treatment down-regulates SDF-1 alpha and CXCR4 expression in multiple myeloma patients, Leuk Res,2009,33(7), 970-3.
[48]D. L. Ou, C. L. Chen, S. B. Lin et al., Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy, J Pathol, 2006,210(3),363-73.
[49]K. L. Moore, Protein tyrosine sulfation:a critical posttranslation modification in plants and animals, Proc Natl Acad Sci U S A,2009,106(35),14741-2.
[50]H. Lemjabbar-Alaoui, A. van Zante, M. S. Singer et al., Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis, Oncogene,29(5),635-46.
[51]N. Wang, Q. L. Wu, Y. Fang et al., Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome,,J Transl Med,2005,3,26.
[52]I. K. Na, C. Scheibenbogen, C. Adam et al., Nuclear expression of CXCR4 in tumor cells of non-small cell lung cancer is correlated with lymph node metastasis, Hum Pathol,2008,39(12), 1751-5.
[53]J. S. Pagano, Epstein-Barr virus:the first human tumor virus and its role in cancer, Proc Assoc Am Physicians,1999,111(6),573-80.
[54]R. A. Harris, A. Yang, R. C. Stein et al., Cluster analysis of an extensive human breast cancer cell line protein expression map database, Proteomics,2002,2(2),212-23.
[55]V. E. Bichsel, L. A. Liotta, and E. F. Petricoin,3rd, Cancer proteomics:from biomarker discovery to signal pathway profiling, Cancer J,2001,7(1),69-78.
[56]G. Niedobitek, N. Meru, and H. J. Delecluse, Epstein-Barr virus infection and human malignancies, Int J Exp Pathol,2001,82(3),149-70.
[57]T. Yoshizaki, Promotion of metastasis in nasopharyngeal carcinoma by Epstein-Barr virus latent membrane protein-1, Histol Histopathol,2002,17(3),845-50.
[58]A. Fendri, A. Khabir, B. Hadhri-Guiga et al., Overexpression of COX-2 and LMP1 are correlated with lymph node in Tunisian NPC patients, Oral Oncol,2008,44(7),710-5.
[59]X. M. Gou, Y. Chen, X. Y. Chen et al, Effects of Epstein-Barr virus latent membrane protein 1(EBV-LMP1) on related factors of metastasis of nasopharyngeal carcinoma cell line CNE1, Ai Zheng 2003,22(5),481-5.
[60]G. R. Yan, W. Luo, X. J. Luo et al., Epstein-Barr virus-encoded latent membrane protein 1 mediates serine-phosphorylation of annexin I by activating protein kinase C, Ai Zheng, 2007,26(7),679-82 ().
[61]H. D. Liu, H. Zheng, M. Li et al., Upregulated expression of kappa light chain by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-kappaB and AP-1 pathways, Cell Signal,2007,19(2),419-27.
[62]B. A. Mainou, D. N. Everly, Jr., and N. Raab-Traub, Epstein-Barr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K, Oncogene,2005,24(46),6917-24.
[63]D. Dudziak, A. Kieser, U. Dirmeier et al., Latent membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-kappaB signaling pathway, J Virol,2003,77(15),8290-8.
[64]Q. Tao, L. S. Young, C. B. Woodman et al., Epstein-Barr virus (EBV) and its associated human cancers-genetics, epigenetics, pathobiology and novel therapeutics, Front Biosci, 2006,11,2672-713.
[65]H. Zheng, L. L. Li, D. S. Hu et al., Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma, Cell Mol Immunol,2007,4(3), 185-96.
[66]Y. Tao, X. Song, Y. Tan et al., Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1, Sci China C Life Sci,2004,47(3), 258-67.
[67]Y. G. Tao, Y. N. Tan, Y. P. Liu et al., Epstein-Barr virus latent membrane protein 1 modulates epidermal growth factor receptor promoter activity in a nuclear factor kappa B-dependent manner, Cell Signal,2004,16(7),781-90.
[68]Z. Cai, J. Tang, X. Li et al., [Expressions of matrix metalloproteinase-9 and interleukin-8 in nasopharygeal carcinoma and association with Epstein-Barr virus latent membrane protein-1], Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2007,21(21),966-9.
[69]H. Takeshita, T. Yoshizaki, W. E. Miller et al., Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2, J Virol, 1999,73(7),5548-55.
[70]G. X. Zeng L, Tang FQ, et al, Metastasis-associated gene in nasopharyngeal carcinoma by comparative genomic hybridization and cDNA array, Chinese Journal of Biochemistry and Molecular Biology,2002, (18),588-593.
[71]M. M. Rosenkilde, Virus-encoded chemokine receptors-putative novel antiviral drug targets, Neuropharmacology,2005,48(1),1-13.
[72]S. Kondo, S. Y. Seo, T. Yoshizaki et al., EBV latent membrane protein 1 up-regulates hypoxia-inducible factor 1alpha through Siahl-mediated down-regulation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells, Cancer Res,2006,66(20),9870-7.
[73]M. Luftig, T. Yasui, V. Soni et al., Epstein-Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKK alpha-dependent noncanonical NF-kappaB activation, Proc Natl Acad Sci U S A,2004,101(1),141-6.
[74]T. Nakayama, R. Fujisawa, D. Izawa et al, Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5, J Virol, 2002,76(6),3072-7.
[75]J. Li, X. S. Zhang, D. Xie et al., Expression of immune-related molecules in primary EBV-positive Chinese nasopharyngeal carcinoma:associated with latent membrane protein 1 (LMP1) expression, Cancer Biol Ther,2007,6(12),1997-2004.
[76]H. Y. W. Liao, X.Y. Li, M. Tang, H.H. Gu, Y. Cao, Doxycycline dependent expression of Epstein-Barr virus latent membrane protein 1 with tet regulating system in nasopharygeal carcinoma cell line, Shengwu Huaxue Yu Shengwu Wuli Xuebao (Shanghai).1999,31(3), 309-312.
[77]N. B. Schwartz, M. Cortes, and L. A. King, Got sulfate? Luring axons this way and that, Chem Biol,2007,14(2),119-20.
[78]K. L. Moore, The biology and enzymology of protein tyrosine O-sulfation, J Biol Chem, 2003,278(27),24243-6.
[79]G. Walsh, and R. Jefferis, Post-translational modifications in the context of therapeutic proteins, Nat Biotechnol,2006,24(10),1241-52.
[80]L. Su, J. Zhang, H. Xu et al., Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells, Clin Cancer Res,2005,11(23), 8273-80.
[81]T. Onoue, D. Uchida, N. M. Begum et al., Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells, Int J Oncol,2006,29(5),1133-8.
[82]T. Kobayashi, K. Honke, S. Gasa et al., Epidermal growth factor elevates the activity levels of glycolipid sulfotransferases in renal-cell-carcinoma cells, Int J Cancer,1993,55(3),448-52.
[83]E. A. Nigg, Nucleocytoplasmic transport:signals, mechanisms and regulation, Nature, 1997,386(6627),779-87.
[84]A. Kaffman, N. M. Rank, E. M. O'Neill et al., The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus, Nature,1998,396(6710),482-6.
[85]R. Ben-Levy, S. Hooper, R. Wilson et al., Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2, Curr Biol,1998,8(19),1049-57.
[86]R. R. Rowland, and D. Yoo, Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences, Virus Res,2003,95(1-2),23-33.
[87]A. Wells, and U. Marti, Signalling shortcuts:cell-surface receptors in the nucleus?, Nat Rev Mol Cell Biol,2002,3(9),697-702.
[88]J. F. Reilly, and P. A. Maher, Importin beta-mediated nuclear import of fibroblast growth factor receptor:role in cell proliferation, J Cell Biol,2001,152(6),1307-12.
[89]S. Y. Lin, K. Makino, W. Xia et al., Nuclear localization of EGF receptor and its potential new role as a transcription factor, Nat Cell Biol,2001,3(9),802-8.
[90]J. B. Cordero, M. Cozzolino, Y. Lu et al.,1,25-Dihydroxyvitamin D down-regulates cell membrane growth-and nuclear growth-promoting signals by the epidermal growth factor receptor, J Biol Chem,2002,277(41),38965-71.
[91]M. A. Olayioye, R. M. Neve, H. A. Lane et al., The ErbB signaling network:receptor heterodimerization in development and cancer, EMBO J,2000,19(13),3159-67.
[92]Robin, The Oncogene Factsbook, San Diego:Acadenic Press,1996 (1996).
[93]P. Duchek, and P. Rorth, Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis, Science,2001,291(5501),131-3.
[94]C. Gutacker, G. Klock, P. Diel et al., Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells, Biochem J,1999,339 (Pt 3),759-66.
[95]Z. Q. Xiao, and A. P. Majumdar, Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging, Am J Physiol Gastrointest Liver Physiol,2000,278(6), G855-65.
[96]R.-T. N. Miller WE, The EGFR as a target for viral oncoproteins, Trends Microbiol,1999,7, 453-458.
[97]T. Y. Tan YN, Song X, et al, Expression of JAK3 in nasopharyngeal carcinoma cell line associated with STAT activation regulated by EB virus encoded protein LMP1, Prog Biochem Biophys Res Commun,2003,30,560-565.
[98]L. Deng, J. Yang, X. R. Zhao et al., Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1, Cell Res, 2003,13(3),187-94.
[99]D. L. Zhao XR, Weng XX, et al, Further study on cyclin D1 expression and function in human nasopgaryngeal carcinoma cell line Zhonghua Zhong Liu Za Zhi,2001,23,373-375.
[100]T. Y. Song X, Zeng L, et al, Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line, Virus Res,2005,113,89-99.
[101]S. C. Wang, Y. Nakajima, Y. L. Yu et al., Tyrosine phosphorylation controls PCNA function through protein stability, Nat Cell Biol,2006,8(12),1359-68.
[102]N. Cabioglu, Y. Gong, R. Islam et al., Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer, Ann Oncol,2007,18(6),1021-9.
[103]R. J. Phillips, J. Mestas, M. Gharaee-Kermani et al., Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1 alpha, J Biol Chem, 2005,280(23),22473-81
[104]T. Y. Tan YN, Song X, et al, Expression of JAK3 in nasopharyngeal carcinoma cell line associated with STAT activation regulated by EB virus encoded protein LMP1, Prog Biochem Biophys,2003,30, 560-565.
[105]C. Porcile, A. Bajetto, S. Barbero et al., CXCR4 activation induces epidermal growth factor receptor transactivation in an ovarian cancer cell line, Ann N Y Acad Sci,2004,1030,162-9.
[1]Christofori G. New signals from the invasive front[J]. Nature,2006,441(7092):444-450.
[2]Hay ED. The extracellular matrix in development and regeneration[J]. An interview with Elizabeth D. Hay. Int J Dev Biol,2004,48(8-9):687-694.
[3]Huber MA, Beug H, Wirth T. Epithelial-mesenchymal transition:NF-kappaB takes center stage[J]. Cell Cycle,2004,3(12):1477-1480.
[4]Thiery JP. Epithelial-mesenchymal transitions in tumour progression[J]. Nat Rev Cancer, 2002,2(6):442-454.
[5]Guarino M. Epithelial-mesenchymal transition and tumour invasion[J]. Int J Biochem Cell Biol,2007,39(12):2153-2160.
[6]Christiansen JJ, Rajasekaran AK. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J]. Cancer Res,2006,66(17):8319-8326.
[7]Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer:do myofibroblasts come also from epithelial cells via EMT[J]?. J Cell Biochem,2007,101(4):830-839.
[8]Bates RC, DeLeo MJ, Mercuri AM. The epithelial-mesenchymal transition of colon carcinoma involves expression of IL-8 and CXCR-1-mediated chemotaxis[J]. Exp Cell Res,2004,299(2): 315-324.
[9]Onoue T, et al. Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells[J]. Int J Oncol,2006,29(5): 1133-1138.
[10]Robson EJ, et al. Epithelial-to-mesenchymal transition confers resistance to apoptosis in three murine mammary epithelial cell lines[J]. Differentiation,2006,74(5):254-264.
[11]Jethwa P, et al. Overexpression of Slug is associated with malignant progression of esophageal adenocarcinoma[J]. World J Gastroenterol,2008,14(7):1044-1052.
[12]Yang J, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis[J]. Cell,2004,117(7):927-939.
[13]Lee TK., et al. Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition[J]. Clin Cancer Res,2006,12(18): 5369-5376.
[14]Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior[J]. Annu Rev Cell Dev Biol,2001,17:463-516.
[15]Orlichenko LS, Radisky DC. Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development[J]. Clin Exp Metastasis.2008;25(6):593-600.
[16]Przybylo JA, Radisky DC. Matrix metalloproteinase-induced epithelial-mesenchymal
transition:tumor progression at Snail's pace[J]. Int J Biochem Cell Biol,2007,39(6): 1082-1088.
[17]JiaoWJ, et al. Effect of endothelin-1 in esophageal squamous cell carcinoma invasion and its correlation with cathepsin B[J]. World J Gastroenterol,2007,13(29):4002-4005.
[18]Rosano L, et al. Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression[J]. Exp Biol Med (Maywood),2006,231(6):1128-1131.
[19]Yoshizaki T. Promotion of metastasis in nasopharyngeal carcinoma by Epstein-Barr virus latent membrane protein-1[J]. Histol Histopathol,2002,17(3):845-850.
[20]Zheng H, et al. Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma[J]. Cell Mol Immunol,2007,4(3):185-196.
[21]Horikawa T, et al. Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma[J]. Cancer Res,2007,67(5):1970-1978.
[22]Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression[J]. Cancer Sci,2007,98(10):1512-1520.
[23]Avizienyte E, et al. Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition[J]. Mol Biol Cell,2004,15(6):2794-2803.
[24]Bachelder RE, et al. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription:implications for the epithelial-mesenchymal transition[J]. J Cell Biol,2005, 168(1):29-33.
[25]Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis[J]. Am J Pathol,2001,159(4):1465-1475.
[26]Brabletz T, et al. Invasion and metastasis in colorectal cancer:epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin[J]. Cells Tissues Organs,2005,179(1-2):56-65.
[2]H. Kulbe, N. R. Levinson, F. Balkwill et al., The chemokine network in cancer-much more than directing cell movement, Int J Dev Biol,2004,48(5-6),489-96.
[3]A. Orimo, P. B. Gupta, D. C. Sgroi et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion, Cell,2005,121(3),335-48.
[4]M. Cotton, and A. Claing, G protein-coupled receptors stimulation and the control of cell migration, Cell Signal,2009,21(7),1045-53.
[5]F. Balkwill, The significance of cancer cell expression of the chemokine receptor CXCR4, Semin Cancer Biol,2004,14(3),171-9.
[6]A. Krohn, Y. H. Song, F. Muehlberg et al., CXCR4 receptor positive spheroid forming cells are responsible for tumor invasion in vitro, Cancer Lett,2009,280(1),65-71.
[7]J. I. Lee, B. H. Jin, M. A. Kim et al., Prognostic significance of CXCR-4 expression in oral squamous cell carcinoma, Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2009,107(5), 678-84.
[8]H. J. Lee, S. W. Kim, H. Y. Kim et al., Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer, Int J Oncol,2009,34(2),473-80.
[9]K. Sasaki, S. Natsugoe, S. Ishigami et al., Expression of CXCL12 and its receptor CXCR4 in esophageal squamous cell carcinoma, Oncol Rep,2009,21(1),65-71,
[10]Y. Lee, S. J. Kim, H. D. Park et al., PAUF functions in the metastasis of human pancreatic cancer cells and upregulates CXCR4 expression, Oncogene,2009.
[11]A. Muller, B. Homey, H. Soto et al., Involvement of chemokine receptors in breast cancer metastasis, Nature,2001,410(6824),50-6.
[12]H. Tsutsumi, T. Tanaka, N. Ohashi et al., Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents, Biopolymers,2007,88(2),279-89.
[13]A. Zlotnik, Chemokines and cancer, Int J Cancer,2006,119(9),2026-9.
[14]J. A. Burger, and T. J. Kipps, CXCR4:a key receptor in the crosstalk between tumor cells and their microenvironment, Blood,2006,107(5),1761-7.
[15]M. Farzan, T. Mirzabekov, P. Kolchinsky et al., Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry, Cell,1999,96(5),667-76.
[16]J. Gutierrez, L. Kremer, A. Zaballos et al., Analysis of post-translational CCR8 modifications and their influence on receptor activity, J Biol Chem,2004,279(15),14726-33.
[17]M. Farzan, G. J. Babcock, N. Vasilieva et al., The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry, J Biol Chem,2002,277(33),29484-9.
[18]C. Seibert, M. Cadene, A. Sanfiz et al., Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence, Proc Natl Acad Sci U S A,2002,99(17),11031-6.
[19]J. R. Bundgaard, J. W. Sen, A. H. Johnsen et al., Analysis of tyrosine-O-sulfation, Methods Mol Biol,2008,446,47-66.
[20]J. Liu, S. Louie, W. Hsu et al., Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease, Am J Respir Cell Mol Biol,2008,38(6),738-43.
[21]C. Seibert, and T. P. Sakmar, Toward a framework for sulfoproteomics:Synthesis and characterization of sulfotyrosine-containing peptides, Biopolymers,2008,90(3),459-77.
[22]P. A. Baeuerle, and W. B. Huttner, Tyrosine sulfation of yolk proteins 1,2, and 3 in Drosophila melanogaster, J Biol Chem,1985,260(10),6434-9.
[23]M. J. Stone, S. Chuang, X. Hou et al., Tyrosine sulfation:an increasingly recognised post-translational modification of secreted proteins, N Biotechnol,2009,25(5),299-317.
[24]J. W. Kehoe, and C. R. Bertozzi, Tyrosine sulfation:a modulator of extracellular protein-protein interactions, Chem Biol,2000,7(3), R57-61.
[25]R. A. Colvin, G. S. Campanella, L. A. Manice et al., CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis, Mol Cell Biol,2006,26(15),5838-49.
[26]F. Monigatti, B. Hekking, and H. Steen, Protein sulfation analysis-A primer, Biochim Biophys Acta,2006,1764(12),1904-13.
[27]Y. Ouyang, W. S. Lane, and K. L. Moore, Tyrosylprotein sulfotransferase:purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins, Proc Natl Acad Sci U S A,1998,95(6), 2896-901.
[28]R. Beisswanger, D. Corbeil, C. Vannier et al., Existence of distinct tyrosylprotein sulfotransferase genes:molecular characterization of tyrosylprotein sulfotransferase-2, Proc Natl Acad Sci U S A,1998,95(19),11134-9.
[29]S. Goettsch, R. A. Badea, J. W. Mueller et al., Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment, J Mol Biol,2006,361(3), 436-49.
[30]S. Hemmerich, D. Verdugo, and V. L. Rath, Strategies for drug discovery by targeting sulfation pathways, Drug Discov Today,2004,9(22),967-75.
[31]S. H. Lee, and M. Hannink, Molecular mechanisms that regulate transcription factor localization suggest new targets for drug development, Adv Drug Deliv Rev,2003,55(6), 717-31.
[32]Y. Tao, X. Song, X. Deng et al., Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1, Exp Cell Res,2005,303(2),240-51.
[33]S. U. Woo, J. W. Bae, C. H. Kim et al., A significant correlation between nuclear CXCR4 expression and axillary lymph node metastasis in hormonal receptor negative breast cancer, Ann Surg Oncol,2008,15(1),281-5.
[34]J. Hu, X. Deng, X. Bian et al., The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma, Clin Cancer Res, 2005,11(13),4658-65.
[35]S. E. Ong, B. Blagoev, I. Kratchmarova et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol Cell Proteomics,2002,1(5),376-86.
[36]E. K. Frow, J. Reckless, and D. J. Grainger, Tools for anti-inflammatory drug design:in vitro models of leukocyte migration, Med Res Rev,2004,24(3),276-98.
[37]L. H. Wang, Q. Liu, B. Xu et al., Identification of nuclear localization sequence of CXCR4 in renal cell carcinoma by constructing expression plasmids of different deletants, Plasmid, 2010,63(1),68-72.
[38]W. H.-m. SONG Li-bing, ZHENG Mu-sheng, et al., Study on tumor heterogeneity of Nasopharyngeal Carcinoma cell line (SUNE 1) [J], Chinese Journal of Cancer,1998,17(5), 324-327.
[39]Y. J. i. SONG Li-bing, WANG Hui-min, et al., Molecular mechanisms of tumorgenesis and metastasis in nasopharyngeal carcinoma cell sublines [J]. Chinese Journal of Cancer, 2002,21(2),158-162.().
[40]A. Albini, Y. Iwamoto, H. K. Kleinman et al., A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res,1987,47(12),3239-45.
[41]L. B. Song, J. Li, W. T. Liao et al., The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells, J Clin Invest,2009,119(12),3626-36.
[42]R. Horuk, Chemokine receptors, Cytokine Growth Factor Rev,2001,12(4),313-35.
[43]A. P. Vicari, and C. Caux, Chemokines in cancer, Cytokine Growth Factor Rev,2002,13(2), 143-54.
[44]M. Przybylski, A. Kozlowska, P. P. Pietkiewicz et al., Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B, Biomed Pharmacother,2009.
[45]L. Kubarek, A. Kozlowska, M. Przybylski et al., Down-regulation of CXCR4 expression by tamoxifen is associated with DNA methyltransferase 3B up-regulation in MCF-7 breast cancer cells, Biomed Pharmacother,2009,63(8),586-91.
[46]A. Zlotnik, Involvement of chemokine receptors in organ-specific metastasis, Contrib Microbiol,2006,13,191-9.
[47]A. M. Oliveira, D. A. Maria, M. Metzger et al., Thalidomide treatment down-regulates SDF-1 alpha and CXCR4 expression in multiple myeloma patients, Leuk Res,2009,33(7), 970-3.
[48]D. L. Ou, C. L. Chen, S. B. Lin et al., Chemokine receptor expression profiles in nasopharyngeal carcinoma and their association with metastasis and radiotherapy, J Pathol, 2006,210(3),363-73.
[49]K. L. Moore, Protein tyrosine sulfation:a critical posttranslation modification in plants and animals, Proc Natl Acad Sci U S A,2009,106(35),14741-2.
[50]H. Lemjabbar-Alaoui, A. van Zante, M. S. Singer et al., Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis, Oncogene,29(5),635-46.
[51]N. Wang, Q. L. Wu, Y. Fang et al., Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma:pattern of expression and correlation with clinical outcome,,J Transl Med,2005,3,26.
[52]I. K. Na, C. Scheibenbogen, C. Adam et al., Nuclear expression of CXCR4 in tumor cells of non-small cell lung cancer is correlated with lymph node metastasis, Hum Pathol,2008,39(12), 1751-5.
[53]J. S. Pagano, Epstein-Barr virus:the first human tumor virus and its role in cancer, Proc Assoc Am Physicians,1999,111(6),573-80.
[54]R. A. Harris, A. Yang, R. C. Stein et al., Cluster analysis of an extensive human breast cancer cell line protein expression map database, Proteomics,2002,2(2),212-23.
[55]V. E. Bichsel, L. A. Liotta, and E. F. Petricoin,3rd, Cancer proteomics:from biomarker discovery to signal pathway profiling, Cancer J,2001,7(1),69-78.
[56]G. Niedobitek, N. Meru, and H. J. Delecluse, Epstein-Barr virus infection and human malignancies, Int J Exp Pathol,2001,82(3),149-70.
[57]T. Yoshizaki, Promotion of metastasis in nasopharyngeal carcinoma by Epstein-Barr virus latent membrane protein-1, Histol Histopathol,2002,17(3),845-50.
[58]A. Fendri, A. Khabir, B. Hadhri-Guiga et al., Overexpression of COX-2 and LMP1 are correlated with lymph node in Tunisian NPC patients, Oral Oncol,2008,44(7),710-5.
[59]X. M. Gou, Y. Chen, X. Y. Chen et al, Effects of Epstein-Barr virus latent membrane protein 1(EBV-LMP1) on related factors of metastasis of nasopharyngeal carcinoma cell line CNE1, Ai Zheng 2003,22(5),481-5.
[60]G. R. Yan, W. Luo, X. J. Luo et al., Epstein-Barr virus-encoded latent membrane protein 1 mediates serine-phosphorylation of annexin I by activating protein kinase C, Ai Zheng, 2007,26(7),679-82 ().
[61]H. D. Liu, H. Zheng, M. Li et al., Upregulated expression of kappa light chain by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-kappaB and AP-1 pathways, Cell Signal,2007,19(2),419-27.
[62]B. A. Mainou, D. N. Everly, Jr., and N. Raab-Traub, Epstein-Barr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K, Oncogene,2005,24(46),6917-24.
[63]D. Dudziak, A. Kieser, U. Dirmeier et al., Latent membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-kappaB signaling pathway, J Virol,2003,77(15),8290-8.
[64]Q. Tao, L. S. Young, C. B. Woodman et al., Epstein-Barr virus (EBV) and its associated human cancers-genetics, epigenetics, pathobiology and novel therapeutics, Front Biosci, 2006,11,2672-713.
[65]H. Zheng, L. L. Li, D. S. Hu et al., Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma, Cell Mol Immunol,2007,4(3), 185-96.
[66]Y. Tao, X. Song, Y. Tan et al., Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1, Sci China C Life Sci,2004,47(3), 258-67.
[67]Y. G. Tao, Y. N. Tan, Y. P. Liu et al., Epstein-Barr virus latent membrane protein 1 modulates epidermal growth factor receptor promoter activity in a nuclear factor kappa B-dependent manner, Cell Signal,2004,16(7),781-90.
[68]Z. Cai, J. Tang, X. Li et al., [Expressions of matrix metalloproteinase-9 and interleukin-8 in nasopharygeal carcinoma and association with Epstein-Barr virus latent membrane protein-1], Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2007,21(21),966-9.
[69]H. Takeshita, T. Yoshizaki, W. E. Miller et al., Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2, J Virol, 1999,73(7),5548-55.
[70]G. X. Zeng L, Tang FQ, et al, Metastasis-associated gene in nasopharyngeal carcinoma by comparative genomic hybridization and cDNA array, Chinese Journal of Biochemistry and Molecular Biology,2002, (18),588-593.
[71]M. M. Rosenkilde, Virus-encoded chemokine receptors-putative novel antiviral drug targets, Neuropharmacology,2005,48(1),1-13.
[72]S. Kondo, S. Y. Seo, T. Yoshizaki et al., EBV latent membrane protein 1 up-regulates hypoxia-inducible factor 1alpha through Siahl-mediated down-regulation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells, Cancer Res,2006,66(20),9870-7.
[73]M. Luftig, T. Yasui, V. Soni et al., Epstein-Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKK alpha-dependent noncanonical NF-kappaB activation, Proc Natl Acad Sci U S A,2004,101(1),141-6.
[74]T. Nakayama, R. Fujisawa, D. Izawa et al, Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5, J Virol, 2002,76(6),3072-7.
[75]J. Li, X. S. Zhang, D. Xie et al., Expression of immune-related molecules in primary EBV-positive Chinese nasopharyngeal carcinoma:associated with latent membrane protein 1 (LMP1) expression, Cancer Biol Ther,2007,6(12),1997-2004.
[76]H. Y. W. Liao, X.Y. Li, M. Tang, H.H. Gu, Y. Cao, Doxycycline dependent expression of Epstein-Barr virus latent membrane protein 1 with tet regulating system in nasopharygeal carcinoma cell line, Shengwu Huaxue Yu Shengwu Wuli Xuebao (Shanghai).1999,31(3), 309-312.
[77]N. B. Schwartz, M. Cortes, and L. A. King, Got sulfate? Luring axons this way and that, Chem Biol,2007,14(2),119-20.
[78]K. L. Moore, The biology and enzymology of protein tyrosine O-sulfation, J Biol Chem, 2003,278(27),24243-6.
[79]G. Walsh, and R. Jefferis, Post-translational modifications in the context of therapeutic proteins, Nat Biotechnol,2006,24(10),1241-52.
[80]L. Su, J. Zhang, H. Xu et al., Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells, Clin Cancer Res,2005,11(23), 8273-80.
[81]T. Onoue, D. Uchida, N. M. Begum et al., Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells, Int J Oncol,2006,29(5),1133-8.
[82]T. Kobayashi, K. Honke, S. Gasa et al., Epidermal growth factor elevates the activity levels of glycolipid sulfotransferases in renal-cell-carcinoma cells, Int J Cancer,1993,55(3),448-52.
[83]E. A. Nigg, Nucleocytoplasmic transport:signals, mechanisms and regulation, Nature, 1997,386(6627),779-87.
[84]A. Kaffman, N. M. Rank, E. M. O'Neill et al., The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus, Nature,1998,396(6710),482-6.
[85]R. Ben-Levy, S. Hooper, R. Wilson et al., Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2, Curr Biol,1998,8(19),1049-57.
[86]R. R. Rowland, and D. Yoo, Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences, Virus Res,2003,95(1-2),23-33.
[87]A. Wells, and U. Marti, Signalling shortcuts:cell-surface receptors in the nucleus?, Nat Rev Mol Cell Biol,2002,3(9),697-702.
[88]J. F. Reilly, and P. A. Maher, Importin beta-mediated nuclear import of fibroblast growth factor receptor:role in cell proliferation, J Cell Biol,2001,152(6),1307-12.
[89]S. Y. Lin, K. Makino, W. Xia et al., Nuclear localization of EGF receptor and its potential new role as a transcription factor, Nat Cell Biol,2001,3(9),802-8.
[90]J. B. Cordero, M. Cozzolino, Y. Lu et al.,1,25-Dihydroxyvitamin D down-regulates cell membrane growth-and nuclear growth-promoting signals by the epidermal growth factor receptor, J Biol Chem,2002,277(41),38965-71.
[91]M. A. Olayioye, R. M. Neve, H. A. Lane et al., The ErbB signaling network:receptor heterodimerization in development and cancer, EMBO J,2000,19(13),3159-67.
[92]Robin, The Oncogene Factsbook, San Diego:Acadenic Press,1996 (1996).
[93]P. Duchek, and P. Rorth, Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis, Science,2001,291(5501),131-3.
[94]C. Gutacker, G. Klock, P. Diel et al., Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells, Biochem J,1999,339 (Pt 3),759-66.
[95]Z. Q. Xiao, and A. P. Majumdar, Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging, Am J Physiol Gastrointest Liver Physiol,2000,278(6), G855-65.
[96]R.-T. N. Miller WE, The EGFR as a target for viral oncoproteins, Trends Microbiol,1999,7, 453-458.
[97]T. Y. Tan YN, Song X, et al, Expression of JAK3 in nasopharyngeal carcinoma cell line associated with STAT activation regulated by EB virus encoded protein LMP1, Prog Biochem Biophys Res Commun,2003,30,560-565.
[98]L. Deng, J. Yang, X. R. Zhao et al., Cells in G2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-kappaB and AP-1, Cell Res, 2003,13(3),187-94.
[99]D. L. Zhao XR, Weng XX, et al, Further study on cyclin D1 expression and function in human nasopgaryngeal carcinoma cell line Zhonghua Zhong Liu Za Zhi,2001,23,373-375.
[100]T. Y. Song X, Zeng L, et al, Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line, Virus Res,2005,113,89-99.
[101]S. C. Wang, Y. Nakajima, Y. L. Yu et al., Tyrosine phosphorylation controls PCNA function through protein stability, Nat Cell Biol,2006,8(12),1359-68.
[102]N. Cabioglu, Y. Gong, R. Islam et al., Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer, Ann Oncol,2007,18(6),1021-9.
[103]R. J. Phillips, J. Mestas, M. Gharaee-Kermani et al., Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1 alpha, J Biol Chem, 2005,280(23),22473-81
[104]T. Y. Tan YN, Song X, et al, Expression of JAK3 in nasopharyngeal carcinoma cell line associated with STAT activation regulated by EB virus encoded protein LMP1, Prog Biochem Biophys,2003,30, 560-565.
[105]C. Porcile, A. Bajetto, S. Barbero et al., CXCR4 activation induces epidermal growth factor receptor transactivation in an ovarian cancer cell line, Ann N Y Acad Sci,2004,1030,162-9.
[1]Christofori G. New signals from the invasive front[J]. Nature,2006,441(7092):444-450.
[2]Hay ED. The extracellular matrix in development and regeneration[J]. An interview with Elizabeth D. Hay. Int J Dev Biol,2004,48(8-9):687-694.
[3]Huber MA, Beug H, Wirth T. Epithelial-mesenchymal transition:NF-kappaB takes center stage[J]. Cell Cycle,2004,3(12):1477-1480.
[4]Thiery JP. Epithelial-mesenchymal transitions in tumour progression[J]. Nat Rev Cancer, 2002,2(6):442-454.
[5]Guarino M. Epithelial-mesenchymal transition and tumour invasion[J]. Int J Biochem Cell Biol,2007,39(12):2153-2160.
[6]Christiansen JJ, Rajasekaran AK. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J]. Cancer Res,2006,66(17):8319-8326.
[7]Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer:do myofibroblasts come also from epithelial cells via EMT[J]?. J Cell Biochem,2007,101(4):830-839.
[8]Bates RC, DeLeo MJ, Mercuri AM. The epithelial-mesenchymal transition of colon carcinoma involves expression of IL-8 and CXCR-1-mediated chemotaxis[J]. Exp Cell Res,2004,299(2): 315-324.
[9]Onoue T, et al. Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells[J]. Int J Oncol,2006,29(5): 1133-1138.
[10]Robson EJ, et al. Epithelial-to-mesenchymal transition confers resistance to apoptosis in three murine mammary epithelial cell lines[J]. Differentiation,2006,74(5):254-264.
[11]Jethwa P, et al. Overexpression of Slug is associated with malignant progression of esophageal adenocarcinoma[J]. World J Gastroenterol,2008,14(7):1044-1052.
[12]Yang J, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis[J]. Cell,2004,117(7):927-939.
[13]Lee TK., et al. Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition[J]. Clin Cancer Res,2006,12(18): 5369-5376.
[14]Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior[J]. Annu Rev Cell Dev Biol,2001,17:463-516.
[15]Orlichenko LS, Radisky DC. Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development[J]. Clin Exp Metastasis.2008;25(6):593-600.
[16]Przybylo JA, Radisky DC. Matrix metalloproteinase-induced epithelial-mesenchymal
transition:tumor progression at Snail's pace[J]. Int J Biochem Cell Biol,2007,39(6): 1082-1088.
[17]JiaoWJ, et al. Effect of endothelin-1 in esophageal squamous cell carcinoma invasion and its correlation with cathepsin B[J]. World J Gastroenterol,2007,13(29):4002-4005.
[18]Rosano L, et al. Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression[J]. Exp Biol Med (Maywood),2006,231(6):1128-1131.
[19]Yoshizaki T. Promotion of metastasis in nasopharyngeal carcinoma by Epstein-Barr virus latent membrane protein-1[J]. Histol Histopathol,2002,17(3):845-850.
[20]Zheng H, et al. Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma[J]. Cell Mol Immunol,2007,4(3):185-196.
[21]Horikawa T, et al. Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma[J]. Cancer Res,2007,67(5):1970-1978.
[22]Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression[J]. Cancer Sci,2007,98(10):1512-1520.
[23]Avizienyte E, et al. Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition[J]. Mol Biol Cell,2004,15(6):2794-2803.
[24]Bachelder RE, et al. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription:implications for the epithelial-mesenchymal transition[J]. J Cell Biol,2005, 168(1):29-33.
[25]Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis[J]. Am J Pathol,2001,159(4):1465-1475.
[26]Brabletz T, et al. Invasion and metastasis in colorectal cancer:epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin[J]. Cells Tissues Organs,2005,179(1-2):56-65.