Regulation Of Vascular Endothelial Growth Factor Expression By EBV-LMP1 Via The JAK/STAT And MAPK/ERK Signaling Pathways

Regulation of Vascular Endothelial Growth Factor Expression by EBV-LMP1 via the JAK/STAT and MAPK/ERK signaling pathwaysVEGF was purified by Gospodarowicz et al(1989) and Ferrara and Henzel(1989) from the conditioned medium of bovine pituitary folliculo Stellate cells, which was subsequently found to be an angiogenic endothelial cell mitogen, VEGF play a significant role in tumor angiogenesis and permeability. VEGF binds to transmembrane receptor tyrosine kinases of endothelial cells. This activates endothelial-cell migration and proliferation, which is necessary for the formation of new blood vessels. Persistent expression of VEGF has been shown in an increasing number of human cancers, including gastric, kidney, breast and lung cancer. This indicates that VEGF is necessary for proliferation, migration and microvascular-tube formation.Nasopharyngeal carcinoma(NPC) is one of epithelial maliganancies with a high incidence in Southern China. NPC is distinctive among the head and neck caricinomas for its marked tendency to metastasis and invasion. It has been shown that at the time of diagnosis, 78.9% of NPC patients already have clinically detectable aggressive metastasis in the regional lymph nodes, so far there is still no effective treatment for NPC at the stage of metastasis. As a result, prognosis is poor and the 5-year survival rate is less than 50%. It has been known that all tumors must undergo angiogenesis or neovascularization in order to aquire nutrients for continued growth and metastatic spread. VEGF is the most important factor of inducers of angiogenesis. Recent studies showed that VEGF expression was elevated in primary NPC and metastatic VS chronic nasopharyngeal inflammation. However, the function and regulation mechanisms of VEGF in the progression and metastasis of NPC were incompletely understood. The most striking feature in the pathogenesis of NPC is its almost universal association with Epstein-Barr virus (EBV) infection. EBV infection in NPC is classified as latency typeâ…¡in which only a limited set of latent gene expression, e.g., EBNA1, EBNA2, EBNA3A, EBNA3C and LMP1, can be detected. Of the EBV-coding products, LMP1 has been proposed as a key molecule involved in cell transformation and subsequently tumor metastasis and invasion by affecting multiple cell metastatic factors. LMP1 CTAR triggers a series of signal transduction pathways. CTAT3 is responsible for the association of JAK3 and activation of the JAK3/STAT signal pathway.Our previous studies indicate that EB virus latent membrane protein I can regulate VEGF expression in the nasopharyngeal carcinoma cell lines via activating stat3 binding ability in VEGF-848 promoter binding site. STAT3, a member of JAK/STAT signaling pathway, is a latent transcriptional factor, which is activated by EBV-LMP1 in NPC. Therefore, as a key regulator of vascular endothelial growth factor (VEGF) expression, which is critical for neovascularization, STAT3 signalling also stimulates tumour angiogenesis. These findings suggest that STAT3 is a molecular target whose inhibition not only broadly prevents tumor cell production of angiogenic factors but also their effects on endothelial effects. And we confirm that there is a close relation among expression level of LMP1, activation of STAT3 signaling, and production of VEGF in invasive and metastatic NPC cells. But the regulation mechanism of VEGF by LMP1 in the metastasis of NPC was not completely understood.STAT3 is known to require phosphorylation on tyrosine 705 for activating and on serine 727 for maximal activation. Our previous work found that LMP1 is capable of activating STAT3 through a complex mechanism involving MAPK/ERK and JAK/STAT phthways which directly phosphorylate STAT3 at Ser727 and Tyr 705, respectively. LMP1 also stimulates STAT3 Tyr705-dependent nuclear accumulation. This has been demonstrated that activation of STAT3 in response to LMP1 is dependent on phosphorylation of both tyrosine and serine amino acid residues. This indicates that JAK3 and ERK are involved in LMP1-induced STAT3 transactivation. Next, we hypothesized that one or both of these two signaling pathways may mediate the elevated VEGF expression in NPC cells.On the basis of confirming the promotion of STAT3(Tyr705, Ser727) and nuclear translocation regulated by LMP1, we further investigated whether VEGF expression might be determined by LMP1-mediated mechanisms using specific inhibitor of LMP1-mediated STAT3 signalling pathways. The VEGF upstream components LMP1,JAK3,ERK and STAT3 are involved in LMP1-induced VEGF expression. To identify the signaling pathway responsible for VEGF expression, five inhibitors were studied: DZ1, WHI-P131, PD98059, STAT3 RNA interference (siRNA) and STAT3 antisense oligonucleotides (ASO), known to specifically inhibit LMP1, JAK3, MEK1 and STAT3 pathways, respectively. Treatment of LMP1-expressing cells with LMP1-targeted DNAzymes DZ1 induced a dramatic decrease of STAT3 phosphorylation as well as STAT transcriptional activities. WHI-P131, a JAK3 kinase inhibitor treatment for 24 hours could effectively inhibit LMP1-induced STAT3 Tyr705 phosphorylation in CNE1-LMP1, whereas STAT3 Ser727 phosphorylation and STAT3 protein level had not changed. LMP1-induced STAT3 Ser 727 phosphorylation in CNE1-LMP1 was abolished in the presence of the MAP/ERK(MEK) inhibitor PD98059 which had no effect on STAT3 Tyr705 phosphorylation and STAT3 protein expression. Furthermore, Luciferase reporter assays show that inhibiting LM 1-mediated upstream of STAT signalling efficiently inhibited the transactivation activatity of STAT induced by LMP1. We found that pretreatment of cells with WHI-P131 or PD98059 impairs the LMP1-induced STAT-dependent transcription activity through the decreased STAT3 phosphorylation. Western blot analysis of total STAT3 protein levels demonstrates that STAT3 antisense and siRNA were able to substantially diminish STAT3 protein expression as well as STAT3 phosphorylation.To determine if LMP1 functions in the regulation of VEGF expression, CNE1-LMP1 cells were treated with DNAzyme DZ1. Western blotting results indicated that DZ1 obiviously downregulate STAT3 phosphorylation in the LMP1-posibive cell line CNE1-LMP1, which has no effect on STAT3 expression. We next measured by ELISA the secretion of VEGF in the cell culture supernatants induced by LMP1. We found that VEGF secretion was abolished. To further confirm the involvement of the JAK3/STAT and MAPK/ERK pathways in LMP1-augmented VEGF expression, WHI-P131, a specific JAK3 kinases inhibitor and PD98059, a MEK1 inhibitor was used to investigate VEGF upregulation induced by LMP1. Treatment of CNE1-LMP1 cells with both inhibitor resulted in a dose-dependent suppression of VEGF induction by the LMP1, which is correlated with a dose-dependent attenuation of STAT3 phosphorylation induced by LMP1. We also checked the secretion of VEGF in the culture medium, LMP1 increased the level of VEGF protein in extracelluar fluid was also suppressed both by WHI-P131 and PD98059. A reduction of endogenous stat3 protein levels as a result of stat3 siRNA or antisense oligonucleotide transfer caused inhibition of VEGF expression and secretion.STAT3 is frequently overexpressed and constitutively activated by tyrosine phosphorylation during malignant transformation. Dispite the clear importance of STAT3 in the proliferation and survival in diverce human cancers, its possible contribution to tumor cell adhesion, motility and invasion is to be further investigated. In this study, we showed that inhibiting LMP1-mediated STAT3 signalling pathway significantly altered cell motility, substratum adhesion, and transmembrane invasion ablity. These results supporting an important role of stat3 signaling pathway induced by LMP1 in NPC metastasis.In conclusion, we demonstrated that LMP1 is capable of activating STAT3 phosphorylation and transcription activity by JAK3/STAT and MAPK/ERK pathways and STAT3 up-regulates VEGF expression through the VEGF gene promoter. JAK3/STAT and MAPK/ERK pathway is obligatory for LMP1-induced VEGF up-regulation amd secretion. As a key regulator of VEGF expression, which is critical for neovascularization, LMP1-mediated STAT3 signalling also stimulates tumour angiogenesis and metastasis. Our findings indicate that LMP1-mediated STAT3 signalling pathway is closely associated with the development, angiogenesis, metastasis of NPC, and STAT3 could be a useful molecular target for the therapertic development of NPC.