Molecular Mechanisms Of Cyclin D1 Gene Regulated By Epstein-Barr Virus Encoded Latent Membrane Protein 1 Via Transcription Factor EGFR And STAT3

Nasopharyngeal carcinoma (NPC) is a malignant tumor with remarkably distinctive geographic distribution and thirdly high morbility in south china. One of the unique features of NPC is its strong association with infection of Epstein-Barr Virus (EBV). Latent membrane protein 1 (LMP1) is the only one with oncogenic properties among EBV encoded proteins and its biological functions has involved in cell transformation, apoptosis, cell cycle locomotion and participating in invasion and metastasis of carcinomas. Our previous studies have been focused on signaling transduction pathways mediated by LMP1, we elucidated that LMP1 was involved in abnormal signaling pathway and by which multiple tumor biological functions brought about.Transcription factor (TF) is important core of signaling transduction research. Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane protein and belongs to receptor tyrosine kinases (RTKs). Recently, studies on nuclear translocation of the EGFR family (erbB-1/EGFR, erbB-3, and erbB-4) have greatly improved the knowledge of the biological function of cell surface receptors. It is reported that high level EGFR have been detected in the nucleus of many cancer cells, including those of skin, breast, cervix, bladder, and ovaries, and EGFR acting as a new transcriptional factor or co-activator affect on the target genes which closely related to cell cycle progressing or cell proliferation, and promote tumorigenesis and development. Signal transducer and activation of transcription 3 (STAT3) is a key molecule through which receptors of multiple cytokines and growth factors perform their function. After its activation with the phosphorylation of tyrosine 705(tyr 705), STAT3 forms dimmers with itself or with STAT1, translocates to nuclei and binds to specific DNA sequences, then, starts the transcription of multiple related downstream genes.Our previous research first found that LMP1 can finely regulated EGFR expression and increased phosphorylation of EGFR by recruiting tumor necrosis factor receptor associated factors (TRAFs) through its carboxterminal activating region 1(CTAR1) in NPC cells; then, discovered that LMP1 could regulate nuclear translocation of EGFR. It is reported that after epidermal growth factor (EGF) stimulate 10 minutes, EGFR could be found in nucleus with laser cofocal microscopy (LCFM). Meanwhile, some authors consider that EGFR educe its promoting proliferation needing to interact with other transcription factors which can directly bind to DNA. It has been previously reported that LMP1 can promote STAT3 705 phosphorylation then to be activated, and activated STAT3 translocate to nuclear immediately and accumulate in nuclear. Therefore, we presume LMP1 may regulate TF EGFR and STAT3 interaction through inducing them nuclear translocation. We chose LCFM to observe whether TF EGFR and STAT3 co-localization exists in NPC cells nuclear controlled under LMP1. The results showed EGFR and STAT3 co-localization was discovered in LMP1 positive expression cells CNE1-LMP1; and by co-immunoprecipitation (COIP)-western blot (WB) assay, it displayed that LMP1 can facilitate the complex combination of EGFR and STAT3; We performed COIP-WB again to part with kytoplasm and nuclear protein, and found that the main subcellular localization of EGFR and STAT3 complex combination together was in nuclei. The results above provided important experiment foundation for understanding the mechanism how LMP1 regulate NF EGFR and STAT3 in NPC cells.Regulatory system of cell cycle is the network in which changes often occur in tumorigenesis, especially for cyclin D1 which always become one of the commonest molecular targets. Cyclin D1 is one of the most important proteins during cell cycle from G1 to S phase in cancer cells. Currently, there is conclusive evidence that cyclin D1 is oncogene which can promote cell proliferation, but its gene transcription was regulated by many factors. Our previous studies showed that LMP1 can activate cyclin D1 expression, and found that EGFR could bind to directly cyclin D1 and cyclin E promoters and trans-activate the transcription of cyclin D1 and cyclin E.In order to investigate whether the target gene is cyclin D1 which LMP1 effect on by modulation of EGFR and STAT3, we used the CNE1 and CNE1-LMP1 NPC cells with LMP1 negative and positive expression as model, transferred cyclin D1 reporter plasmids to CNE1 and CNE1-LMP1 cells with liposome transient transfection methods, and luciferase reporter assays were carried out. Meanwhile, we also performed another reporter assays after transfering DZ1 which is deoxyribozyme /DNAzyme specificity for LMP1 to block LMP1 expression. It was exhibited that cyclin D1 promoter activity in CNE1-LMP1 was obviously higher than in CNE1 cells; the luciferase reporter activity transfected with DZ1 significantly lower than without transfection in CNE1-LMP1 cells. The results revealed that LMP1 could effectively control TF EGFR and STAT3 to trans-activate cyclin D1. Further reporter analysis showed that each group of reporter activity was markedly lower after transfection with EGFR siRNA and STAT3 siRNA into CNE1-LMP1 cells, compared with controlled group, P value<0.05. To explore whether LMP1 can regulate the transcription of cyclinD1 mRNA via modulating EGFR/STAT3, we designed and synthetized the cDNA primers of cyclin D1 and real-time PCR were carried out. The results suggested that LMP1 can impact the expression of cyclin Dl mRNA via EGFR and STAT3.Emerging evidences indicated that many membrane receptors can nuclear translocation, such as EGFR, HER-2, Fibroblast growth factor receptor (FGFR), HER-3. The mechanisms of how membrane receptors affect on gene targets have not been fully elucidate. Recent advances and our research let us think about:Is there the existence of EGFR and STAT3 consensus sequence in cyclin D1 promoter in NPC cells? What are the mechanisms in which LMP1 regulate cyclin D1 transcription through inducing EGFR and STAT3 bind to their respective binding site or consensus binding site in cyclin D1 promoter? We first analyzed the role that EGFR,STAT3 bind to each binding site of the promoter region in cyclin D1 and trans-activation of cyclin D1 modulated by LMP1. We found that LMP1 can enhance the EGFR and DNA binding activity of the promoter region of cyclin D1 only in CNE1-LMP1 cells detected by electrophoretic mobility shift assay(EMSA); Because previous works have confirmed that some small molecular inhibitors can block the activation of main location of phosphorylation of EGFR and STAT3, and nuclear translocation modulated by LMP1, we used small molecular inhibitors to aim directly at RTK AG1478 to obstruct EGFR phophorylation pathway and to restrict EGFR nuclear translocation, and the data showed that EGFR-DNA binding activity of the promoter region of cyclin Dl attenuated after being treated with AG1478 with EMS A in same cells. Likewise, we identified that STAT3-DNA binding activity of the promoter region of cyclin D1 was increased regulated by LMP1, and decreased after being treated with small molecular inhibitors aimed to STAT3, WHI-P131 and PD98059, to inhibit its phophorylation with EMS A in CNE1-LMP1 cells. Then, further reporter tests displayed that the cyclinDl promoter activity induced by LMP1 via EGFR and STAT3 could be inhibited differently degree by small molecular inhibitors which repressed the phophorylation of EGFR Tyr 1173, STAT3 Tyr 705 and Ser 727. These finding from right and reverse sides indicated the mechanism that LMP1 can enhance EGFR/STAT3 binding activity to their respective DNA site in the region of cyclin D1 promoter, and increase cycin D1 transcription.According to molecular biology and information technology, and referring to literatures, we presume that there is a putative consensus DNA sequence of EGFR/STAT3 in the region of cyclin D1 promoter. We conjectured that the nucleotides, TTCTATGAA was the putative consensus sequence and only found one such nucleotides sequence after checking promoter region of cyclin D1. PCR primers were designed according to the nucleotides sequence and Chromatin immunoprecipitation (CHIP), PCR, agarose gel electrophoresis assays were performed. The data suggested that the consensus DNA sequence of EGFR/STAT3 existed in the promoter region of cyclin D1 in CNE1-LMP1 and CNE1 cells in vivo. This consensus sequences may be the common feature of biological structure in NPC cells. To further understanding the LMP1 effect on the EGFR/STAT3 binding with consensus DNA sequences in cyclin D1 promoter region, we designed and synthesized biotin labeled probes containing consensus sequences with EGFR/STAT3 binding in cyclin D1 promoter region. Results showed that the binding activity of neucleoprotein with wild biotin labeled probes in CNE1-LMP1 cells was markedly higher than in CNE1 cells, and it revealed that LMP1 can regulate EGFR/STAT3 binding ability with consensus DNA in cyclin D1 promoter region through inducing EGFR cooperation with STAT3. To further confirm the role of consensus DNA in cyclin D1 promoter region, applying reporter test and point mutation technique in which we mutated the key nucleotides in the consensus sequence of EGFR/STAT3 in the region of cyclin D1 promoter to lead to TF EGFR/STAT3 could not bind with, the reporter results displayed that transcription activity of cyclin D1 promoter after point mutation obviously decreased about 1 fold compared with point mutation before, P value<0.05. The data suggested that LMP1 can induce the synergism of EGFR and STAT3 to trans-activate the activation of cyclin D1 promoter.In conclusion, the present study demonstrate that the co-localization of EGFR and STAT3 in the LMP1 positive expression NPC cells; it mainly occurred in nuclei of NPC cells in which the association of complex combination between EGFR and STAT3 was modulated by EBV-encoded oncoprotein LMP1; The main target gene that LMP1 effects on by regulating EGFR and STAT3 is cyclin D1; and LMP1 can activate the promoter activity of cyclinDl through mediating EGFR,STAT3 to bind to their consensus sequence DNA site, as well as to bind to their respective sites in promoter region of cyclin D1.This project for the first time observed that LMP1 can induce TF EGFR and STAT3 co-localization in the nuclei of NPC cells, and provides structure foundation for the integration of EGFR and STAT3 which are two different pathways activated by LMP1; it for also the firs time discovered that there is a consensus sequences binding site of EGFR/STAT3 in cyclin D1 promoter region in NPC cells, by which LMP1 can enhance EFGR/STAT3 binding ability with this binding site and trans-activating promoter activity of cyclin D1. These new discoveries elucidate the new mechanisms of EGFR and STAT3 participating in the abnormal progression of cell cycle from a new field of view of proteins interaction, hence, it also provides experimental evidences for understanding the function of EBV important oncoprotein LMP1, and for finding new treating targets of molecular treatment of NPC.