Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

Abstract

The principal Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1) is strongly associated with nasopharyngeal carcinoma (NPC), a prevalent cancer in China. The epidermal growth factor receptor (EGFR) is important in carcinogenesis, as it is a ubiquitously expressed receptor tyrosine kinase. Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis. Our previous study demonstrated that the nuclear EGFR could bind to the cyclin D1 promoter directly in the presence of LMP1, and the correlation between EGFR and STAT3 in NPC remains to be further explored. Here, we have shown that the interaction of EGFR and STAT3 increased in the nucleus in the presence of LMP1. LMP1 promoted both EGFR and STAT3 binding to the promoter region of cyclin D1, in turn, enhancing the promoter activity of cyclin D1. Furthermore, we demonstrated that both transcriptional activity and mRNA levels of cyclin D1 were decreased by small molecule interference of EGFR and STAT3 activity. These findings may provide a novel linkage between the EGFR and STAT3 signaling pathways and the activation of cyclin D1 by LMP1 in the carcinogenesis of NPC.

Figure 1

LMP1 affected the interaction of EGFR and STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-EGFR antibody (A) or anti-STAT3 antibody (B) and analyzed by Western blotting with a STAT3 and EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG). ®-actin were used as an internal control of Inuput. The bottom panels show the 50 μg of input materials. IP: immunoprecipitation, IB: immunoblot, kDa: kilodalton.

Figure 2

LMP1 induced co-localization of EGFR and STAT3 in the nucleus. Endogenous association of EGFR (A) with STAT3 (B) in NPC cells without or with LMP1 expression. Equal amounts of fractionated cellular proteins were immunoprecipitated with an anti-EGFR or anti-STAT3 antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, STAT3, nucleolin, and α-tubulin are shown as loading and fractionation controls. N: nuclear fraction, C: cytosolic fraction, IB: immunoblot.

Figure 3

Identification of an EGFR and STAT3 response element in the cyclin D1 promoter. (A) Schematic diagram of mutant cyclin D1 promoter constructs are shown. The expansion for EGFR and STAT3 binding site illustrates the wild-type sequence and frames the nucleotides replaced by mutations. (B-C) Dual luciferase-reporter assays were performed in LMP1-negative and LMP-positive CNE1 cells after co-transfection of a wild type or mutant cyclin D1 promoter-reporter construct, plasmids expressing wild-type EGFR or STAT3, and a Renilla luciferase transfection control plasmid. The fold induction by EGFR and STAT3 is displayed as the ratio of promoter activity obtained with wild-type compared to the DNA-binding mutant. (mean ± SD, n = 3, *p < 0.05, **p < 0.01). mt: mutation, wt: wild-type.

Figure 4

Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, *p < 0.05) (C) WHI-P131, PD98059 and AG1478 inhibited the activity of cyclin D1 induced by stable expression of LMP1. CNE1-LMP1 cells were transfected with a cyclin D1 promoter-reporter construct and a Renilla luciferase plasmid as an internal control. Twenty-four hrs. after transfection, the cells were treated with WHI-P131, PD98059, AG1478 or 0.1% DMSO for 2 hrs. The cells were harvested at 26 hrs. after transfection and subjected to the luciferase assay. An empty firefly reporter vector served as a control (n = 3). * p < 0.05. (D) WHI-P131, PD98059 and AG1478 inhibited the expression of cyclin D1 induced by stable expression of LMP1. The cells were harvested for Western Blot at 8 hrs. after the treatment of WHI-P131, PD98059, AG1478 or 0.1% DMSO. β-actin was served as an internal control.

Figure 5

LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro. (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.

Figure 6

Cyclin D1 expression is reduced in CNE1-LMP1 cells after treatment with EGFR siRNA and STAT3 siRNA. (A) Dual luciferase-reporter assays were performed in CNE1-LMP1 cells after co-transfection with either control siRNA (siControl), EGFR siRNA (siEGFR), or STAT3 siRNA (siSTAT3) in addition to cyclin D1 promoter-reporter constructs and a Renilla luciferase transfection control plasmid. Firefly luciferase was measured and normalized to Renilla luciferase activity. The fold change in cyclin D1 expression by the indicated siRNA is displayed in each case. The control siRNA served as a non-targeting control. (mean ± SD, n =3, *p < 0.05) (B) The cells were incubated with medium containing the indicated siRNAs for 72 h. Total RNA was isolated from the cells and subjected to real-time PCR, using specific primers designed to amplify cyclin D1. β-actin mRNA served as an internal control. (mean ± SD, n =3, *p < 0.05, **p < 0.01). (C) Western Blot was performed in CNE1-LMP1 cells after co-transfection with the indicated siRNAs for 72 h. β-actin was served as an internal control. (D) FACS was performed in CNE1 and CNE1-LMP1 cells after co-transfection with the indicated siRNAs for 72 h. The data are presented from three independent experiments.