HPV-18 E6 Oncoprotein and Its Spliced Isoform E6*I Regulate the Wnt/β-Catenin Cell Signaling Pathway through the TCF-4 Transcriptional Factor

Abstract

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/β-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/β-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and β-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with β-catenin to promote cell proliferation.

Keywords: HPV-18 E6; HPV-18 E6*I; TCF-4 transcription factor; Wnt/β-catenin signaling.

Figure 1

E6 and E6*I proteins induce TCF-4 transcriptional activity. (A) Expression of E6 and E6*I proteins was analyzed 48 h post-transfection in C33A cells, by western blot. (B) 18E6WT, 18E6SM, 18E6*I, 16E6, and β-catenin expressing vectors were transfected as indicated, with TOPFLASH (TCF-4 reporter plasmid) and β-galactosidase reporter plasmids in C33A cells. Luciferase reporter activity was measured 48 h post-transfection. Luciferase activities were compared with the empty vector or β-catenin plasmid. (C) Cyclin D1 and (D) Axin2 gene expression was evaluated by qPCR in E6 transfected cells. The means and ±SD of three independent experiments are depicted in each graph. Student t test was performed to evaluate the significant differences, the values are represented as * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 2

HPV-18 E6 and E6*I increase β-catenin and TCF-4 total protein levels. C33A cells were transfected with E6WT, E6SM, and E6*I expressing vectors. 48 h post-transfection, total cell lysates were analyzed by western blot. (A) β-catenin immunoblot and (B) densitometric analysis; (C) TCF-4 immunoblot; and (D) densitometric analysis. Data from three independent experiments were collected and graphed showing the mean and ±SD. t student analysis was performed, * p < 0.05, ** p < 0.001 and *** p < 0.0001 vs. empty vector values.

Figure 3

E6 proteins do not alter subcellular distribution of β-catenin or TCF-4. C33A cells were transfected with plasmids encoding 18E6WT, 18E6SM, or 18E6*I as indicated. 48 h post-transfection cells were fixed, and immunofluorescence stain was performed using specific antibodies against β-catenin (A) or TCF-4 (B) (Red) and FLAG (Green). Cells were also stained with DAPI (Blue) to visualize the nuclei. Images were acquired by confocal microscope. Data from three independent experiments were collected with a 63× objective oil immersion lens. Scale bar size 5 μm.

Figure 4

E6 and E6*I proteins interact with β-catenin and the transcriptional factor, TCF-4, in vivo and in vitro. C33A cells were transfected with different E6 expressing vectors, and 48 h post-transfection, protein lysates were obtained. (A) β-catenin and (B) TCF-4 were immunoprecipitated with the appropriate antibodies. The immuno-complexes were analyzed by Western blot using anti-β-catenin and anti-TCF-4 antibodies to detect the immunoprecipitated protein, and with an anti-FLAG to detect E6 proteins. Image shows a representative experiment of three performed. For comparison, 10% of protein used for immunoprecipitation (input) and the precipitation with an irrelevant IgG antibody (isotype) are shown. An overexposure of E6 proteins in shown in panel A. (C) Purified GST-18E6, GST-18E6*I, and GST-16E6 recombinant proteins were incubated with C33A protein extracts, while GST purified protein was used as a control. Immunoblots were performed using anti-β-catenin and anti-TCF-4 antibodies. 10% of protein extract was used as input. Lower panel shows Ponceau S red staining of a representative nitrocellulose membrane. Asterisks (*) show the E6 recombinant proteins.

Figure 5

HPV-18 E6 proteins augment TCF-4 stability. C33A cells were transfected with E6 expressing plasmids. 48 h post-transfection, 200 µg/mL of cycloheximide was added to the culture medium. Protein extracts were obtained at 0, 6, and 12 h after treatment. (A) A representative immunoblot is shown with the different treatments. In non-E6 transfected cells, the TCF-4 levels were diminished at 6 and 12 h post-treatment, in contrast to E6 expressing cells, where TCF-4 levels remained without change at 6 and 12 h. (B) Graph showing the data as the mean and ± SD of three independent experiments. One-way ANOVA and a Tukey’s post-hoc test, ** p < 0.001 versus empty vector values.

Figure 6

E6 proteins increase nuclear TCF-4 protein levels. (A) Representative immunoblot of TCF-4 and E6 proteins in nuclear and cytoplasmic soluble fractions of C33A cells transfected with E6 expressing plasmids. Lamin BI and GAPDH proteins were used as nuclear and cytoplasmic load controls, respectively. (B) A densitometric analysis of relative nuclear TCF-4 levels shows an increase of TCF-4 protein levels in the presence of the E6 proteins. Data from three independent experiments were collected and graphed showing the mean and ± SD. t student analysis was performed, * p < 0.05 vs. empty vector values.

Figure 7

E6 and E6*I of HPV-18 interact with the Sp5 promoter. C33A cells were co-transfected with β-catenin and E6 expressing plasmids as indicated: (A) E6 proteins increase Sp5 relative expression as shown by qPCR analysis; * p < 0.05, ** p < 0.01, compared to the empty vector; (B) Chromatin immunoprecipitation assay (ChIP) shows that 18E6 binds to the Sp5 promoter in vivo. Anti-HA antibody was used to detect E6-HA tagged protein, and anti-TCF-4 and anti-IgG antibodies were used as positive and isotype controls, respectively. 10% of input was analyzed. *** p < 0.001, of E6-HA compared to the empty vector. (C) C33A cells were transfected with 18E6WT, 18E6SM, or 18E6*I expressing plasmids, and 48 h post-transfection, a DNA pull-down assay was performed. As expected, in all the samples, TCF-4 interact with the Sp5 promoter probe in vitro. Interestingly, both E6 full-length and E6*I form a complex with the Sp5 promoter. The p53 transcriptional factor was used as a negative control since it does not bind to the Sp5 promoter. (D) Scheme showing the suggested interactions of E6 and E6*I of HPV-18 with the TCF-4 dependent promoter, proposing a possible mechanism of Wnt cell signaling regulation by E6 proteins.

Figure 8

HPV-18 E6 and E6*I alone or in combination with β-catenin increase cell proliferation. C33A cells were transfected with the indicated plasmids, and 24 h post-transfection were seeded into a 96 well plate. Then, experiments were assessed after 72 h either by (A) MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) or (B) Crystal violet assays. Data from three independent experiments were collected and graphed showing the mean and ± SD. t student analysis was performed, * p < 0.05, ** p < 0.001 and *** p < 0.0001 vs. empty vector values.

Figure 9

HPV-18 E6AsAi and E6Af variants modulate TCF4 transcriptional activity. (A) HPV-18 E6AsAi and E6Af protein expression in C33A transfected cells. (B) C33A cells were transfected with E6AsAi, E6Af, alone or combined with β-catenin expressing plasmids, and co-transfected with TCF-4 transcriptional reporter plasmid (TOPFLASH) and β-galactosidase reporter vector as indicated. (C) Cyclin D1 and (D) Axin2 gene expression was analyzed by qPCR. The means and ±SD of three independent experiments are depicted in each graph. Student t test was performed to evaluate the significant differences, the values are represented as * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 10

HPV-18 E6 variants interact in vivo with β-catenin and TCF-4. C33A cells were transfected with the E6 variant plasmids. 48 h post-transfection, cell lysates were collected and immunoprecipitated with anti-β-catenin (A,B) and anti-TCF-4 (C,D). Immunoblots show an interaction of E6 (A,C) and E6*I (B,D) with both proteins. Overexposures of β-catenin and E6 proteins are shown in panel B and C, respectively. Representative images are shown from three experiments performed. 10% of protein used for immunoprecipitation is indicated as input, and an irrelevant antibody was used (isotype).

Figure 10

HPV-18 E6 variants interact in vivo with β-catenin and TCF-4. C33A cells were transfected with the E6 variant plasmids. 48 h post-transfection, cell lysates were collected and immunoprecipitated with anti-β-catenin (A,B) and anti-TCF-4 (C,D). Immunoblots show an interaction of E6 (A,C) and E6*I (B,D) with both proteins. Overexposures of β-catenin and E6 proteins are shown in panel B and C, respectively. Representative images are shown from three experiments performed. 10% of protein used for immunoprecipitation is indicated as input, and an irrelevant antibody was used (isotype).