Transcriptional factor 3 binds to sirtuin 1 to activate the Wnt/β-catenin signaling in cervical cancer.

Transcriptional factor 3 (TCF3, also termed E2A), first reported to exert crucial functions during lymphocyte development, has been revealed to participate in the pathogenesis of human cancers. The aim of this work was to investigate the function of TCF3 in cervical cancer (CC) and the molecular interactions. The bioinformatics prediction suggested that TCF3 was highly expressed in CC and linked to poor prognosis. Increased TCF3 expression was identified in CC cell lines, and its downregulation reduced proliferation and migration of CC cells in vitro as well as growth of xenograft tumors in vivo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the TCF-3-related genes and genes showed differential expression between CC and normal tissues were mainly enriched in the Wnt/β-catenin pathway. TCF3 bound to sirtuin 1 (SIRT1) promoter for transcriptional activation, and SIRT1 promoted deacetylation and nuclear translocation of β-catenin in CC. SIRT1 overexpression blocked the role of TCF3 silencing and restored cell proliferation in vitro and tumor growth in vivo. Treatment with XAV-939, a β-catenin inhibitor, significantly suppressed the cell proliferation and tumor growth induced by SIRT1 overexpression. In conclusion, this study demonstrates that TCF3 augments progression of CC by activating SIRT1-mediated β-catenin signaling.

Highlights

TCF3 is possibly associated with unfavorable prognosis of CC.

TCF3 downregulation reduces activity of CC cells in vitro.

TCF3 binds to the promoter of SIRT1 to activate its transcription.

TCF3 bind to SIRT1 to promote nuclear accumulation of β-catenin.

TCF3/SIRT1/β-catenin mediates CC development in vivo.

Introduction

Cervical cancer (CC) ranks the fourth most common malignancy and the fourth leading cause of cancer-related death in females globally [1]. Human papillomavirus (HPV) infection remains the major cause for over 99% of CC cases [2]. The virus induces activation of oncoproteins including E5, E6, and E7, which prolongs cell-cycle progression, delays differentiation and suppresses apoptosis of host keratinocyte cell to induce carcinogenesis [2]. Fortunately, CC is now preventable via human papillomavirus (HPV) vaccination in young adolescent girls and cervical screening, and the incidence rate is projected to be significantly decreased in the coming years [3]. However, the morbidity of CC in low-income regions remains high [4]. Approximately 80% cases occur in developing nations, and the mortality is reported to be 10-fold higher than that in developed nations [5]. For the diagnosed patients, approximately 13% of them were found at advanced stages at the first diagnosis [6]. For patients with metastatic disease, the 5-year survival rate was 16.5%, but the rate of the localized cancer was 91.5% in Europe [7]. Patients at early stage or with locally advanced disease are candidates for receiving conventional therapies including surgery, radiotherapy, and chemotherapy, but standard treatment for metastatic disease is limited due to the heterogeneous manifestations [6]. Therefore, there is an urgent need in the development of novel therapeutic options for CC.

Transcriptional factor 3 (TCF3, also termed E2A) belongs to the basic helix loop helix (bHLH) proteins which encode two alternatively spliced variants E12 and E47 [8]. TCF3 protein exerts its cell type- or tissue-specific functions by forming a homodimer or a heterodimer with other bHLH proteins [9]. TCF3 was at first suggested to exert crucial functions during lymphocyte development [10], and studies have reported its involvement in the pathogenesis of several human malignancies, such as prostate cancer [11], breast cancer [12] and nasopharyngeal carcinoma [13]. Elevated expression of TCF3 has been associated with poor prognosis in CC [14]. However, TCF3 has also been reported to function as a tumor suppressor in endometrial cancer [15]. The exact role of TCF3 in CC aroused our interest. In the present study, the integrated bioinformatic analyses suggested that there is a potential molecular cascade comprising TCF3, sirtuin 1 (SIRT1) and catenin beta 1 (CTNNB1, β-catenin) possibly implicated in the development of CC. SIRT1 belongs to the SIRTs family [16]. It is a nicotinamide adenine dinucleotide 1-dependent class-III histone deacetylase (HDAC) which participates in key biological processes, such as metabolism, oncogenesis, and tumor progression [17,18]. SIRT1 modifies deacetylation of many substrates involved in aging and disease [19-21]. Restoration of SIRT1 has been reported to be correlated with deacetylation and nuclear translocation of β-catenin [22]. β-catenin is a core member of the Wnt signaling pathway, and the evolutionary Wnt/β-catenin pathway plays significant roles in embryonic development, but its hyperactivation is crucial for tumorigenesis, including the development of CC [23]. Taken together, we hypothesized that there might be a TCF3/SIRT1/β-catenin axis that is involved in the pathogenesis of CC. We aimed to explore the expression profiles of TCF3, SIRT1, and β-catenin in CC and determine their roles in proliferation, migration, invasiveness, and apoptosis of two CC cell lines by performing gain- and loss-of-function studies.

Materials and methods

Pan-cancer gene expression analysis

The pan-cancer gene expression analysis was performed via the Oncomine database (https://www.oncomine.org/resource/login.html). Differential gene expression between CC and normal tissues was analyzed using two Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) datasets. The GSE7803 dataset comprises 10 normal cervical squamous epithelium samples and 21 invasive squamous cell carcinoma samples. The GSE9750 dataset comprises that 33 primary CC samples and 24 normal cervical epithelium samples. The distribution image of TCF3 in CC tissues was downloaded from the Human Protein Atlas system (https://www.proteinatlas.org/). The levels and prognostic values of genes in patients were obtained via The Cancer Genome Atlas (TCGA, https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga).

Cell culture and treatment

Human CC cell lines CaSki, SiHa, C-33A, SW756, and HeLa, and a human immortalized cervical epithelial cell-line End1/E6E7 were procured from American Type Culture Collection (Manassas, VA, USA). The C-33A, SW756, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). CaSki cells were incubated in Roswell Park Memorial Institute-1640 (Sigma-Aldrich). All media contained 10% inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Jiangsu, China). The culture condition was 37°C with 5% CO2.

Small interfering (si) RNA of TCF3 (si-TCF3), overexpression plasmid of SIRT1 (SIRT1-OE) and the negative control (NC) plasmids were provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The plasmids were transfected into cells using a Lipofectamine 3000 kit (Thermo Fisher Scientific) at a ratio of 1:2.5 (RNA: Lipofectamine 3000). After 48 h, the cells were treated with 10 μM EX527 (MedChemExpress, Co., Ltd., NJ, USA), a SIRT1 inhibitor, for 24 h to suppress SIRT1 activity, or treated with a β-catenin inhibitor XAV-939 (2 μM, MedChemExpress) for 24 h to suppress β-catenin accumulation in nucleus. Cells treated with dimethyl sulfoxide (DMSO) were set to controls.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated using the TRIzol Reagent (Thermo Fisher Scientific). Reverse transcription was conducted using a PrimeScript RT Reagent Kit (Takara Biotechnology Ltd., Dalian, China). Thereafter, real-time qPCR was conducted using the TB Green® Premix Ex TaqTM II kit (Takara) in accordance with the instruction manual. The 2−ΔΔCt method was used to evaluate gene expression. The primer sequences are listed below: TCF3: 5’-CCAGACCAAACTGCTCATCCTG-3’ (forward), 5’-TCGCCGTTTCAAACAGGCTGCT-3’ (reverse); SIRT1: 5’-TAGACACGCTGGAACAGGTTGC-3’ (forward), 5’-CTCCTCGTACAGCTTCACAGTC-3’ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5’-GCGAGATCCCGCTAACATCA-3’ (forward); 5’-CTCGTGGTTCACACCCATCA-3’ (reverse).

Colony formation assay

Cells (2 × 102) were cultured in each well of 12-well plates supplemented with complete medium. The medium was renewed every week. After 15 days, colonies formed by cancer cells were fixed with formaldehyde and stained with crystal violet (0.2 g/L). The count of cell colonies was calculated under the microscope [24].

Transwell assay

Migration and invasiveness of cells were determined using the Millicell cell culture inserts (8 μm in diameter; Merck Millipore, Darmstadt, Germany). Cells were seeded in apical chambers at 1 × 105 cells per well, to which 200 μL serum-free medium was added. The basolateral chambers were added with 500 μL 10% FBS-supplemented DMEM for migration induction. Invasiveness of cells was examined in a similar manner with the additional pro-coating of Matrigel on the Transwell apical chambers. After incubation at 37°C for 48 h, migratory or invasive cells at the lower membranes were fixed and stained with 0.5% crystal violet. The counts of migratory or invasive cells in five random fields were calculated under the microscope [25].

Flow cytometry

Apoptosis of cells was examined using flow cytometry. After transfection, exponentially growing cells were collected and resuspended in binding buffer. The Annexin-V-FLUOS (Roche Ltd., Basel, Switzerland) was used to identify apoptotic cells. The cells were stained with Annexin V-fluorescein isothiocyanate/propidium iodide at 23°C avoiding light exposure for 15 min. The fluorescence signal was obtained using FACSCanto (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo 8.7.1 (Ashland, OR, USA) [26].

Dual luciferase reporter gene assay

The protein sequence of TCF3 was obtained from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/), secondary structure was predicted from SMART (http://smart.embl-heidelberg.de/), and the tertiary structure was downloaded from the structure summary MMDB database (https://ncbiinsights.ncbi.nlm.nih.gov/tag/mmdb/). The MMDB ID was 179,555, which was obtained via the X-ray diffraction by Wojnarowicz PM et al. [27]. The pmir-GLO vector containing firefly luciferase (LUC) reporter gene was transfected into tumor cells. After 48 h, the activity of the firefly luciferase and renilla luciferase (REN) was determined. The ratio of promoter activity between groups was calculated by the ratio of LUC/REN activity. For the analysis of TCF3 transcriptional activity, the coding sequence of TCF3 was inserted to VP16. The dual reporter vector contained LUC and internal control REN. The activity of LUC and REN luciferase was examined using a dual luciferase reporter assay kit (Promega Corp., Madison, WI, USA) and a Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific). At least six transient detections were performed each time [28].

Bioinformatic analyses

A protein–protein interaction (PPI) network was constructed via the STRING system (https://string-db.org/) to explore the TCF3-related genes. The PPI network was established and labeled using the Cytoscape software with the confidence value set at > 0.6. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, conducted using the R package (NIH), were calculated via a ClusterProfiler Package (Bioconductor, Seattle, WA, USA) and visualized by a Barplot Package (version 3.4.0, R). Differentially expressed genes (DEGs) between CC and normal tissues in GSE7803 and GSE9750 datasets were determined using the empirical bayes method using the LIMMA Package (Bioconductor). P value after Benjamini-Hochberg adjustment was used to evaluate the significance of differential expression. DEGs were identified with foldchange > 2 and adj. p < 0.05 as the cutoff values. Binding region between SIRT1 and TCF3 was calculated via ALGGEN (http://alggen.lsi.upc.es/cgi-bin/promo_v3), and the sequence of SIRT1 promoter was acquired from the UCSC system (https://genome.ucsc.edu/index.html).

Chromatin immunoprecipitation (ChIP)-qPCR

The ChIP assay was performed using the EZ-ChIP kit (Millipore Corp., Billerica, MA, USA). In brief, the cells were treated with 37% methanol for protein crosslinking and then treated with 0.125 M glycine to terminate the reaction. After that, the chromatin-protein complexes were reacted with 5 μg anti-TCF3 (sc-416, 1:500, Santa Cruz Biotechnology) and anti-SIRT1 (sc-74465, 1:300, Santa Cruz Biotechnology) for immunoprecipitation. Thereafter, the abundance of promoter fragments in the immunoprecipitated complexes was examined using qPCR [29].

Western blot analysis

Cells were lysed in sodium dodecyl sulfate buffer to extract total protein. The protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loaded onto nitrocellulose membranes. The membranes were hybridized with the primary antibodies against SIRT1 (ab110304, 1:1200, Abcam Inc., Cambridge, MA, USA), β-catenin (13–8400, 1:1,000, Thermo Fisher Scientific), TCF3 (sc-416,1:1000, Santa Cruz Biotechnology), acetyl Lysine (1:1,000, ab22550, Abcam), Lamin B1 (1:1,000, 33–2000, Thermo Fisher Scientific), and GAPDH (G8795, 1:1,200, Sigma-Aldrich) overnight at 4°C, and then with horseradish peroxidase-conjugated immunoglobulin G (IgG, ab205719, Abcam) at 23°C for 2 h. The chemiluminescence signal was developed using the enhanced chemiluminescence reagent and quantified using Image J [30].

Xenograft tumors in nude mice

Thirty female BALB/c nude mice (4–6 weeks old) were procured from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and cultured in a temperature-(22-25°C) and humidity-(40-50%) maintained condition. Stably transfected CC cells (1 × 106) were injected into the right flank of mice through subcutaneous injection. The volume of the xenograft tumors was calculated weekly: volume = length × width2/2. After four weeks, the mice were sacrificed to collect the tumors. After weighed, the tumors were embedded in paraffin for histological analysis. All animal procedures were ratified by the Animal Ethics Committee of Affiliated Cancer Hospital of Zhengzhou University (Approval No. 2020–031-001) and performed according to the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA) [31].

Immunohistochemistry (IHC)

Paraffin-embedded xenograft tumor tissues were cut into 5-μm sections. The sections were dewaxed in xylene, rehydrated in alcohol, microwaved for antigen retrieval and treated with H2O2 to block the activity of the endogenous peroxidase. Thereafter, the sections were incubated with anti-Ki-67 (ab16667, 1:300, Abcam) at 4°C overnight and then with the secondary antibody (ab205718, 1:1,000, Abcam) for 1 h at 37°C. The staining was developed using 2,4-diaminobutyric acid. After that, the sections were stained with hematoxylin, dehydrated, and sealed. The portion of positively stained cells in total cells was observed under the microscope [32].

Statistical analysis

SPSS22.0 (IBM Corp. Armonk, NY, USA) was used for data analysis. Three repetitions were performed. Measurement data were presented as the mean ± standard deviation (SD). Differences were analyzed by the unpaired t test, or by one- or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Post-statistical analysis was conducted by Log rank test. P < 0.05 represents statistically significant difference.

Results

TCF3 has been reported to play key roles in the carcinogenesis of many human malignancies, and its upregulation in CC has been found as well. In this work, we aimed to define the expression pattern and biological functions of TCF3 in the malignant phenotype of CC cells and its interaction with SIRT1 and β-catenin. We obtained the putative binding sites of TCF3 to SIRT1 and SIRT1 to β-catenin and validated the interactions via ChIP-qPCR assays. SiRNA of TCF and overexpression vector of SIRT1, and a β-catenin-specific inhibitor XAV-939 were used to examine their functions in the proliferation, migration and invasiveness, apoptosis, and xenograft tumor formation ability of CC cells.

TCF3 is possibly related to unfavorable prognosis of CC

First, according to the pan-cancer system, TCF3 was suggested to show a high-expression profiling in multiple cancer types (Figure 1a). Thereafter, two GEO datasets GSE7803 and GSE9750 comprising CC tissues and normal tissue samples were downloaded. In these two datasets, TCF3 mRNA was suggested to be highly expressed in CC tumor tissues compared to the normal tissues (Figure 1b). Next, the TCF3 distribution in CC tumor tissues was evaluated via The Human Protein Atlas system. It was suggested that TCF3 was highly expressed in CC tumor tissues, whereas poorly expressed in normal tissues (Figure 1c). Thereafter, the expression and prognostic value of TCF3 in CC were explored in TCGA-CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma) database which contained the whole-genome RNA-seq data of 262 patients with CESC and the clinical information such as survival events and follow-up time. It was indicated that TCF3 expression was increased in patients with CESC (Figure 1d) and its high level was significantly associated with poor prognosis (Log-rank p = 0.0186; hazard ratio = 2.176) (Figure 1e). In the procured cell lines, the TCF3 expression was significantly elevated in all CC cells, particularly in SiHa and HeLa cells, versus the End1/E6E7 cells (figure 1f). This result showed a similar trend with the bioinformatics analysis results above and indicated that TCF3 is possibly related to the progression and unfavorable prognosis of CC.

Figure 1.

TCF3 is possibly linked to unfavorable prognosis of CC. A, expression of TCF3 in pan cancers; B, expression of TCF3 in GSE7803 and GSE9750 datasets; C, distribution of TCF3 in CC tissues and normal cervical tissues in The Human Protein Atlas system; D, TCF3 expression in TCGA-CESC; E, association of TCF3 expression with the patient survival in TCGA-CESC; F, TCF3 mRNA expression in CC cell lines (CaSki, C-33A, SiHa, SW756 and HeLa) and in normal End1/E6E7 cells detected by RT-qPCR (*p < 0.05; **p < 0.01; one-way ANOVA). Data are expressed as the mean ± SD.

TCF3 downregulation reduces proliferation, migration and invasiveness, and resistance to death of CC cells in vitro

Since TCF3 was upregulated in CC tissues, si-TCF3 was administrated into HeLa and SiHa cells. The successful TCF3 knockdown in cells was detected by RT-qPCR (Figure 2a). After that, silencing of TCF3 significantly decreased the count of colonies formed by HeLa or SiHa cells (Figure 2b). The Transwell assays demonstrated that the migration of cells was decreased after si-TCF3 transfection (Figure 2c). Likewise, the invasiveness of cells was suppressed by si-TCF3 (Figure 2d). The flow cytometry on cell apoptosis indicated that the apoptosis rate of HeLa and SiHa cells was significantly elevated after TCF3 silencing (Figure 2e).

Figure 2.

TCF3 downregulation reduces proliferation, migration and invasiveness, and resistance to death of CC cells in vitro. A, TCF3 expression in HeLa and SiHa cells after si-TCF3 transfection detected by RT-qPCR (*p < 0.05, two-way ANOVA); B, proliferation activity of HeLa and SiHa cells examined by colony formation assay (*p < 0.05, two-way ANOVA); C, migration activity of HeLa and SiHa cells measured by Transwell assay (*p < 0.05, two-way ANOVA); D, invasiveness of HeLa and SiHa cells examined by Transwell assay (*p < 0.05, two-way ANOVA); E, HeLa and SiHa apoptosis examined by flow cytometry (*p < 0.05, two-way ANOVA). Data are expressed as the mean ± SD.

TCF3 plays a transcriptional activation role in CC cells

The sequence analysis of TCF3 protein validated that TCF3 contained bHLH motifs (Figure 3a), which can form homodimers to play a transcriptional activation role in biological regulation [33]. To examine whether TCF3 has the transcriptional activation activity, the full-length cDNA of TCF3-VP16 (effector) and the VP16 transcriptional activation domain (control) were inserted into the pmiR-GLO reporter vectors. The constructs were transfected into CC cells (Figure 3b). Compared to the control VP16, the TCF3-VP16 construct showed increased luciferase activity, indicating TCF3 might further activate the VP16 activity (Figure 3c). The transcription activity of TCF3 was further examined by the luciferase assay. The firefly luciferase (LUC) reporter gene at the PGK promoter was used as the internal control, and the renilla luciferase (REN) reporter gene at the SV40 promoter was used as the internal control as well (Figure 3d). It was found that compared to effects of pGLO alone, pGLO-TCF3 significantly enhanced the luciferase activity of the LUC reporter vector (Figure 3e). These results, collectively, suggest that TCF3 functions as a transcriptional activator in CC cells.

Figure 3.

TCF3 plays a transcriptional activation role in CC cells. A, the structural domain of TCF3 determined by protein sequencing; B, construction of pmir-GLO luciferase reporter vectors containing TCF3 and VP16; C, effect of TCF3 on the activity of the luciferase reporter examined by the luciferase assay (*p < 0.05, two-way ANOVA); D, construction of pGLO-TCF3 vector; E, transcriptional regulation activity of TCF3 validated via the luciferase assay (*p < 0.05, unpaired t test). Data are expressed as the mean ± SD.

TCF3 binds to the promoter of SIRT1

Gene clusters closely correlated with TCF3 were analyzed, and a PPI network was constructed, in which 14 TCF3-related genes which had an over 0.6 correlation-coefficient with TCF3 were identified (Figure 4a). The subsequent functional annotation and enrichment analyses of these gene clusters indicated that the TCF3-related genes were mainly enriched in the cell proliferation and apoptosis pathways, especially in the Wnt/β-catenin signaling pathway (Figure 4b). In the bioinformatic analyses using the GSE7803 and GSE9750 datasets mentioned above, 31 genes were commonly upregulated whereas 12 genes were decreased in both datasets (Figure 4c-d). The functional annotation and enrichment analyses also indicated that the DEGs were mainly enriched in the cell proliferation and apoptosis-related pathways, particularly in the Wnt/β-catenin signaling pathway (Figure 4e). We therefore speculated that TCF3 possibly mediates specific signaling pathways involving cell proliferation and apoptosis, including the Wnt/β-catenin signaling, to regulate the cellular behaviors in CC. Specific TCF3-mediated gene axis might be involved during the process. Among the TCF-3 related genes, SIRT1 aroused our attention. An interaction among TCF3, SIRT1, and CTNNB1 has been identified in Figure 4a. These three genes are all related to cell proliferation and apoptosis-related pathways including the Wnt/β-catenin. The bioinformatic analysis also indicated that only the SIRT1 promoter had binding sites with TCF3 (figure 4f). To validate this, the TCF3-specific antibody was prepared for ChIP-qPCR assay. Compared to the control IgG, anti-TCF3 significantly enriched the promoter fragments of SIRT1 in the immunoprecipitates (Figure 4g). Taken together, the results in Figure 3 and Figure 4 indicate that, as a transcriptional factor, TCF3 binds to the promoter sequence of SIRT1 to activate its transcription.

Figure 4.

TCF3 binds to the promoter of SIRT1. A, a PPI network of the TCF3-related gene clusters; B, GO and KEGG pathway enrichment analyses of the TCF3-related genes; C, volcano maps for the DEGs in the GSE7803 and GSE9750 datasets; D, a Venn diagram for common DEGs in two datasets; E, GO and KEGG pathway enrichment analyses based on the DEGs; F, putative binding site between TCF3 and the SIRT1 promoter; G, binding between TCF3 and the SIRT1 promoter validated by the ChIP-qPCR assay (*p < 0.05, two-way ANOVA). Data are expressed as the mean ± SD.

TCF3 binds to SIRT1 to mediate nuclear accumulation of β-catenin

The expression profiling of SIRT1 in CC and its correlation with patients were then explored in TCGA-CESC database as well. SIRT1 was suggested highly expressed in CESC and linked to poor prognosis of patients (Figure 5a-b). Data in TCGA-CESC also showed that TCF3 had a positive association with SIRT1 expression (r = 0 .720) (Figure 5c). Data in the GSE7803 and GSE9750 datasets, likewise, indicated that the SIRT1 expression was increased in the CC tumor tissues compared to the normal tissues (Figure 5d), which showed a positive correlation with TCF3 (Figure 5e). To further evaluate the correlation between SIRT1 and β-catenin, ChIP-qPCR assay was performed, which confirmed that overexpression of SIRT1 increased the abundance of β-catenin promoter fragments enriched by anti-SIRT1 in HeLa and SiHa cells (figure 5f). Western blot assay also indicated that overexpression of SIRT1 in cells weakened acetylation of β-catenin and promoted its nuclear accumulation. However, after EX527 treatment, the β-catenin acetylation was promoted and its nuclear accumulation was reduced (Figure 5g). These results indicated that TCF3 binds to SIRT1, which further promotes the nuclear accumulation of β-catenin.

Figure 5.

TCF3 binds to SIRT1 to promote β-catenin nuclear accumulation. A, expression of SIRT1 in TCGA-CESC; B, prognostic value of SIRT1 in CC in TCGA-CESC; C, correlation between TCF3 and SIRT1 in CC tumor tissues in TCGA-CESC; D, expression of SIRT1 in CC tissues in GSE7803 and GSE9750 datasets; E, correlation between TCF3 and SIRT1 in CC in GSE7803 and GSE9750 datasets; F, binding relationship between SIRT1 and β-catenin examined by ChIP-qPCR (*p < 0.05, two-way ANOVA); G, acetylation and nuclear accumulation of β-catenin in CC cells (*#p < 0.05, two-way ANOVA; * compared to SIRT1-NC; # compared to DMSO). Data are expressed as the mean ± SD.

Interactions between TCF3/SIRT1/β-catenin in CC cells

The SIRT1 and β-catenin expression in cells was examined. The western blot analysis showed that the SIRT1 expression and the nuclear accumulation of β-catenin in HeLa and SiHa cells were increased (Figure 6a). However, the SIRT1 expression and the nuclear accumulation of β-catenin in cells were reduced upon TCF3 knockdown (Figure 6b). Next, cells transfected with si-TCF3 were further transfected with SIRT1-OE, and cells transfected with SIRT1-OE were further administrated with an endogenous nuclear β-catenin inhibitor XAV-939 for rescue experiments. It was found that artificial upregulation of SIRT1 in cells elevated β-catenin expression, but XAV-939 treatment did not affect the expression of SIRT1, indicating the SIRT1/β-catenin axis is a unidirectional regulatory axis (Figure 6c). Thereafter, it was found that the colony formation ability of cells suppressed by si-TCF3 was restored by SIRT1 overexpression, but the proliferation of cells was suppressed again after XAV-939 treatment (Figure 6d). The migration ability of cells, according to the Transwell assay, was restored by SIRT-OE and weakened by XAV-939 (Figure 6e). Upregulation of SIRT1 also reduced the apoptosis of HeLa and SiHa cells induced by si-TCF3, but further XAV-939 treatment increased the portion of apoptotic cells again (figure 6f). These results indicated that SIRT1 overexpression blocked the role of TCF3 silencing in CC cells, and β-catenin degradation blocked the role of SIRT1-OE again.

Figure 6.

Interactions between TCF3/SIRT1/β-catenin in CC cells. A, SIRT1 expression and β-catenin nuclear accumulation in CC cell lines (CaSki, C-33A, SiHa, SW756 and HeLa) and normal End1/E6E7 cells examined by western blot analysis (*p < 0.05, two-way ANOVA); B, SIRT1 expression and β-catenin nuclear accumulation in HeLa and SiHa cells transfected with si-TCF3 determined by western blot assay (*p < 0.05, two-way ANOVA); C, expression of SIRT1 and nuclear accumulation of β-catenin in HeLa and SiHa cells after SIRT1-OE transfection and XAV-939 treatment examined by western blot assay (*#p < 0.05, two-way ANOVA, * compared to si-TCF3 + SIRT1-NC; # compared to SIRT1-OE + DMSO); D, cell proliferation determined by colony formation assay (*#p < 0.05, two-way ANOVA, * compared to si-TCF3 + SIRT1-NC; # compared to SIRT1-OE + DMSO); E, migration of cells determined by Transwell assay (*#p < 0.05, two-way ANOVA, * compared to si-TCF3 + SIRT1-NC; # compared to SIRT1-OE + DMSO); F, apoptosis rate in HeLa and SiHa cells detected by flow cytometry (*#p < 0.05, two-way ANOVA, * compared to si-TCF3 + SIRT1-NC; # compared to SIRT1-OE + DMSO). Data are expressed as the mean ± SD.

TCF3/SIRT1/β-catenin mediates CC development in vivo

Animal experiments were conducted to validate the interactions of TCF3/SIRT1/β-catenin in vivo. Stably transfected HeLa cells were injected into mice to induce xenograft tumors. TCF3 silencing in HeLa cells also suppressed xenograft tumor growth in vivo. Further upregulation of SIRT1 significantly induced the tumor growth in mice, but XAV-939 treatment significantly blocked the role of SIRT1 and reduced the tumorigenesis activity of CC cells in vivo (Figure 7a). Likewise, downregulation of TCF3 in HeLa cells significantly reduced the weight of xenograft tumors on the 28th day. However the tumor weight was increased after further overexpression of SIRT1 in cells, and reduced after further administration of XAV-939 in cells (Figure 7b). The expression of a proliferation marker Ki67 in xenograft tumor tissues was examined to determine the function of TCF3/SIRT1/β-catenin in tumor cell proliferation in vivo. The IHC results indicated that TCF3 silencing significantly reduced the expression of Ki67 in tumor tissues. But the expression of Ki67 was significantly restored after SIRT1 overexpression but reduced after β-catenin degradation (Figure 7c).

Figure 7.

TCF3/SIRT1/β-catenin mediates CC development in vivo. A, growth rate of xenograft tumors induced by HeLa cells in nude mice (*#@p < 0.05, two-way ANOVA; * compared to si-TCF3-NC; # compared to si-TCF3+ SIRT1-NC; @ compared to SIRT1 + DMSO); B, weight of the xenograft tumors on the 28th day (*#@p < 0.05, one-way ANOVA; * compared to si-TCF3; # compared to si-TCF3 + SIRT1-NC; @ compared to SIRT1 + DMSO); C, expression of Ki-67 in xenograft tumors examined by IHC (*#@p < 0.05, one-way ANOVA; * compared to si-TCF3; # compared to si-TCF3+ SIRT1-NC; @ compared to SIRT1 + DMSO). In each group, n = 5. Data are expressed as the mean ± SD.

Discussion

CC remains a huge healthy challenge due to the insufficient coverage of HPV vaccination, and the median survival time of patients at advanced stage is low at only 8 to 13 months [34]. With the aid of advanced bioinformatics tools, researchers in this field have identified increasing number of key molecules or prognostic biomarkers for CC [35]. In the present study, the authors report a novel TCF3/SIRT1/β-catenin cascade that is implicated in the tumor development of CC, which may offer new thoughts in the management of this malignancy.

While proper activity of the E proteins-mediated transcriptional networks is crucial for functionality of the immune system, aberrant expression or function of E proteins can lead to a multitude of defects from impaired lymphocyte development and immunodeficiency to aberrant function, autoimmunity, and cancers [36]. Aberrant activation of TCF3 (E2A), most commonly, was correlated with the risk and onset of lymphoblastic leukemia [37]. Associations between TCF3 and other human malignancies, such nasopharyngeal carcinoma [13], breast cancer [38], colorectal cancer [39], blastemal Wilms tumors [40], have been identified as well. In this study, the integrated bioinformatic analyses via Oncomine, GSE datasets, and TCGA-CESC database indicated that TCF3 was upregulated in multiple cancers, including CC, and its high expression was predicted to be correlated with poor outcome of patients. Increased expression of TCF3 was then identified in the acquired CC cell lines versus normal cells. Interestingly, TCF3 has been reported as a downstream target of signal transducer and activator of transcription 3 [41], which is upregulated in HPV-positive CC cells and allows cellular proliferation, viral replication, and tumorigenesis [42,43]. This might represent a major reason for TCF3 upregulation in CC cells. As aforementioned, TCF3 has been demonstrated correlating with poor prognosis of patients with CC in a clinical research [14]. In experimental cellular models, downregulation of TCF3 by microRNAs has been observed to suppress growth and metastasis of CC cells [44,45]. Therefore, silencing of TCF3 using siRNA in HeLa and SiHa cells was performed in this study, which weakened proliferation, migration, invasiveness, and resistance to death of the CC cells. These cells also had a lower growth rate in vivo when forming xenograft tumors. These results validated the tumor-promoting role of TCF3 in CC.

TCF3 plays a transcriptional activation role when forming homodimers with other bHLH proteins [33], and the subsequent dual-luciferase assay indicated that TCF3 played a transcriptional activating role in CC cells. TCF3 has been reported to activate transcription of distinct oncogenes such as FAM201A [12], LINC00152 [46], and matrix metalloproteinase-9 [47] to promote cancer progression. In this study, pathway enrichment analyses based on the TCF3-related genes and the DEGs in CC indicated that TCF3 possibly regulated the Wnt/β-catenin pathway, and the SIRT1 gene was likely involved in this regulatory cascade according to the established PPI network. Intriguingly, TCF3 has been reported to interact with HDAC3 and regulates transcription of downstream microRNA-101 to promote progression of Burkitt lymphoma [48]. Here, we confirmed the binding relationships between TCF3 and SIRT1 promoter and between SIRT1 and β-catenin promoter using ChIP-qPCR assays. Among the HDACs closely linked to the tumorigenesis process and cancer metastasis [49], SIRT1 is found in the nucleus and mainly targets histones and transcription factors for epigenetic regulation [50]. SIRT1 has been reported to deacetylate the DNA repair protein Werner helicase to promote its interaction with HPV E1-E2-replicating DNA [51,52]. SIRT1 has been demonstrated to be upregulated in HPV-positive CC as well and plays important function in counteracting antiviral immunity, with its knockdown inducing massive death of CC cells [53]. As expected, we found that overexpression of SIRT1 blocked the role of TCF3 silencing and induced growth and dissemination of CC cells. As a deacetylase, SIRT1 has been reported to activate the Wnt/β-catenin pathway by inducing β-catenin accumulation in nucleus through deacetylation regulation [22,54]. Aberrant Wnt/β-catenin signaling activation is well known to exert crucial functions in tumorigenesis and therapy response by triggering cell proliferation and differentiation and cancer stem cell renewal [55]. Therefore, increased nuclear accumulation of β-catenin by SIRT1 has been observed to increase oncogenesis and self-renewal of cancer stem cells [56]. Likewise, a recent study revealed that SIRT1 upregulation increased stemness and radio-resistance of cancer stem cells by triggering the deacetylation and nuclear translocation of β-catenin [57]. Activation of the Wnt/β-catenin is also frequently involved in the malignant development of CC [58], leaving it as a candidate target for CC treatment [23]. Intriguingly, TCF3 has also been reported to interact with the β-catenin as well for its pro-migration ability [41]. However, the role of TCF3/SIRT1/β-catenin cascade in CC has not been investigated before. Our experimental results revealed that SIRT1 overexpression restored the malignant phenotype of CC cells blocked by TCF3 silencing, but treatment with the β-catenin-specific inhibitor XAV-939 hampered the malignant behaviors of cells increased by SIRT1 again.

Conclusion

In summary, this study validates that TCF3 functions as an oncogene in CC by inducing cell growth and metastasis through increasing SIRT1-mediated deacetylation and nuclear translocation of β-catenin. However, undoubtedly there would be more molecules or pathways that interact with TCF3/β-catenin involved in CC carcinogenesis [59]. We would like to focus on this issue in our later researches. Anyway, the findings of the present work may shed new lights in the field of CC treatment that TCF3 or SIRT1 may serve as prognostic biomarkers or therapeutic targets for CC management.

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