CTCF regulates the FoxO signaling pathway to affect the progression of prostate cancer

Abstract The present research focuses on the influence of CCCTC‐binding factor (CTCF) on prostate cancer (PC) via the regulation of the FoxO signalling pathway. A bioinformatics analysis was conducted to screen out target genes for CTCF in LNCaP cells and to enrich the relevant pathways in LNCaP cells. It was found that the FoxO pathway was enriched according to the ChIP‐seq results of CTCF. The expression of CTCF, pFoxO1a, FoxO1a, pFoxO3a and FoxO3a was tested by RT‐qPCR and Western blot. Inhibition of CTCF could lead to the up‐regulation of the FoxO signalling pathway. The rates of cell proliferation, cell invasion and apoptosis were examined by MTT assay, cell invasion assay and flow cytometry under different interference conditions. Down‐regulation of CTCF could suppress cell proliferation, cell invasion and facilitate cell apoptosis. Lastly, the effect of CTCF on tumour growth was determined in nude mice. Inhibition of CTCF regulated the FoxO signalling pathway, which retarded tumour growth in vivo. In conclusion, CTCF regulates the FoxO signalling pathway to affect the progress of PC.

There are more than 20 000 binding sites in the CTCF genome; therefore, the regulatory actions of CTCF are quite complex and depend on the specific DNA sequence and interacting factors at CTCF binding sites. 9 The distribution of CTCF binding sites in the genome relates to gene density, with approximately 46% of sites lying in intergenic regions, 20% near transcriptional start sites, 22% in introns and 12% in exons. 10 CTCF is a nuclear protein, which is widespread across cell types. Dysfunction of CTCF can epigenetically alter many cancer-related genes. Recent genome-wide assays have demonstrated that the transcription factor CTCF can link chromatin domains through long-distance interactions between distal genomic regions, suggesting a critical role in chromatin conformation. 11 FoxO proteins, including FoxO1a and FoxO3a, are evolutionarily conserved transcription factors that are involved in multiple fundamental cellular activities, acting in transcriptional activities related to cell proliferation, apoptosis and stress response. [12][13][14][15][16] Numerous therapies can induce cell growth arrest and apoptosis through activation of FoxO transcription factors in PC cells. 17 However, upexpression of FoxO has inhibited tumorigenesis in xenograft models in nude mice. [18][19][20][21][22][23][24] Therefore, reactivation of FoxO based on its tumour-suppressant properties is considered a very promising therapy for PC. Since FoxO proteins have been found to be critical mediators of apoptosis, we hypothesized that FoxO expression or its transcriptional activity could be an important event in changing the progression of PC. Therefore, we studied the relationship between CTCF and FoxO signalling. To assess the rates of cell proliferation, cell invasion and apoptosis, an MTT assay, cell invasion assay and flow cytometry were performed under different interference conditions. The flow cytometry detected the effect of CTCF on tumour growth in nude mice. Tumour tissues were selected by conventional pathologic criteria, and the histopathology was further confirmed by microscopic examination.

| Transfection
Scramble siRNA was used as a control (GenePharma, Shanghai, China). CTCF siRNA was obtained from GenePharma, Shanghai, China. The transfection procedure was performed using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer's protocols. 25  The results were determined using the 2 −ΔΔCt method. GAPDH was used as a control for normalization, and the data were calculated and analysed with Rotor-Gene Real-Time Analysis software 6.0 (Corbett Research, Mortlake, Australia).

| Western blot
Samples containing 20 μg of total protein were separated on 8%-12% SDS-PAGE gels according to the different molecular weights and then transferred onto nitrocellulose membranes (Whatman, Germany) in transfer buffer using a Mini Trans-Blot Cell (Bio-Rad) at 400 mA for 2 hours. The membranes were blocked by incubating them in 5% nonfat milk in TBS-T for 1 hour at room temperature.
Proteins were detected using specific rabbit polyclonal anti-CTCF

| MTT assay
Cell viability was measured using microculture tetrazolium test (MTT) from Sigma-Aldrich as described in the manufacturer's manual.
Briefly, the cells that had been transfected for 0, 12, 24 and 48 hours were added to 20 μL of 5 mg/mL MTT and then incubated at 37°C for 4 hours. The medium was removed, and 150 μL of the MTT solution was added to the cells for a further incubation of 15 minutes at room temperature with shaking.

| Cell invasion assay
Cell invasion was assessed using the Transwell assay. LNCaP and PC-3 cells were harvested after trypsinization and washed with a serum-free media containing 0.1% bovine serum albumin. Cells were diluted to 5 × 10 5 cells/mL, and 100 μL of the cell suspension was seeded on the top of Matrigel invasion chambers (8 μm pore size, cat no. 354480; Corning). The lower chamber was filled with 600 μL of medium containing 10% FBS. After being incubated for 24 hours at 37°C, cells from the upper chamber were gently removed with a cotton swab.
The filter was fixed with 4% paraformaldehyde for 15 minutes at room temperature, then stained with 500 μL of 0.1% crystal violet for 15 minutes, and washed with PBS, and the cells were counted under a light microscope to determine the level of cell invasion. Each cell type was assayed in triplicate, and all the experiments were repeated three times.

| Wound healing assay
LNCaP and PC-3 cells transfected with siNC, siCTCF were cultured to obtain 80%-90% monolayer confluency. A wound was created by scraping the cells using a 10 μL plastic pipette tip, and the old F I G U R E 1 ChIP-seq analysis. A and B, Gene binding site of transcription factor CTCF was enriched near TSS region in PrEC and LNCaP cells. C, Feature distribution of CTCF genome. D. Details of CTCF binding to TSS region medium was replaced with fresh medium. Cells were photographed under a microscope equipped with a camera at different points in time.

| Flow cytometry
The number of apoptotic cells was calculated using an Alexa Fluor ® 488 annexin V/Dead Cell Apoptosis Kit with Alexa ® Fluor 488 annexin V and PI for flow cytometry (Invitrogen) as described in the manufacturer's manual. The protocol was carried out according to a previously described method. 26 Cells were analysed by fluorescenceactivated cell sorting using Becton Dickinson FACSCalibur cell sorter and CellQuest software. The results show that the percentage of apoptotic cells was relevant to the total cell number.

| Tumour formation in nude mice
Nude mice (4-5 weeks old, 14-16 g) were purchased and housed in barrier facilities with a 12 hours/12 hours light/dark cycle. 27

| Bioinformatics analysis
Analysis of ChIP-seq data showed the gene binding site of transcription factor CTCF was enriched near the TSS region in PrEC and LNCaP cells. The enriched peak in LNCaP cells was higher than that in PrEC cells, which revealed that CTCF and CTCF-related genes might have a strong connection with PC ( Figure 1A,B). The feature information of the CTCF genome is shown in Figure 1C. Details of CTCF binding to the TSS region are shown in Figure 1D.

| Gene functional analysis by ChIP-seq
To determine the possible binding region of CTCF in different species such as humans, mice and rats, the motif was predicted on a website (Figure 2A). KEGG analysis of genes from ChIP-seq analysis was performed on DAVID ( Figure 2B). GO analysis of genes from ChIP-seq analysis was also performed on DAVID. According to the analyses, CTCF was related to biological processes, such as signal transduction, and was located in synapses, cell junctions and many other areas. Furthermore, it could exert multiple functions such as growth factor binding and GABA-A receptor activity ( Figure 2C-E).
Multiple signalling pathways were involved, and investigating the FoxO signalling pathway was regarded as our research objective.

| CTCF was dysregulated in LNCaP
To determine the expression of CTCF in tumours and adjacent tissues, PrEC, LNCaP and PC-3 cells, qPCR and Western blot tests were F I G U R E 2 Gene functional analysis by ChIP-seq. A, Motif of CTCF predicted on website. Possible binding region was compared among humans, mice and rats. B, KEGG analysis of genes from ChIP-seq analysis was performed on DAVID. C-E, GO analysis of genes from ChIP-seq analysis was performed on DAVID SHAN ET AL.

| 3133
conducted. The results showed that compared with tissues in cells adjacent to the tumours, the mRNA of CTCF was richly expressed in tumour tissues ( Figure 3A). The expression of CTCF was up-regulated in LNCaP and PC-3 cells compared with PrEC ( Figure 3B). Western blot tests showed that the expression of CTCF, pFOXO1a and pFOXO3a was up-regulated, while that of FOXO1a and FOXO3a was down-regulated ( Figure 3C) in LNCaP and PC-3 cells.

| CTCF promoted the proliferation of PC cells by regulating the FoxO signalling pathway
CTCF regulates the FoxO signalling pathway to affect the progression of PC. RT-qPCR revealed that siCTCF inhibited the expression of CTCF distinctly ( Figure 4A). MTT assays showed that the inhibition of CTCF could retard the proliferation of LNCaP and PC-3 cells ( Figure 4B). Western blot tests showed that the protein expression of CTCF was inhibited after siCTCF treatment. FoxO1a and FoxO3a are tumour suppressing genes, which deactivate after phosphorylation. After the treatment of siCTCF, the expression of FOXO1a and FOXO3a was up-regulated, while that of pFOXO1a and pFOXO3a was down-regulated ( Figure 4C). After the application of siCTCF, cell invasion was significantly reduced ( Figure 5A), tumour metastasis was suppressed ( Figure 5B) and cell apoptosis was promoted in LNCaP and PC-3 cells ( Figure 5C).

| siCTCF retarded tumour growth in vivo
siNC and siCTCF were injected into nude mice, and the tumour tissues are shown in Figure 6A. As seen in Figure 6B, tumour volume in the siNC group had been increasing rapidly, while the growth in tumour volume in the siCTCF group slowed down starting at week 3. At week 4, the tumour volume in the siCTCF group was quite F I G U R E 3 Expression of CTCF. A, RT-qPCR showed that the expression of CTCF mRNA was up-regulated in tumour tissues compared to tissues adjacent to tumours. B, RT-qPCR showed that the expression of CTCF mRNA was up-regulated in LNCaP and PC-3 cells compared to PrEC. C, Western blot indicated that the expression of CTCF, pFOXO1a and pFOXO3a was high in LNCaP and PC-3 cells, while that of FOXO1a and FOXO3a was low. PrEC is a prostate epithelial cell line; LNCaP and PC-3 are prostate cancer cell lines. *P < 0.05, **P < 0.01, compared with PrEC group smaller than in the siNC group. Tumour weight in the siCTCF group was quite lighter in the siNC group ( Figure 6C). After siCTCF treatment, Western blot revealed that the expression of CTCF, pFOXO1a and pFOXO3a was down-regulated, while that of FOXO1a and FOXO3a was up-regulated in tumour issues ( Figure 6D).

| DISCUSSION
In this study, analysis of ChIP-seq data showed that the gene binding Prostate cancer is the second leading cause of cancer-related death among men in the United States. 2 The factors involved in the progression of PC are not yet fully understood. The correlative factors associated with the regulation of epigenetic marks of PC include the CCCTC-binding factor (CTCF). 7 CTCF is an evolutionarily conserved nuclear protein which is fundamental in regulatory activities in numerous cell types. 8 There are more than 20 000 binding sites in the CTCF genome; therefore, the regulatory actions of CTCF are quite complex and depend on the specific DNA sequence and interacting factors at CTCF binding sites. 9 In our previous study, analysis of ChIP-seq data showed that the gene binding site of transcription factor CTCF was enriched near the TSS region in both PrEC and LNCaP cells. The enriched peak in LNCaP cells was higher than in PrEC, which suggested that CTCF and CTCF-related genes might have a strong connection to PC. This result indicated that compared with tissues adjacent to the tumour, the mRNA of CTCF was highly expressed in tumour tissues. The expression of CTCF was up-regulated in LNCaP cells compared with PrEC.
Research found that every zinc finger mutation enabled CTCF binding to a subset of target sites within the promoters/insulators of certain genes, which is involved in the reduction of cell proliferation. KEGG analysis of genes from ChIP-seq analysis found that CTCF was involved in the FoxO signalling pathway. Studies on PC patients also found the increased cytoplasmic expression of phosphorylated F I G U R E 6 Tumour growth in vivo. A, Tumour tissues in nude mice were collected. B, Tumour weight was reduced by siCTCF. C, Line chart of tumour volume D. Western blot revealed the expression of CTCF, pFOXO1a, FOXO1a, pFOXO3a and FOXO3a in tumour tissues. **P < 0.01, compared with siNC group. ##P < 0.01, compared with siNC group FoxO3a (Ser253), which relates to disease progression. 32 In addition, a decrease in FoxO function is frequently observed with several human cancers. 15,33 Data indicate that chemical compounds such as resveratrol could induce apoptosis and growth arrest through activation of FoxO transcription factors. 17 Moreover, the forced expression of FoxO has been confirmed to inhibit tumorigenesis in xenograft models in nude mice. [19][20][21][22][23][24] In our present study, after siCTCF treatment, the expression of pFOXO1a and pFOXO3a was up-regulated, while that of FOXO1a and FOXO3a was down-regulated in LNCaP cells. After siCTCF treatment, Western blot showed that the protein expression of CTCF in tumour tissues in nude mice was inhibited, the expression of FOXO1a and FOXO3a was up-regulated, while that of pFOXO1a and pFOXO3a was down-regulated.
Tumour growth was evidently inhibited in nude mice with PC. The therapy approach of targeting CTCF seems to be promising for the treatment of PC by regulating the FoxO signalling pathway and further retarding cancer progression. Additionally, there have been an increasing number of studies on the use of combination therapies to treat cancer using DNA repair mechanisms. [34][35][36] Research has shown that the inactivation of AKT results in dephosphorylation and activation of FOXO transcription factors, which is involved in mediating cell cycle arrest, DNA repair and apoptosis. 31,32 It would be feasible to make a combination therapy via DNA repair progression.
In our study, we identified an interaction between CTCF and the FoxO signalling pathway in PC progression. However, some questions remain. The specific mutations of the gene binding regions of CTCF and the importance of the role of CTCF and the FoxO signalling pathway in PC cells warrant further study.
In summary, our research proved that CTCF could retard PC progression by altering the FoxO signalling pathway in vitro and in vivo.
These data indicate that CTCF may serve as a potential therapeutic target for PC.

CONF LICT OF I NTEREST
Authors declare no conflicts of interest for this article.

AUTHOR CONTRIBU TI ON
Zhengfei Shan, Yongwei Li, Shengqiang Yu, Jitao Wu conceived and designed of the work and acquisition, analysed the data and drafted the article; Chengjun Zhang, Yue Ma, Guimin Zhuang analysed the data and revised the article critically for important intellectual content; Jiantao Wang, Zhenli Gao, Dongfu Liu performed the experiments and analysed the data. All authors read and approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content.