Forkhead Box S1 Inhibits the Progression of Hepatocellular Carcinoma

Introduction

Liver cancer is considered one of several cancers with the highest incidence rates worldwide,¹ and HCC accounts for 80% of liver cancer cases.² Despite advances in treatments such as surgery and ablation,² liver cancer is still the fourth leading cause of death among all cancers,³ and thus, the molecular mechanisms of this lethal disease must be elucidated.

Forkhead box (FOX) proteins are an evolutionarily conserved family of transcriptional regulatory proteins that are closely related to the carcinogenesis and development of cancer.⁴ FOXS1, a member of this family, has been reported to be closely associated with stomach cancer and promotes cell proliferation and the epithelial–mesenchymal transition (EMT) in gastric cancer.⁵ Additionally, some researchers found that FOXS1 inhibits the proliferation, motility, and EMT of gastric cancer cells.⁶ FOXS1 is also associated with the differentiation of fibroblasts to myofibroblasts⁷ and the prognosis of patients with aggressive and metastatic breast cancer.⁸ Moreover, FOXS1 plays an important role in integrating and processing neuronal signals that are important for energy conversion and motor function.⁹ However, the mechanism underlying its effect on hepatocellular carcinoma has not been elucidated. In this study, we overexpressed FOXS1 in HCC cells to evaluate its function and found that FOXS1 suppressed the proliferation, motility and EMT of HCC cells by modulating the hedgehog (Hh) pathway.

Materials and Methods

Clinical Samples and Cell Lines

We collected 40 pairs of liver tumors and adjacent tissues from patients treated at The First Affiliated Hospital of Chongqing Medical University. Patients were excluded if they receive chemotherapy or radiation before surgery. Our study was approved by the Ethics Committee of Chongqing Medical University and that this was conducted in accordance with the Declaration of Helsinki. We obtained written informed consent from each patient after providing information about the study, and patients signed the informed consent forms. The normal hepatocyte cell line (HL-7702) and human HCC lines (SMMC-7721, Hep-3B, Hep-G2, SMMC-7402, and Huh-7) were purchased from the Institute of Chinese Academy of Sciences. SMMC-7721, Hep-3B, Hep-G2, SMMC-7402 and Huh-7 cell lines were cultured in DMEM (Corning Incorporated, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Waltham, MA, USA). The HL-7702 cell line was cultured in RPMI 1640 medium (Corning Incorporated, Corning, NY, USA) supplemented with 10% FBS. All cells were cultured in an incubator with an atmosphere of 95% O₂ and 5% CO₂ at 37°C.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from tissues and cells using TRIzol^® reagent (Takara Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Reverse transcription and qRT-PCR were performed as previously described. The FOXS1 mRNA level was examined using real-time PCR. The sequences of the primers were as follows: FOXS1-forward, 5ʹ-AGCCCAAAGAGATTTCCACGC-3 and FOXS1-reverse, 5ʹ-CTGCTCCCGATGCCTGATTC-3ʹ.

Western Blot Analysis

Total proteins were extracted from HCC tissue samples or cells with RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) containing protease inhibitors. Protein concentrations were measured with BCA protein detection kit (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amounts of proteins were separated on 8% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with skimmed milk (dissolved in PBS) for 2 h, the membranes were subsequently probed using antibodies against β-actin (Cell Signaling Technology, USA, 1:10,000), FOXS1 (Sigma, 1:1000), vimentin (Cell Signaling Technology, USA, 1:1000), E-cadherin (Cell Signaling Technology, USA, 1:1000), N-cadherin (Cell Signaling Technology, USA, 1:1000), Slug (Cell Signaling Technology, USA, 1:1000), Hedgehog receptor protein patched homolog 1 (Cell Signaling Technology, USA, 1:1000), Sonic Hedgehog (Cell Signaling Technology, USA, 1:1000), smoothened (Cell Signaling Technology, USA, 1:1000) and glioma-associated oncogene homolog 1 (Cell Signaling Technology, USA, 1:1000) and glioma-associated oncogene homolog 1 (Cell Signaling Technology, USA, 1:1000) overnight at 4°C. The membranes were then washed with Tris-buffered saline containing Tween and incubated with an HRP-conjugated anti-rabbit antibody at 37°C for 2 h. Finally, the protein bands on the membranes were observed with an Odyssey Scanning System.

Immunohistochemistry (IHC)

We collected 40 human liver tumor tissues and adjacent tissues. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin and sliced (3 μm). The samples were subjected to high-pressure antigen retrieval in pH 6.0 citrate buffer for 5 min, blocked with a buffer containing 10% bovine serum albumin for 60 min, and incubated with primary antibodies overnight at 4°C followed by an incubation with the peroxidase-conjugated anti-rabbit secondary antibody (1:500).

Transfection

Huh-7 and Hep-3B cells were infected with Lentivirus5-GFP-FOXS1. Transfected cells were cultured in complete media containing puromycin (1 μg/mL) for 72 h to generate a stable cell line overexpressing GFP-FOXS1. The methods were performed in accordance with the corresponding guidelines and regulations.

Cell Proliferation Assay

Huh-7 and Hep-3B cells were plated in 96-well plates at a density of 5×10³/well. The Cell Counting Kit-8 (CCK8) cell proliferation assay was conducted according to the manufacturer’s protocol. Absorbance was measured at a wavelength of 450 nm.

Transwell and Wound Healing Assays

Transwell chambers were used to analyze the ability of cells to migrate and invade. A total of 5×10⁴ cells in serum-free culture medium were directly and uniformly distributed in the well for the migration assay. Similarly, the invasion assay was performed almost identically to the migration assay, except that the upper chamber was first precoated with Matrigel according to the manufacturer’s instructions. For both assays, medium containing 15% FBS was added to the lower chamber as a chemoattractant. The cells were incubated in an atmosphere containing 5% CO₂ for 12 (migration) or 16 h (invasion). The cells on the upper surface of the chamber were removed by wiping the membrane with a cotton swab, and invaded cells were fixed with 4% formaldehyde and stained with a 0.5% crystal violet solution. Cells were grown to confluence in 6-well plates. Six wounds were created in each well with sterile straw heads. The cells were washed with PBS and then cultured with DMEM. After 0 or 24 h of incubation, the same location in the wells was photographed to assess migration.

In vivo Xenograft Mouse Model

This experiment conforms to the the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and has been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval number: 2020–645). Athymic nude mice (4 weeks old) were obtained from the Experimental Animal Center of Chongqing Medical University. Mice were subcutaneously injected with 5×10⁶ HCC cells. Tumor size was monitored with calipers weekly. Mice were sacrificed 30 days after the injection, and subcutaneous tumor formation was observed.

Statistical Analysis

Data were analyzed using GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 20.0 software and are reported as means ± standard errors of the means (SEMs). T-tests or analysis of variance were used for statistical comparisons. *P <0.05 was considered statistically significant.

Results

FOXS1 Was Expressed at Lower Levels in Tumor Tissues and Associated with a Poor Prognosis

We conducted qRT-PCR, Western blotting and IHC to detect the levels of the FOXS1 mRNA and protein in paired samples of HCC and normal adjacent tissues.The FOXS1 mRNA was expressed at lower levels in HCC tissues than in the corresponding para-carcinoma tissues (Figure 1A). As shown in Figure 1B, the tumor tissue also expressed the FOXS1 protein at a significantly lower level. Immunohistochemical staining also revealed lower FOXS1 expression in HCC samples compared with adjacent normal liver samples (Figure 1C and D). Then, we detected FOXS1 expression in several human HCC cell lines. Compared with normal liver cells, the FOXS1 mRNA and protein were expressed at significantly lower levels in HCC cell lines (Figure 1E and F).

Figure 1 FOXS1 expression in HCC. (A) The FOXS1 mRNA is expressed at lower levels in tumor tissues than in adjacent normal liver tissues among the 26 paired HCC tissue and normal tissue samples. ***P<0.001 compared with normal tissues. (B) Western blot showing the level of the FOXS1 protein in 8 paired HCC and normal tissues. (C and D) Representative image of FOXS1 IHC staining in HCC tissues (100× and 400× magnification). **P<0.01 compared with normal tissues. (E) qRT-PCR analysis of the expression of the FOXS1 mRNA in the normal human liver cell line and five HCC cell lines. **P<0.01, ***P<0.001 vs HL-7702 cells. (F) Western blot showing the levels of the FOXS1 protein in the normal human liver cell line HL-7702 and five HCC cell lines (Huh-7, SMMC-7721, BEL-7402, Hep-3B and Hep-G2). All experiments were performed in triplicate.

Finally, we collected 40 pairs of HCC tissues and their adjacent para-carcinoma tissues and assessed the clinicopathological features of patients to further examine the association between FOXS1 expression and clinicopathological factors. FOXS1 expression was related to the TNM stage (Table 1), suggesting that FOXS1 expression levels may be a biomarker for liver cancer.

Table 1 Clinicopathological Characteristics of 40 Patients with HCC

Overexpression of FOXS1 Suppresses the Proliferation and Colony Formation of HCC Cells

We overexpressed FOXS1 in two cell lines, Hep-3B and Huh-7, to elucidate the function of FOXS1 in HCC cells (Figure 2A–D). CCK8 and colony formation assays were performed to analyze cell proliferation. The cell growth curve showed a significant decrease in the proliferation of the experimental group compared with the control group (Figure 2E and F). In addition, overexpression of FOXS1 significantly reduced the number of colonies formed by the two cell lines (Figure 2G and H).

Figure 2 FOXS1 inhibited the proliferation of HCC cells. (A–D) PCR and Western blot assays showing FOXS1 expression in transfected cells. ***P<0.001. (E and F) Cell proliferation was determined using a CCK8 assay. **P<0.01 and ***P<0.001 compared with NC (Huh-7) or NC (Hep-3B). (G and H) A lower level of colony formation was observed in the overexpression group than in the control group. *P<0.05 compared with NC (Huh-7) and **P<0.01 compared with NC (Hep-3B).

FOXS1 Inhibits the Migration and Invasion of HCC Cells

Transwell and wound healing experiments were conducted to investigate the effects of FOXS1 on the migration and invasion of HCC cells. FOXS1 overexpression inhibited the migration and invasion of Hep-3B and Huh-7 cell lines (Figure 3A and B). Similarly, in these two cell lines, the wound healing ability was lower than the control group when FOXS1 was overexpressed (Figure 3C and D). In addition, the EMT of Hep-3B and Huh-7 cells was inhibited by FOXS1 overexpression, as determined by the levels of E-cadherin, N-cadherin and other EMT-related proteins (Figure 3E).

Figure 3 FOXS1 overexpression in HCC cells suppressed cell migration, invasion, and EMT. (A and B) Transwell assays were performed to evaluate the migration and invasion of FOXS1-overexpressing cells. (100× magnification). **P<0.01 compared with NC (Huh-7) or NC (Hep-3B). (C and D) Wound healing assays were performed to detect migration (100× magnification). *P<0.01 compared with NC (Huh-7) or NC (Hep-3B). (E) Levels of EMT-related proteins were detected using Western blotting.

FOXS1 Alters the EMT in HCC via the Hh Signaling Pathway

The Hh signaling pathway is closely related to tumor formation.¹⁰ Prior to this study, FOXS1 was reported to be associated with the Hh pathway.¹¹ As shown in the figure, the levels of Sonic Hedgehog (Shh), Hedgehog receptor protein patched homolog 1 (PTCH1), smoothened (SMO) and glioma-associated oncogene homolog 1 (Gli1) were significantly decreased in the FOXS1-overexpressing Huh-7 cell line (Figure 4A). We used a SMO agonist to activate the Hh signaling pathway and further confirm whether FOXS1 modulates the EMT through the Hh signaling pathway. SAG induced SMO expression in FOXS1-overexpressing Huh-7 cells, and cell metastasis and invasion were significantly increased (Figure 4B–E). As shown in Figure 4F, SMO and GLI1 expression were significantly upregulated in the cell lines treated with the agonist, while E-cadherin levels were decreased and vimentin levels were increased. Thus, FOXS1 modulates the EMT through the Hh signaling pathway.

Figure 4 FOXS1 inhibited the EMT in HCC cells via the Hh signaling pathway. (A) Western blots showing the levels of the Gli1, Pith1, Smo, Shh, E-cadherin, and vimentin proteins. (B and C) The abilities of cells to migrate and invade were determined using Transwell assays (100× magnification). *P<0.05 and **P<0.01. (D and E) Wound healing assays were used to detect cell migration (100× magnification). *P<0.05 and **P<0.01. (F) Western blots showing the levels of the Gli1, Smooth, E-cadherin, and vimentin proteins.

FOXS1 Inhibits Tumor Growth in vivo

A subcutaneous xenograft mouse model was used to assess the effect of FOXS1 on tumor growth in vivo. Huh-7 cells overexpressing FOXS1 and negative control cells were injected into the backs of nude mice. The tumor size was measured for 4 weeks (Figure 5A), and all the mice were sacrificed after 4 weeks. The tumor size in the experimental group were significantly smaller than in the control group (Figure 5B). Based on these results, FOXS1 inhibited tumor growth. In addition, the expression of SMO, GLI1, E-calcitonin and Vimentin was detected using Western blotting (Figure 5C).

Figure 5 Effects of FOXS1 on the growth of Huh-7 cells in vivo. (A) Xenograft tumor formation in nude mice from the NC and FOXS1 overexpression groups is shown. **P<0.01. (B) The effect of FOXS1 on the tumor size is presented. (C) Protein levels of FOXS1, EMT markers, and Hh pathway markers in xenograft tissues were detected using Western blotting.

Discussion

Despite the rapid development of treatments, the prognosis of patients with HCC remains poor, due to high recurrence and drug resistance rates.¹² The metastasis of HCC also clearly decreases therapeutic efficacy in patients with HCC.¹³ Therefore, additional studies are urgently needed to obtain a better understanding of the mechanisms underlying the occurrence, development, and metastasis of HCC.

The liver plays an important role in the body. Hepatic transcription factors also play important roles in regulating the specific functions of the liver.^4,5 Hepatic transcription factors include members of the FOX family, the alkaline leucine zipper family, the PP-homologous domain family (but not HNF1) and the nuclear orphan receptor family.¹⁴ Among these families, liver-enriched FOX transcription factors exert a significant effect on the development of HCC.^15,16 FOXS1 plays a critical role in tumors.^5,6,8 Wang reported FOXS1 overexpression in gastric cancer tissue through a mechanism mediated by GLI1 and miR-125a-5p, which promotes gastric cancer cell proliferation and the EMT.⁵ Interestingly, Lu discovered that FOXS1 expression is downregulated in GC, which promotes cell proliferation and metastasis by activating the Wnt/β-catenin pathway.⁶ Previous research reports that FOXS1/GLI1 interact in some tumors, and high FOXS1 expression is associated with a better prognosis.¹⁷ However, the definitive contribution of FOXS1 to the invasion and migration of HCC remains unclear. In the current study, we detected FOXS1 expression and observed a decrease in HCC tissues and cells. Then, we evaluated the effect of FOXS1 on the invasion and migration of HCC and subsequently explored the underlying mechanisms. FOXS1 inhibited the invasion and migration of Huh7 and Hep-3B cells, and FOXS1 also suppressed cell proliferation. Finally, FOXS1 overexpression inhibited the Hh signaling pathway, and when a SMO agonist was used to activate the Hh pathway, the effect of FOXS1 expression was partially reversed. Additionally, tumor growth was significantly reduced upon FOXS1 overexpression in nude mice, similar to the phenomena observed in vitro. Based on these results, FOXS1 may be a potential tumor suppressor.

The EMT is reported to play key roles in metastasis and invasion and may lead to the early metastasis of tumors.¹⁸ The EMT occurs during embryonic development to allow cells to transition between epithelial and mesenchymal states, and the transition to mesenchymal states modifies the expression of adhesion molecules in cells to promote their migration and metastasis.¹⁸ In addition, the EMT plays an important role in many biological processes, including wound healing, embryonic development, and fibrosis.^19–21 Down-regulation of epithelial markers (E-cadherin) and up-regulation of mesenchymal markers (N-cadherin and vimentin) are the characteristics of the EMT,²² which play an important role in the early metastasis of cancer. The EMT plays an important role in HCC development.²³ However, the relationship between FOXS1 and the EMT in HCC remains unclear. Here, overexpression of FOXS1 reduced E-cadherin expression, increased N-cadherin, vimentin and Slug expression, and inhibited cell invasion and migration in HCC.

The Hh signaling pathway is associated with the development and homeostasis of several organs.^24,25 Hyperactivity associated with mutations of proteins in the Hh signaling pathway is also related to many malignancies.¹⁷ The Hh pathway has been shown to be associated with the initiation and maintenance of tumor growth, drug resistance and metastatic spread.¹⁷ For example, Hh signaling increases the proliferation and metastasis of human pancreatic cancer cells.²⁶ Activation of the Hh pathway plays an important role in the development of prostate cancer and its progression.²⁷ When FOXS1 was overexpressed, the expression of transcription factors related to Hh signaling were reduced. When an agonist of the Hh pathway was added to the treated cells, the invasion and metastasis were partially rescued, further confirming that FOXS1 modulated the EMT of HCC through the Hh pathway. FOXS1 may inhibit the expression of Gli1 by binding to the promoter region of Gli1, suggesting that Gli1 and FOXS1 may bind and inhibit each other’s expression, thus affecting the Hh pathway. In conclusion, FOXS1 participated in the EMT by modulating Hh signaling and inhibited HCC proliferation, indicating that FOXS1 plays a key role in HCC progression. Thus, FOXS1 might be a promising therapeutic target in HCC.