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Introduction

Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer with high rates of recurrence and mortality (1). Several studies reported that various genetic factors, such as gene mutations and epigenetic modifications, as well as environmental factors, are involved in HCC development (2). Despite improvements in the diagnosis and treatment of HCC in recent decades, HCC prognosis is poor and there are limited effective treatments (3). Tumor metastasis is a leading cause of poor prognosis for patients with HCC (4); therefore, determining the underlying biological mechanisms promoting HCC metastasis is crucial for the development of diagnostic and therapeutic interventions for HCC.

β-Klotho (KLB) is a single-pass transmembrane protein and is predominantly expressed in the liver, pancreas and white adipose tissues (5). The extracellular region of KLB consists of two internal repeats that share significant structural homology with family 1 glycosidase members, but lack intrinsic enzymatic activity (5,6). Human KLB is essential for the high-affinity binding of fibroblast growth factor (FGF)-19 and FGF21 to their cognate FGF receptors (FGFRs) (7). Upon food intake, the gut secretes FGF19, which binds to the FGFR4/KLB complex in hepatocytes to accelerate the metabolic response to feeding (8). By contrast, the liver secretes FGF21, the starvation hormone, which binds to the FGFR1c/KLB complex in adipocytes to induce metabolic responses under fasting conditions (9). It has been indicated that aberrant expression of FGF19-FGFR4 is a metastatic driver of HCC (10) and that KLB amplified FGF19-FGFR4 signaling in HCC (11). However, the activation of FGF21-KLB signaling and its role in HCC metastasis have remained to be fully elucidated.

Epigenetic regulations, such as DNA methylation, histone modification and microRNA regulation, are heritable and stable changes in gene expression that do not alter the DNA sequence (12,13). Epigenetic mechanisms have been reported to have crucial roles in tumor metastasis, including HCC metastasis (14). Histone acetylation is a widely studied post-translational epigenetic regulation that facilitates the dissociation of DNA and histone octamers and allows transcription factor binding to specific sites on the DNA to initiate gene transcription (15). Histone acetylation and deacetylation are conducted by histone acetyltransferases and histone deacetylases (HDACs), respectively (16). Previous studies demonstrated that HDACs have a critical role in tumor metastasis; therefore, epigenetic drugs may serve as effective therapeutic interventions against liver cancers (17,18). However, since epigenetic modifications occur in a cell or gene-specific manner, identification of the key genes of acetylation modifications is essential for the development of HCC treatments.

The present study found that KLB expression was increased in HCC tissues and was associated with HCC metastasis. In addition, KLB knockdown with simultaneous FGF21 overexpression promoted epithelial-mesenchymal transition (EMT) and HCC cell motility. Furthermore, that HDAC3 was indicated to be a potential deacetylase for KLB and treatment with HDAC3 inhibitor led to KLB inactivation, resulting in the blockade of FGF21-KLB signaling, further increasing the expression of EMT induction-related genes in HCC cells. Taken together, the present results indicate that aberrant acetylated modification of FGF21-KLB signaling contributes to EMT and HCC metastasis. Therefore, FGF21-KLB signaling may serve as a potential therapeutic target for HCC treatment.

Materials and methods

Cell culture and treatment

The human liver cancer cell lines LM3, HepG2, PLC/PRF/5, Li-7 and Huh7 and the murine HCC cell line Hepa1-6 were provided by the Hepatobiliary Institute of Nanjing University, while the normal hepatocyte cell line MIHA was provided by the Shanghai Institute of Cell Biology. Unless otherwise specified, all the experiments were conducted using the Huh7 cell line. Short tandem repeat profiling was conducted to authenticate all the cell lines used in the present study. The cells were cultured in DMEM or RPMI-1640 supplemented with 10% FBS (all from Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin, and maintained at 37°C and 5% CO2 in a humidified incubator. When the cell density reached 60–70%, the cells were treated with HDAC3 inhibitors, RGFP966 (cat. no. S7229; Selleck Chemicals) or Trichostatin A (TSA; cat. no. S1045; Selleck Chemicals).

Clinical specimens

HCC tissues and the corresponding adjacent normal tissues were provided by the Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China) with written informed consent from the patients concerned. The present study was approved by the local ethics committee. Western blot (WB) and immunohistochemistry (IHC) assays were used to evaluate the protein levels in the HCC tissues, as described previously (19). The following primary antibodies were purchased from Abcam: anti-KLB (WB, 1;1,000 dilution; IHC, 1:200 dilution; cat. no. ab106794) and anti-β-catenin (WB, 1:1,000 dilution; IHC, 1:400 dilution; cat. no. ab223075).

Bioinformatics analysis

Transcript levels of KLB in different cancer tissues and their corresponding normal tissues were analyzed by the ONCOMINE (https://www.oncomine.org/) and Tumor IMmune Estimation Resource (TIMER) (https://cistrome.shinyapps.io/timer/) databases. The transcriptomic data of KLB in normal and HCC samples were obtained from the Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/tcga) and its expression in HCC samples was further determined based on tumor grade, individual cancer stage, TP53 mutation status and nodal metastasis status using the UALCAN database. The Kaplan-Meier plotter (https://kmplot.com/) was used to analyze the prognostic value of the KLB gene in patients with HCC. Gene set evaluation analysis (GSVA) (https://www.aclbi.com/static/index.html#/) of TCGA data was used to determine the potential biological function of KLB.

Small interfering RNA (siRNA) assay

siRNAs were used to alter the expression of KLB and HDAC3 in the Huh7 cell line. The siRNAs with the highest silencing efficiency were screened and used for gene function research. After screening, the Huh7 cells (60–70% confluency in a 6-well plate) were treated with KLB- or HDAC3-siRNAs (si-KLB1, 5′-CGCUAUAGGAAUACAAUGUTT-3′; si-KLB2, 5′-GCUUCAAGCAAUAAGGUUATT-3′; and si-HDAC3, 5′-CACAAAUACGGAAAUUACUTT-3′) and the corresponding control-siRNAs (TSINGKE Biological Technology), which were encapsulated by the INTERFERin reagent (Polyplus-transfection SA). The sequence of the control siRNA was not provided by the supplier.

Lentivirus transductions

To inhibit KLB expression, the Huh7 cell line was infected with lentiviral vectors carrying short hairpin RNA against KLB (sh-KLB; target sequence, 5′-GCTTCAAGCAATAAGGTTA-3′). To overexpress FGF21, the huh7 cell line was transfected with lentiviral vectors carrying the FGF21 gene (GeneChem; sequences provided in supplementary data). Empty lentiviral vectors were used as internal controls.

WB and co-immunoprecipitation (co-IP) assays

Proteins were extracted from the HCC tissues and cell samples using RIPA buffer (KeyGEN BioTECH) with phenylmethylsulfonyl fluoride, according to the manufacturer's instructions, and the WB and co-IP assays were conducted as described previously (20). Anti-KLB (WB, 1:1,000 dilution; cat. no. ab106794), anti-E-cadherin (WB, 1:2,000; cat. no. ab40772), anti-claudin-1 (WB, 1:2,000; cat. no. ab211737), anti-MMP9 (WB, 1:2,000; cat. no. ab76003), anti-β-catenin (WB, 1:1,000; cat. no. ab223075), anti-laminB1 (WB, 1:5,000; cat. no. ab108536), anti-β-actin (WB, 1:1,000; cat. no. ab8226) and anti-GAPDH (WB, 1:10,000; cat. no. ab181602) were purchased from Abcam; anti-vimentin (WB, 1:20,000; cat. no. 60330-1-Ig) and anti-HDAC3 (WB, 1:1,000; cat. no. 10255-1-AP) were purchased from ProteinTech; and anti-acetylated lysine (WB, 1:1,000; cat. no. 9441) was purchased from Cell Signaling Technology, Inc. The bicinchoninic acid assay was conducted to determine the protein concentration. Each experiment was replicated thrice. The results of the WB assay were processed by Image J software (National Institutes of Health, version 1.8.0).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from the HCC cells using TRIzol™ (Thermo Fisher Scientific, Inc.) and subjected to RT-qPCR analysis using the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), as described previously (20). β-Actin was used as the internal reference gene and the 2−ΔΔCq method was used to evaluate the relative mRNA expression (21). The PCR primers were designed by Realgene (Nanjing, China) and are listed in Table I.

Table I. Primers for quantitative real-time PCR.

Table I.

Primers for quantitative real-time PCR.

Gene name	Forward primer	Reverse primer
β-actin	5′-ATTGCCGACAGGATGCAGAA-3′	5′-GCTGATCCACATCTGCTGGAA-3′
KLB	5′-TCTGTCATCCTGTCAGCACTT-3′	5′-CCAGTCCCAATACCCCAGAAAAA-3′
AXIN2	5′-CAACACCAGGCGGAACGAA-3′	5′-GCCCAATAAGGAGTGTAAGGACT-3′
MMP7	5′-GAGTGAGCTACAGTGGGAACA-3′	5′-CTATGACGCGGGAGTTTAACAT-3′
C-MYC	5′-GGCTCCTGGCAAAAGGTCA-3′	5′-CTGCGTAGTTGTGCTGATGT-3′
TCF7	5′-CTGGCTTCTACTCCCTGACCT-3′	5′-ACCAGAACCTAGCATCAAGGA-3′
β-Catenin	5′-AAAGCGGCTGTTAGTCACTGG-3′	5′-CGAGTCATTGCATACTGTCCAT-3′
E-cadherin	5′-CGAGAGCTACACGTTCACGG-3′	5′-GGGTGTCGAGGGAAAAATAGG-3′
N-cadherin	5′-TCAGGCGTCTGTAGAGGCTT-3′	5′-ATGCACATCCTTCGATAAGACTG-3′
SLUG	5′-CGAACTGGACACACATACAGTG-3′	5′-CTGAGGATCTCTGGTTGTGGT-3′
ZEB1	5′-GATGATGAATGCGAGTCAGATGC-3′	5′-ACAGCAGTGTCTTGTTGTTGT-3′
FGF21	5′-CTGTGGGTTTCTGTGCTGG-3′	5′-CCGGCTTCAAGGCTTTCAG-3′

[i] KLB, β-klotho; FGF21, fibroblast growth factor 21; TCF-7, transcription factor-7; ZEB1, zinc finger E-box binding homeobox 1.

Cell viability, EdU, apoptosis and colony-formation assays

Cell viability, EdU and apoptosis assays were conducted using a cell counting kit-8 (cat. no. C0037), the BeyoClick™ EdU kit (cat. no. C0075S) and an Annexin V-PE kit (C1065S; Beyotime Institute of Biotechnology), respectively, following the manufacturer's instructions. For the colony-formation assay, si-KLB-treated cells and negative control cells (n=500 each) were incubated in a 12-well plate and cultured for 2 weeks. Thereafter, the cells were fixed with methanol for 30 min and stained with 0.1% crystal violet for 30 min at room temperature. Finally, the colonies containing ≥50 cells were counted.

Wound-healing, cell-migration and cell-invasion assays

Wound-healing, cell migration and cell invasion assays were conducted as described previously (22). For the cell migration and invasion assays, migrated or invaded cells were counted using a microscope in six random fields. For the wound-healing assay, cell migration was calculated as the percentage of wound closure.

Immunofluorescence (IF) assay

The IF assay was conducted as described previously (20). In brief, treated cells were seeded in 24-well plates and incubated for 24 h. Thereafter, the cells were fixed using 4% paraformaldehyde and washed thrice with PBS. The cells were then treated with anti-vimentin antibodies (IF, 1:500 dilution; cat. no. 60330-1-Ig, ProteinTech), followed by incubation with goat anti-mouse IgG H&L (Alexa Fluor® 488; IF, 1:500 dilution; cat. no. ab150113; Abcam). The cells were then observed under a fluorescent microscope (Leica Microsystems GmbH) and the images were obtained.

Animal model

5×106 Huh7 cells were injected into male BALB/c nude mice (GemPharmatech; age, 3–4 weeks; weight, 16–20 g; n=6 per group) through the tail vein to generate the mouse lung metastasis model. The mice were housed with 5 mice per cage on a 12-h light/dark cycle, controlled humidity (50–60%) and temperature (24–26°C) and had free access to food and water (23). After 6 weeks, the mice were anesthetized and euthanized by intraperitoneal injection of pentobarbital sodium 150–200 mg/kg. The lungs were excised, imaged and embedded in paraffin. Tumor metastases were analyzed by hematoxylin and eosin staining according to standard procedures. The Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China) approved the animal experiments.

Statistical analysis

GraphPad Prism v8.02 (Dotmatics) was used to conduct data analyses and to calculate the P-value. Student t-test and one-way analysis of variance (ANOVA) with Dunnett's post-hoc test were used for comparisons between two or multiple groups, respectively. P<0.05 was considered to indicate a statistically significant difference.

Results

KLB expression is elevated in HCC and associated with HCC metastasis

KLB, a vital component of the endocrine FGFR complex, is involved in tumor progression in several cancers. To obtain KLB expression profiles in human cancers, pan-cancer KLB expression data were retrieved from the ONCOMINE and TIMER databases. As presented in Fig. 1A and B, KLB expression was lower in a majority of cancer tissues, including breast cancer, colorectal cancer, lung cancer and pancreatic cancer, compared with their corresponding normal tissues. However, KLB expression was markedly increased in HCC tissues compared to the corresponding normal tissues (Fig. 1B). Differential-expression analysis of the TCGA-liver hepatocellular carcinoma data using paired and unpaired t-tests revealed that KLB expression was significantly increased in the HCC tissues compared with the normal tissues (Fig. 1C and D). Immunoblotting and IHC staining were conducted to assess the protein levels of KLB in 10 human HCC tissues and their corresponding adjacent non-cancerous tissues, to further confirm KLB expression in HCC. The results revealed that KLB protein levels were markedly higher in the HCC tissues (Fig. 1E), which is consistent with previously published results (11). In addition, KLB expression was also evaluated in various HCC cell lines (LM3, HepG2, PLC/PRF/5, Li-7 and Huh 7) and a normal hepatocyte cell line (MIHA), and the results suggested that the mRNA and protein levels of KLB were significantly upregulated in Huh7, HepG2 and Li-7 compared with MIHA (Fig. 1F and G). Furthermore, KLB expression was observed to gradually increase with tumor grade (Fig. 1H). Altogether, these findings confirmed that KLB expression is high in HCC tissues and cell lines.

Figure 1. KLB expression is elevated in HCC and associated with HCC metastasis. (A and B) Pan-cancer KLB expression analysis using (A) ONCOMINE and (B) Tumor IMmune Estimation Resource databases. (C and D) The transcriptomic data of KLB in HCC tissues and adjacent normal tissues were obtained from the TCGA database. Analysis of the KLB expression levels in HCC tissues and adjacent normal tissues using (C) unpaired and (D) paired t-tests. (E) Representative images of KLB expression obtained by western blot analysis of HCC tissues. (F) Western blot and (G) quantitative real-time PCR analyses indicated increased KLB expression in most HCC cell lines. (H) Representative images of immunohistochemistry staining of KLB in different stages of human HCC tissues compared with the adjacent normal tissues (scale bars: Upper panel, 100 µm; lower panel, 50 µm). (I) The non-alcohol-consuming HCC patients were subjected to overall survival analysis based on KLB expression level. (J) KLB expression in HCC according to tumor grade, disease stage, TP53 mutation status and nodal metastasis status. **P<0.01, ***P<0.001. ns, not significant; KLB, β-klotho; HCC, hepatocellular carcinoma; N, normal tissue; C, cancer tissue; HR, hazard ratio; TCGA, The Cancer Genome Atlas; ACC, adrenocortical carcinoma; BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

To determine the clinical significance of increased KLB expression in HCC, a survival analysis was performed using Kaplan-Meier plotter. It was indicated that, although KLB is elevated in HCC, its high expression was associated with a favorable prognosis in non-alcohol-consuming patients with HCC (Fig. 1I), thus implying that KLB may serve as a protective modulator in HCC. These results suggest that high expression of KLB may be considered a prognostic biomarker for a specific population of patients with HCC. Further survival analysis of KLB gene expression in non-alcohol-consuming HCC patients with and without vascular invasion revealed that KLB was a specific prognostic marker for patients with HCC without vascular invasion (Fig. S1A and B). To further determine the expression of KLB in HCC development, KLB expression was analyzed according to tumor grade, disease stage, TP53 mutation status and lymph node metastasis status. As presented in Fig. 1J, KLB expression increased continuously with HCC development, suggesting that KLB has an important role in HCC development and metastasis.

Knockdown of KLB promotes HCC cell migration and invasion

To elucidate the role of KLB in HCC progression, a KLB-knockdown Huh7 cell line was generated (Fig. S1C). Consistent with the results of a previous study (11), the EdU, apoptosis, CCK-8 and colony-formation assays revealed that KLB knockdown reduced cell proliferation and induced cell apoptosis in Huh7 cells (Fig. 2A-D). These findings demonstrate the involvement of KLB in the pathogenesis of HCC and highlight the need to investigate its role in enhancing the FGF19-FGFR4 signaling pathway. However, wound-healing migration, Transwell migration and Matrigel invasion assays in Huh7 and Hepa1-6 cell lines revealed that KLB knockdown enhanced HCC cell motility (Fig. 2E-H). This may partly explain why high expression of KLB is associated with a favorable prognosis in patients with HCC without vascular invasion.

Figure 2. Knockdown of KLB promotes HCC cell migration and invasion. (A) EdU (scale bar, 50 µm), (B) flow cytometry, (C) cell counting kit-8 and (D) colony-formation assays (scale bar, 15 mm diameter) of Huh7 cells after KLB knockdown. (E and F) Transwell migration and invasion assays of Huh7 cells after 48 h of si-KLB treatment. (E) Representative images (scale bar, 100 µm) of Transwell migration and invasion assays of Huh7 cells after KLB knockdown. (F) Representative images (scale bar, 100 µm) of Transwell migration and invasion assays of Hepa1-6 cells after KLB knockdown. (G) Wound closure (wound-healing assay) was monitored at 0, 24 and 48 h after wounding (scale bar, 100 µm) in KLB knockdown Huh7 cells. (H) Wound closure (wound healing assay) was monitored at 0, 24 and 48 h after wounding (scale bar, 100 µm) in KLB knockdown Hepa1-6 cells. *P<0.05, **P<0.01, ***P<0.001. ns, not significant; KLB, β-klotho; HCC, hepatocellular carcinoma; NC, negative control; si-KLB, small interfering RNA targeting KLB; OD, optical density.

KLB is identified as a novel upstream regulator of β-catenin signaling

GSVA was performed to further explore the underlying biological function of KLB in HCC and the results demonstrated that KLB is closely associated with β-catenin signaling (Fig. 3A). In addition, it was found that KLB knockdown significantly inhibited the mRNA and protein expression of several critical genes in the β-catenin signaling pathway, including β-catenin, C-MYC, MMP7, transcription factor (TCF)-7 and phosphorylated glycogen synthase kinase 3β-S9 in Huh7 cells; however, the expression of nuclear β-catenin was not obvious (Fig. 3B and C). These results further verified the accuracy of the enrichment analysis, thus suggesting that KLB is possibly involved in the β-catenin-mediated signaling activation in HCC. IHC staining of the HCC tissues and their corresponding normal tissues revealed a high correlation between KLB and β-catenin gene (CTNNB1) expression, consistent with the results of the gene expression profiling interactive analysis (Fig. 3D and E).

Figure 3. KLB is identified as a novel upstream regulator of β-catenin signaling. (A) GSVA revealed that KLB expression was closely associated with β-catenin signaling. (B) Quantitative real-time PCR and (C) western blot assays indicated that KLB knockdown inhibited the expression of β-catenin signaling-related genes. (D) Immunohistochemistry analysis (scale bars, 50 µm) and (E) gene expression profiling interactive analysis of hepatocellular carcinoma tissues and their corresponding normal tissues showed a strong correlation between KLB and CTNNB1 expression. *P<0.05, **P<0.01. ns, not significant; KLB, β-klotho; GSVA, Gene Set Variation Analysis; NC, negative control; si-KLB, small interfering RNA targeting KLB; CTNNB1, β-catenin; GSK, glycogen synthase kinase; TCF-7, transcription factor-7.

FGF21-KLB signaling inhibits HCC metastasis via the β-catenin signaling pathway

Aberrant FGF19-FGFR4-KLB signaling is a metastatic driver for HCC (10), while the function of FGF21-KLB signaling in HCC metastasis remains to be clarified. Therefore, to explore the role of FGF21-KLB signaling in regulating HCC metastasis, Huh7 cells were transduced with lentivirus carrying the FGF21 gene to induce stable expression of FGF21 (Fig. S1D). Several studies demonstrated that β-catenin expression increased, while E-cadherin expression decreased in diabetic FGF21-knockout mice (24). In the present study, it was found that enhanced FGF21 expression inhibited the transcription of AXIN2, CTNNB1, C-MYC and TCF7. However, KLB knockdown in FGF21-overexpressing cells (Fig. S1E and F) caused increased expression of CTNNB1 and TCF7 (Fig. 4A and B), suggesting that FGF21 is an upstream mediator of the β-catenin signaling pathway and that its regulation is dependent on KLB expression. Thereafter, it was examined whether FGF21-KLB induced HCC metastasis via the EMT and it was found that FGF21 overexpression enhanced the protein level of E-cadherin (essential for the establishment of stable adherent junctions), which is downregulated during EMT (Fig. 4D). Subsequently, the mRNA levels of E-cadherin and several EMT inducers, including N-cadherin, Slug and zinc finger E-box binding homeobox 1 (ZEB1), were measured in both FGF21-overexpressing and sh-KLB-treated FGF21-overexpressing cells (Fig. 4C). Compared with FGF21-overexpressing cells, sh-KLB-treated FGF21-overexpressing cells had reduced mRNA levels of E-cadherin and increased mRNA levels of Slug and ZEB1 (key transcriptional repressors of E-cadherin). Furthermore, compared with the FGF21-overexpressing cells, claudin-1 [a repressor of E-cadherin (25)] was increased, while zonula occludens (ZO)−1 (a member of the ZO proteins that constitute tight junctions) was decreased in the sh-KLB-treated FGF21-overexpressing cells (Fig. 4E). Meanwhile, IF staining revealed an upregulation of vimentin expression in the sh-KLB-treated FGF21-overexpressing cells compared with the FGF21-overexpressing cells (Fig. 4F). Furthermore, the wound-healing migration, Transwell migration and Matrigel invasion assays revealed that the sh-KLB-treated FGF21-overexpressing cells had an enhanced migration and invasion ability compared with the FGF21-overexpressing cells (Fig. 4G,H). To confirm the effects of FGF21-KLB on the tumorigenicity of HCC in vivo, a lung metastasis model was established. Consistent with previous results, sh-KLB-treated FGF21-overexpressing cells had an enhanced in vivo metastatic ability compared with the FGF21-overexpressing cells (Fig. 4I and J). Taken together, these results suggest that FGF21 suppresses HCC cell metastasis through the β-catenin signaling pathway, while KLB knockdown promotes HCC cell motility under FGF21 overexpression.

Figure 4. FGF21-KLB signaling inhibited hepatocellular carcinoma (HCC) metastasis in vitro and in vivo via the β-catenin signaling pathway. (A and B) The mRNA levels of AXIN2, CTNNB1, c-MYC, TCF7 and MMP7 in the indicated cells. (C) The mRNA levels of E-cadherin, N-cadherin, Slug and ZEB1 in the indicated cells. (D) The protein levels of E-cadherin and claudin-1 in the indicated cells. (E) The protein levels of ZO-1, vimentin and claudin-1 in the indicated cells. (F) Immunofluorescence assay to detect vimentin expression in the indicated cells (scale bar, 100 µm). (G) Representative images (scale bar, 100 µm) of Transwell migration and invasion assays of the indicated cells. (H) Wound-healing assay of the indicated cells. Wound closure was monitored at 0, 24 and 48 h after wounding (scale bar, 100 µm). (I and J) Representative HE images from the lung metastasis models established by injection of (I) FGF21-overexpressing and (J) sh-KLB-treated FGF21-overexpressing cells (scale bar, 100 µm). *P<0.05, **P<0.01, ***P<0.001. ns, not significant. FGF21, fibroblast growth factor 21; KLB, β-klotho; CTNNB1, β-catenin; NC, negative control; sh-KLB, short hairpin RNA targeting KLB; TCF-7, transcription factor-7; CAD, cadherin; ZO-1, zonula occludens 1; ZEB1, zinc finger E-box binding homeobox 1.

HDAC3 inhibitor-mediated acetylated modification of KLB facilitates the activation of HCC metastasis-promoting genes

The methylation level of the KLB promoter decreased gradually with an increase in tumor grade and disease stage and was associated with TP53 mutation status and lymph node metastasis status (Fig. S1H). It was hypothesized that the epigenetic modification of KLB may contribute to its high expression in HCC. A previous study demonstrated that HDAC3 catalyzes the acetylation of Klotho, a paralog of KLB (26); however, its role in KLB modification has not yet been determined. Therefore, to determine the regulatory effects of epigenetic changes on KLB expression, HCC cells were treated with TSA (a broad-spectrum HDAC inhibitor) and RGFP966 (an HDAC3-specific inhibitor). The results demonstrated that KLB expression decreased in a dose-dependent manner in TSA and RGFP966-treated HCC cells (Fig. 5A and B). Furthermore, it was found that the KLB promoter was indeed acetylated and that its acetylation was significantly increased after treatment with TSA and RGFP966, suggesting that KLB is acetylated in HCC (Fig. 5C and D).

Figure 5. HDAC3 inhibitor-mediated acetylated modification of KLB facilitates the activation of HCC metastasis-promoting genes. (A and B) KLB expression decreased after treatment with (A) TSA (broad-spectrum HDAC inhibitor) and (B) RGFP966 (HDAC3-specific inhibitor) in a dose-dependent manner. (C and D) KLB acetylation levels increased after treatment with (C) TSA and (D) RGFP966 in a dose-dependent manner. The acetylation status was determined by western blot assay using anti-AcK antibodies. (E) Immunoprecipitation assay of the indicated cell extracts using anti-KLB antibodies and IgG (control). Acetylated KLB was detected by western blot assay using AcK. (F) The protein levels of KLB, MMP9 and vimentin were detected in the indicated cells. (G) The protein levels of ZO-1 and vimentin were detected in the indicated cells. (H) Wound-healing assay demonstrated the effect of RGFP966 on HCC cell migration with simultaneous FGF21 overexpression (scale bar, 100 µm). **P<0.01. FGF21, fibroblast growth factor 21; HDAC3, histone deacetylase 3; AcK, acetylated lysine; HCC, hepatocellular carcinoma; KLB, β-klotho; TSA, trichostatin A; ZO-1, zonula occludens 1; si-HDAC3, small interfering RNA targeting HDAC3; Ctrl, control; NC, negative control; IP, immunoprecipitation; IB, immunoblot.

Thereafter, a co-IP assay was performed to determine whether HDAC3 is able to regulate the acetylation status of KLB. The si-HDAC3-transfected Huh7 cells (Fig. S1G) were subjected to IP analysis using anti-KLB, followed by WB analysis with anti-acetylated-lysine antibodies, to detect the levels of acetylated KLB. The results revealed that KLB acetylation was significantly increased in the si-HDAC3-treated group compared with the untreated group (Fig. 5E). Subsequently, to determine the functional significance of HDAC3-mediated acetylation of KLB, the effects of decreased HDAC3 on KLB-knockdown and KLB-overexpressing cells were analyzed. As presented in Fig. 5F, treatment with either si-KLB or HDAC3 inhibitor increased the expression of vimentin and MMP9, while their combined application resulted in increased vimentin and MMP9 expression. However, these effects were reversed by KLB overexpression. Furthermore, the HDAC3 inhibitor induced a significant decrease in ZO-1 expression and an increase in vimentin expression, which was able to be inhibited by FGF21 overexpression (Fig. 5G). In addition, the wound-healing experiments demonstrated that the addition of HDAC3 inhibitor significantly enhanced HCC cell migration under FGF21 overexpression (Fig. 5H). Based on the above results, it was hypothesized that HDAC3 inhibitor inhibits KLB expression by aberrant acetylated modification, resulting in the blockade of FGF21-KLB signaling, thereby increasing the expression of EMT-inducing genes.

Discussion

Recurrence and distant metastasis are common in patients with advanced HCC and their management poses a significant challenge (27). Aberrant activation of the receptor tyrosine kinase (RTK) signaling has been found in HCC. In addition, the clinical benefits of sorafenib further demonstrate the efficacy of targeting multiple RTK pathways in HCC (28). However, another study found that sorafenib treatment accelerates HCC metastasis (29). While the US Food and Drug Administration has recently approved several novel drugs for HCC treatment, the majority of these are multikinase inhibitors, similar to sorafenib, and are thus less likely to inhibit HCC metastasis. Therefore, a comprehensive understanding of the repertoire of pathways that are frequently and selectively upregulated in HCC may facilitate the identification of novel therapeutic targets for HCC treatment.

β-Catenin is elevated in HCC tissues and its mutations are frequently identified in HCC, suggesting that β-catenin may be critical for HCC development and progression (30). It has been widely reported that aberrant activation of the β-catenin signaling pathway is closely associated with tumor invasion and metastasis and involved in tumor microenvironment formation (31). Although the use of β-catenin pathway-targeting molecules is unlikely to produce a viable antitumor effect, identification of the factors upstream of the β-catenin pathway may help in the identification of novel therapeutic targets for HCC treatment. In the present study, KLB was identified as a novel upstream regulator of β-catenin. However, the underlying mechanisms by which KLB and β-catenin promote HCC metastasis require further investigation.

FGF21, a stress-induced hormone, interacts with FGFR-1 and KLB on the target cells via endocrine, paracrine and autocrine pathways, and subsequently exerts its biological effects by regulating energy balance and glucose-lipid balance (32). FGF21 is primarily expressed in hepatocytes, adipose tissue and pancreas, and sufficient FGF21 directly reduces hepatic lipid accumulation in an insulin-independent manner, and inhibits lipolysis in white adipose tissues, further reducing circulating free fatty acid levels (33). Administration of FGF21 in rodents and non-human primates led to the alleviation of several obesity-related metabolic complications, including reduced fat mass and hyperglycemia, insulin resistance, dyslipidemia, cardiovascular disease and nonalcoholic steatohepatitis (NASH), by reducing oxidative stress and lipid peroxidation (34). Furthermore, FGF21 prevents the development of advanced pathologies, such as pancreatic ductal adenocarcinoma and HCC (35). A study from 2006 found that overexpression of FGF21 in hepatocytes led to a delay in the development of chemically-induced liver tumors (36); however, the underlying mechanisms were not known at the time. Recent studies have confirmed that low FGF21 levels may activate Toll-like receptor 4-interleukin-17A signaling pathway in hepatocytes to promote NASH-HCC transformation, revealing the crucial role of FGF21 in liver cancer (37). Furthermore, high serum FGF21 levels are associated with worse survival in patients with HCC, suggesting its use as a novel metabolism-related prognostic biomarker for HCC (38). Therefore, the FGF21 signaling pathway may serve as a potential prognostic factor, as well as a therapeutic target for HCC. The application of several FGF21 analogs has led to significant improvements in dyslipidemia, liver fat fraction and serum markers of liver fibrosis in patients with NASH in the preclinical stages (32). However, FGF21 has limited indications for clinical use due to its poor pharmacokinetic and biophysical properties. Therefore, discerning the pathways associated with FGF21-mediated regulation of HCC will facilitate the development of effective HCC treatments. FGF21 is considered an emerging therapeutic target for NASH and related metabolic diseases and may provide a new perspective on the role of high KLB expression in the prognosis of non-alcohol-consuming patients with HCC. In the present study, the role of FGF21-KLB signaling in the regulation of HCC metastasis was revealed from an epigenetic standpoint. Mechanistically, FGF21 suppressed HCC metastasis by inhibiting β-catenin signaling-mediated EMT, while acetylation-driven suppression of KLB promoted HCC cell motility under FGF21 overexpression. These findings suggest that FGF21-KLB signaling may serve as a potential biomarker and a therapeutic target for HCC.

EMT is a key process in tumor metastasis. Increasing evidence has suggested that aberrant acetylation of EMT-related genes is involved in tumorigenicity and HCC metastasis (39). HDAC-mediated histone acetylation inhibits E-cadherin expression or induces mesenchymal protein expression to facilitate HCC migration and invasion, thereby promoting HCC metastasis (40). Therefore, HDAC inhibitors serve as attractive targets for cancer treatment. Panobinostat, a novel hydroxamic acid-derived HDAC inhibitor, has demonstrated promising anticancer effects by inhibiting HCC growth and metastasis (41). However, other studies found that HDAC inhibitors promote the expression of Snail and induce EMT in HCC cells, thus limiting the clinical outcome of HDAC inhibitor-based therapies in HCC (42,43). In the present study, it was revealed that HDAC3 is a potential deacetylase for KLB. It was further demonstrated that HDAC3 inhibitor-mediated acetylation modification downregulated KLB expression, causing a blockade of FGF21-KLB signaling, further increasing the expression of EMT induction-related genes. Consistently, a previous study reported that HDAC3 deficiency promotes liver cancer through a defect in H3K9ac/H3K9me3 transition (44). Since different target genes of HDAC inhibitors may lead to distinct effects, further identification of the key genes involved in HDAC-mediated acetylation modifications may lead to the development of effective therapeutic interventions for HCC.

There are certain limitations to the present study. First, most of the in vitro experiments in the present study were conducted on Huh7 cells; however, HCC cells are highly heterogeneous and further studies on other HCC cell lines are required to verify the results of the present study. Furthermore, KLB serves as a co-receptor for both FGF21 and FGF19; thus, the present findings may have been a result of KLB knockdown-mediated blockade of FGF19 signaling. For instance, upregulation of the FGF15 (FGF19 orthologue in mice)/FGFR4 signaling in a lipid metabolism disorder mouse model promoted HCC development by activating EMT and Wnt/β-catenin signaling, while activation of the FGF21/KLB signaling induced the downregulation of β-catenin signaling, which may explain why nuclear β-catenin expression is not apparent after KLB knockdown (45,46). In the present study, only FGF21-overexpressing cells and sh-KLB-treated FGF21-overexpressing cells were used to observe the effect of KLB knockdown on FGF21 signaling, and these results need to be verified further in subsequent studies.

In summary, the present study revealed the role of KLB in the regulation of EMT in HCC from an epigenetic perspective. In addition, FGF21 was indicated to exert its anti-HCC metastatic role in a KLB-dependent manner. Furthermore, HDAC3-mediated suppression of KLB was found to accelerate HCC-cell migration and invasion by blocking FGF21 signaling (Fig. 6), which may serve as a potential therapeutic target for HCC treatment.

Figure 6. A schematic model generated by Figdraw illustrating the role of FGF21-KLB signaling in the regulation of EMT-related genes and β-catenin signaling pathway. FGF21, fibroblast growth factor 21; HDAC3, histone deacetylase 3; HCC, hepatocellular carcinoma; KLB, β-klotho; EMT, epithelial-mesenchymal transition; GSK, glycogen synthase kinase; TCF, transcription factor; prom, promotion; Ac, acetyl group.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

The graphical figure was generated by Figdraw (www.figdraw.com).

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 82270646), the Fundamental Research Funds for the Central Universities (grant no. 0214-14380510), the Nanjing Health Science and Technology Development Project for Distinguished Young Scholars (grant no. JQX19002), Project of Modern Hospital Management and Development Institute, Nanjing University and Aid Project of Nanjing Drum Tower Hospital Health, Education & Research Foundation (grant no. NDYG2022057), fundings for Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (grant no. 2022-LCYJ-PY-35) and the Chen Xiao-Ping Foundation for the Development of Science and Technology of Hubei Province, China (grant no. CXPJJH121001-2021073).

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

JX and ZZ conducted the experiments and collected the data; GW, YC, RA and SX analyzed the data; and HR and WG conceived the study, checked and confirmed the authenticity of the raw data, and prepared the manuscript. All the authors have read and approved the final draft of the manuscript.

Ethics approval and consent to participate

Human HCC tissues were obtained in accordance with the Declaration of Helsinki, and all of the patients concerned provided written informed consent. Clinical experiments were approved by the Medical Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China; no. 2019-257-02). Animal experiments were performed in accordance with the international guidelines; they were approved by the Animal Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School University (Nanjing, China; no. 2021AE01021).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References