CTCF-activated FUCA1 functions as a tumor suppressor by promoting autophagy flux and serum α-L-fucosidase serves as a potential biomarker for prognosis in ccRCC

Abstract

Notably, clear cell renal cell carcinoma (ccRCC) is characterized by a distinct metabolic tumor phenotype that involves the reprogramming of multiple metabolic pathways. Although there is increasing evidence linking FUCA1 to malignancies, its specific role and downstream signaling pathways in ccRCC remain poorly understood. Here we found that FUCA1 expression was significantly downregulated in ccRCC tissues, which also predicts poor prognosis of ccRCCpatients. Moreover, enhancing FUCA1 expression resulted in reduced invasion and migration of ccRCC cells, further indicating its protective role. CHIP-qPCR and luciferase assays showed that CTCF was an upstream transcription factor of FUCA1 and could reverse the effects caused by FUCA1 inactivation. The change in FUCA1 led to changes in the results of various autophagy-related proteins and the mRFP-GFP-LC3 dual fluorescence system, indicating that it may play a role in the fusion stage of autophagy. Protein-protein interaction analysis revealed that FUCA2 exhibited the closest interaction with FUCA1 and strongly predicted the prognosis of ccRCC patients. Additionally, serum AFU encoded by FUCA2 could serve as a valuable predictor for survival in ccRCC patients. FUCA1 suppresses invasion and migration of ccRCC cells, with its activity being modulated by CTCF. FUCA1 regulates the autophagy process in ccRCC cells by influencing the fusion between autophagosomes and lysosomes. FUCA2 shares similarities with FUCA1, and elevated serum AFU levels along with increased expression of FUCA2 are indicative of a favorable prognosis in ccRCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-024-03502-2.

Introduction

Renal cell carcinoma (RCC) is among the ten most frequently diagnosed cancers worldwide and is the third most prevalent urological cancer. In particular, its incidence has been increasing in Western countries [1–3]. It is notable that RCC is associated with a poor prognosis, representing an annual threat to approximately 372,000 lives globally [4].

RCC encompasses three primary subtypes, with clear cell renal cell carcinomas (ccRCC) constituting the majority (~ 75%) and arising from the proximal tubular epithelium as a highly aggressive form of cancer [5, 6]. In clinical practice, approximately 70% of newly diagnosed ccRCC patients present with localised or locally advanced disease, for which surgical intervention represents the main curative strategy. Nevertheless, disease recurrence still occurs in 20–40% of patients with locally advanced ccRCC. Despite the availability of therapeutic options such as axitinib (a VEGF receptor tyrosine kinase inhibitor) and nivolumab (an immune checkpoint blocker), which have been approved for the treatment of ccRCC over the past decade, achieving satisfactory results has proven challenging [7, 8]. The response rate and five-year survival remain poor in patients with advanced ccRCC [2]. Given this current scenario, it is crucial to elucidate potential genes associated with the pathogenesis of ccRCC.

Aberrant glycosylation is frequently associated with the initiation and progression of cancer, reflecting cancer-specific alterations in glycan metabolic pathways. These alterations encompass modifications to the expression of glycosyltransferases and glycosidases, which are involved in the biosynthesis and catabolism of glycoconjugates [9, 10]. The FUCA1 gene, situated on chromosome 1p (1p34), encodes a lysosomal enzyme designated FUCA1, which is capable of removing terminal l-fucose residues present in glycoproteins [11, 12]. Concurrently, FUCA1 and FUCA2 collectively constitute the α-L-fucosidase (AFU), which is predominantly situated in the tissues and plasma [12]. The first indication that FUCA1 was a relevant gene came from the observation that its deficiency is associated with an autosomal recessive lysosomal storage disorder known as fucosidosis. This disorder is characterised by progressive neurological deterioration and mental retardation [13]. Recent studies have shown that FUCA1 is significantly associated with several types of cancers, such as colorectal cancer, thyroid cancer, glioma and oesophageal squamous cell carcinoma, and plays a dual role in these cancers [14–17], Ezawa et al. investigated the involvement of FUCA1 in the downstream regulation of P53, leading to tumour suppression while also contributing to the repression of the EGFR signalling pathway [11]. Xu et al. reported that silencing FUCA1 could suppress glioma progression by enhancing autophagy and inhibiting macrophage infiltration [16]. However, the precise impact of FUCA1 expression on ccRCC patients and the underlying mechanism remain incompletely understood. The CCCTC-binding factor (CTCF) is a multifunctional protein that plays fundamental roles in physiological regulatory activities, including transcriptional activation/repression, isolation, and imprinting [18]. Studies have shown that CTCF’s ‘master weaver’ function can inhibit transcription by acting as an insulator at topologically associated domain (TAD) boundaries or as an enhancer blocker [19–21]. Meanwhile, CTCF could also activate transcription when it acts as a transcription factor at promoters or as a promoter-enhancer looping factor [22, 23].

Autophagy, or macroautophagy, is a type II programmed cell death pathway that plays a crucial role in maintaining cellular homeostasis. It enables the degradation and recycling of cellular components, which is essential for the proper functioning of cells and the organism as a whole [24]. The role of autophagy in tumourigenesis can be both suppressive and promotive, and is often influenced by specific oncogenic mutations and the cellular context [24]. The autophagy gene BECN1 has been observed to undergo frequent monoallelic deletion in human breast, ovarian, and prostate cancers. In mice, this gene functions as a haploinsufficient tumour suppressor. These findings lend support to the hypothesis that autophagy may have anticancer potential [25–27]. Previous studies have identified multiple mutations in the phosphatidylinositol 3-kinase pathway and MTOR that may inhibit autophagy in ccRCC [28]. Moreover, it has been demonstrated by several research groups that inducers of autophagy can effectively suppress the development of ccRCC [29–31]. In light of these findings, we postulate that autophagy may exert a tumour-suppressive role in ccRCC.

The present study demonstrated a significant down-regulation of FUCA1 in ccRCC, shedding light on the enhancement of autophagic flux by upregulating FUCA1, which in turn inhibits the progression of ccRCC. Moreover, our findings unveiled the crucial role of the CTCF transcription factor as a master upstream regulator of FUCA1, thereby promoting its expression. These novel findings not only establish the pivotal role of FUCA1 in ccRCC but also present it as a potential therapeutic target for patients with this disease.

Materials and methods

Patients and samples

A total of 19 patients who had undergone surgical treatment for clear-cell renal cell carcinoma (ccRCC) at Qilu Hospital were included in the present study. The primary tumour samples and non-cancerous adjacent renal tissues (NTs) were collected and stored at − 85 °C until use. The usability of the NTs and tumour tissue samples was evaluated by two independent pathologists. All patients provided informed consent to participate in the study, which was conducted in accordance with the ethical guidelines set out in the Declaration of Helsinki and approved by the Ethics Committee of Shandong University Qilu Hospital. The clinicopathological information of patients with ccRCC was retrospectively collected and analysed through the electronic medical records at Qilu Hospital of Shandong University from January 2015 to December 2018. A total of 456 patients with ccRCC met the inclusion criteria following the application of the exclusion criteria (Fig. S1). The patients who met the eligibility criteria were subsequently monitored for the purpose of conducting a survival analysis. A subset of 401 ccRCC patients from this group was included in the effective follow-up cohort.

Serum AFU measurement

A 6 ml venous blood sample was collected after a 12-hour fast without any clinical intervention. The blood was collected into tubes containing coagulant. The sample was then centrifuged at 2000 rpm for 8 min, and the serum was separated using a Roche Cobas8000 automated analyser (Roche, Switzerland) to determine AFU concentration.

TCGA and GEO databases analysis

The ccRCC cohorts were downloaded from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/tcga) to analyse the expression of FUCA1 and CTCF in ccRCC and their influence on prognosis of ccRCC patients. The dataset GSE22541 was derived from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) to further verify the influence of FUCA1 on ccRCC prognosis. The dataset GSE13478 were retrieved from GEO to study the correlation between FUCA1 and pulmonary metastases in ccRCC. The JASPAR (http://jaspar.genereg.net/) was used to analyse the CTCF binding sites.

Gene set enrichment analysis

In order to explore the biological signalling pathway, Gene set enrichment analysis (GSEA) was performed in the high FUCA1 expression and the low FUCA1 expression groups respectively. Gene sets with nominal p-values < 0.05, |NES|>1 and FDR q < 0.25 were considered statistically significant.

Protein–protein interaction network construction

GeneMANIA (http://www.genemania.org) is an interactive and intuitive website for constructing protein-protein interaction (PPI) network, which generates hypotheses about gene function prediction and detects genes with similar functions [32]. We used GeneMAMA to explore the function of FUCA1.

Cell lines and culture

786O, A498, Tk10, Caki1 and Caki2 ccRCC cancer cells were purchased from CellScource China. Cells were used within 15 passages in each designed experiment. They were routinely tested for mycoplasma contamination. All cell culture media contained 10% fetal bovine serum (ExCell Bio, China), 1% penicillin and streptomycin (KeyGen Biotech, China). All cell lines were incubated at 37 °C in 5% CO₂.

Western blotting

ccRCC tumour tissue or cultured cells were mixed in lysis buffer containing protease inhibitors, subjected to centrifugation at an appropriate speed for 15 min at 4 °C, and the protein concentration in the resulting supernatant was measured using a BCA protein quantification kit (Solarbio, China) according to the manufacturer’s protocol. The precipitated proteins were fractionated by 10% SDS/PAGE and transferred to PVDF membrane (Millipore, USA) for 2 h. The membranes were then blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against overnight at 4 °C. The membranes were then washed with PBS and incubated with secondary antibodies for 80 min at room temperature. Finally, protein bands were detected using ECL assay kit (Millipore, USA) and quantified using Image J software (National Institutes of Health). GAPDH (mouse, 1:1000, Santa Cruz Biotechnology) was used as an internal control. The primary antibodies employed included FUCA1 (1:1000 dilution; Proteintech), CTCF (1:1000 dilution; Proteintech), P62 (1:1000 dilution; Proteintech), LC3-I and LC3-II (1:1000 dilution; Proteintech). The secondary antibodies utilized were goat anti-mouse IgG and goat anti-rabbit IgG.

Immunohistochemical (IHC) staining

Formaldehyde-fixed paraffin-embedded (FFPE) ccRCC tumour tissues were sectioned at 3 μm. Following baking at 60 °C, FFPE tissue sections were decarbonised in xylene and rehydrated in a graded ethanol series. Antigen retrieval was then performed using citrate buffer in a microwave oven for 15 min, followed by natural cooling to room temperature. The samples were then incubated with 3% H2O2 for 10 min and non-specific sites were blocked with 3% bovine serum albumin (BSA, ZSGB-BIO, Beijing, China) for 30 min at 25 °C. The samples were then incubated with primary antibodies overnight at 4 °C. On the following day, the sections were incubated with appropriate secondary antibodies for 30 min. Finally, the reactions were detected using a DAB detection kit (DAB + ZSGB-BIO, China) and, if appropriate, the staining was terminated and counterstained with haematoxylin. The primary antibodies used were FUCA1 (ABclonal, Wuhan, China) and CTCF (ABclonal, Wuhan, China).

Wound healing assays

Tumour cell migration was assessed using a wound healing assay. The tumour cells were inoculated into 6-well plates, with a cell confluence exceeding 90%. A scratch was made in the cell monolayer using the blunt end of a 10-µl pipetting gun, and the width of the scratch between different wells remained consistent. Following the scratching, the well was washed three times with PBS and then supplemented with serum-free medium for 20 h. Cell migration was then observed by taking images at 0 h and 24 h after wounding. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to measure the migration distance.

Migration and invasion assay

To assess the migration and invasive abilities of ccRCC cells, transwell chambers with an 8 μm pore size (Corning, Tewksbury, USA) were used. For the transwell migration assay, cells were inoculated into the upper chambers of 24-well transwell inserts (5 × 104 cells/well) and maintained in serum-free medium, while the corresponding lower chambers were filled with 10% FBS medium. After 24 h, the migrated cells were stained with 1% crystal violet staining solution. Cells that migrated to the lower surface were further counted under a microscope and photographed. For the invasion assay, the upper transwell chambers were coated with 50 µl Matrigel (Becton Dickinson, Franklin Lakes, NJ) for 4 h. After 24 h, the non-invaded cells in the upper inserts were removed and the invasive cells were imaged and quantified using the same method described above.

Animal studies

Six-week-old male athymic BALB/c mice (Hunan SJA Laboratory Animal Company) weighing between 20 and 24 g were used to establish an orthotopic tumour model. Luciferase-expressing Renca cells and FUCA1-Renca cells (1 × 10⁵) were injected into the subrenal capsule of the left kidney of BALB/c mice (four mice per group). After one week, the mice were euthanised by cervical dislocation. The growth of the tumours was recorded in vivo using bioluminescence imaging (BLI). The mice were provided with standard food and water ad libitum. All mice were housed in an environment where temperature and humidity could be controlled, and a 12 h light/dark cycle was maintained. Studies on animals were conducted in accordance with relevant guidelines and approved by the Shandong University Qilu Hospital Ethics Committee.

Chromatin immunoprecipitation (ChIP)-qPCR analysis

Magna ChIP Protein A + G Magnetic Beads (PERFEMIKER) was used for chromatin immunoprecipitation. The 786O cells were fixed with 1% formaldehyde for 10 min at 25 °C. The cells were then incubated with glycine for 5 min to terminate the interaction. The cells were washed twice with PBS, and then collected by centrifugation following the addition of SDS lysis buffer and protease inhibitors. Subsequently, the cross-linked chromatin was subjected to 10 cycles of sonication (30s each). Cross-linked chromatin was immunoprecipitated with antibodies against CTCF (ABclonal, Wuhan, China) and rabbit IgG antibodies. IgG antibodies were included as negative controls. The DNA fragments were pulled down by the antibody against CTCF. Precipitated DNA was analysed by quantitative PCR and all values were normalized. Primers used in this study are documented in Table S1.

Luciferase reporter assay

Two different dual renilla/firefly luciferase containing plasmids were purchased: one containing the FUCA1-WT 3′UTR and the other containing the FUCA1-MUT 3′UTR (Abiotech, Jinan, China). Briefly, the cells were seeded in 24-well plates at a concentration of 1.5 × 105 per well. The next day, the cells were transfected with the indicated luciferase reporter plasmid and the appropriate plasmids. After 48 h, the dual-luciferase reporter assay kit (Beyotime, Shanghai, China) was used to evaluate the activities of firefly luciferase and Renilla luciferase. At least three independent experiments were carried out.

GFP-mRFP-LC3 staining

The GFP-mRFP-LC3 lentivirus was purchased from Jikai Biological Technology, Shanghai, China. The 786O and TK10 cell lines, cultured on coverslips were transfected with pcDNA3.1-FUCA1 + or siFUCA1 and its control for 24 h. Subsequently, the cells were transfected with GFP-mRFP-LC3 lentiviral for 12 h. The cells were then observed by fluorescence microscopy. The presence of yellow dots indicated the formation of autophagosomes, while red dots indicated the formation of autophagic lysosomes.

Confocal microscopy analysis

The autophagy of ccRCC cells was detected by fluorescence confocal microscopy. Cells were fixed in 4% paraformaldehyde for 25 min after 4 h of EBSS starvation. Subsequently, the cells were incubated overnight with primary rabbit anti-LC3B antibody (Proteintech, 1:500) and rat anti-LAMP2 (Proteintech, 1:500). Then, the cells were incubated with Fluor 555-conjugated donkey anti-rabbit IgG (Beyotime, 1: 500). Alexa Fluor 488-conjugated goat anti-mouse IgG (Beyotime, 1:500), and nuclei were counterstained with DAPI (Beyotime, 1:500). Imaging was carried out using the confocal microscope (Zeiss 710), and immunoreactivity manifested specific green or red fluorescence.

Transmission electron microscopy (TEM)

The cells were fixed in 1.6% glutaraldehyde (EMS, USA) in 0.1 M phosphate buffer immediately following the removal of the medium. Then, the samples were rinsed with phosphate buffer and post-fixed in osmium tetroxide (1%) for 1 h. The samples were embedded in epoxy resin after being dehydrated through a graded series of ethanol. The 70 nm sections were stained with 2% uranyl acetate and lead citrate. The TEM was performed using a HITACHI, H-7800 Transmission electron microscopy (Hitachi HT-7800, Japan).

Chloroquine assay

A solution of 10 µM chloroquine (CQ; 20 µM in use; Abmole, Houston, TX, USA) was added to the culture medium of the control group and 786O cell and TK-10 cell lines overexpressing FUCA1, with the objective of inhibiting autophagy. As previously described, autophagy was evaluated by Western blot assay using cell lysates. Additionally, three independent experiments were conducted under identical conditions.

Statistical analyses

All statistical analyses were conducted using IBM SPSS Statistics version 24 (IBM, Armonk, NY). The data presented here were obtained from at least three independent experiments and are expressed as means ± standard deviations. A log-rank test was employed for the purpose of performing survival analyses. Kaplan–Meier plots were used to visualize overall survival (OS), progression-free interval (PFI), and disease-free survival (DFS). Multivariate Cox regression analyses were employed to ascertain the impact of diverse predictor variables on OS and PFI. The Spearman’s correlation test was utilised to evaluate the relationship between numerical variables. Furthermore, statistical significance was determined at the *P < 0.05, **P < 0.01, ***P < 0.001 levels.

Results

Downregulation of FUCA1 expression is observed in ccRCC tumors and predicts poor prognosis based on public database

The initial comparison of FUCA1 mRNA expression levels was conducted between tumours, normal tissues and corresponding para-cancerous tissues using the KIRC item from TCGA. A markedly reduced expression of FUCA1 was evident in tumour specimens relative to normal and adjacent non-cancerous tissues (Fig. 1A and B). To gain further insight into the role of FUCA1 in ccRCC, we proceeded to examine the differences in FUCA1 levels among ccRCC patients with varying clinical stages and histological classifications. It is noteworthy that a pronounced elevation in FUCA1 expression was observed in patients with T1&T2 and M0 stage in comparison to those with T3&T4 and M1 stage (Fig. 1C and D). Furthermore, lowered FUCA1 expression was evident in patients with advanced neoplasm staging and higher histological grading (Fig. 1E and F). The results of the survival analysis indicated that ccRCC patients with elevated FUCA1 expression exhibited a more favourable prognosis than those with lower expression, as evidenced by data from the TCGA database (Fig. lG-lH). This finding was further validated using the GSE22541 dataset (Fig. 1). Multivariate Cox regression analysis incorporating other clinical factors demonstrated that FUCA1 expression could be considered an independent prognostic indicator for ccRCC patients regarding OS and PFI (Fig. 1J and K).

Fig. 1.

Expression and survival analysis of FUCA1 in ccRCC. (A) Comparison of FUCA1 expression between normal renal tissues and ccRCC tissues based on TCGA database. (B) Comparison of FUCA1 expression between ccRCC tissues and paired adjacent normal renal tissues based on TCGA database. (C-F) Analysis of FUCA1 expression in different clinical subgroups of ccRCC patients (C: T1&2 vs. T3&4; D: M0 vs. M1; E: Stage I&II vs. Stage III&IV; F: G1&2 vs. G3&4). (G-H) Kaplan-Meier estimates for OS and PFI in ccRCC patients from the TCGA database, stratified by the 50th percentile of FUCA1 expression values. (I) Kaplan-Meier estimates for DFS in ccRCC patients from the GSE22541 dataset, stratified by the 50th percentile of FUCA1 expression values. (J) Multivariate Cox regression analysis evaluating the association between FUCA1 level and OS in ccRCC patients from the TCGA database. (K) Multivariate Cox regression analysis evaluating the association between FUCA1 level and PFI in ccRCC patients from the TCGA database. FUCA1, α-L-fucosidase1; ccRCC, clear cell renal cell carcinoma; TCGA, the cancer genome atlas; KM, kaplan meier curve; OS, Overall survival; DFS, Disease free survival; PFI, Progression free interval. **P < 0.01,*** P < 0.001

Differential protein expression of FUCA1 in tumor and normal renal tissues

To validate the downregulation of FUCA1 in ccRCC, we obtained tumour and paired normal specimens from 15 ccRCC patients at Qilu Hospital and performed a western blot experiment to assess its expression levels in neoplastic and normal tissues. Our experimental results provide evidence that the expression of FUCA1 is decreased in tumour tissues compared to normal tissues (Fig. 2A), which strongly supports our previous conclusion. In addition, we used an IHC method to quantify FUCA1 expression as the optical density of FUCA1 staining in two types of tissue from four patients and found lower OD values in tumours (Fig. 2B). This observation is in agreement with our Western blot findings. Overall, our study hassuccessfully confirmed the downregulation of FUCA1 expression in ccRCC through the integration of both bioinformatics analysis and experimental approaches.

Fig. 2.

Expression of FUCA1 in ccRCC tissues. (A) The protein expression levels of FUCA1 were validated by western blot analysis in ccRCC patients (n = 15). (B) Immunohistochemistry was performed to detect the expression intensities of FUCA1 in normal renal tissues and ccRCC specimens. FUCA1, α-L-fucosidase1;CTCF, CCCTC binding factor. *P < 0.05; ***P < 0.001

FUCA1 is involved in the regulation of cellular migration and invasion in ccRCC cells

In the preceding text, a decrease in FUCA1 expression was observed in ccRCC patients with distant metastatic lesions. The objective was to thoroughly investigate the impact of FUCA1 on the characterisation of ccRCC. Correlation analysis revealed a negative association between FUCA1 expression and the number of lung metastases, with higher levels observed in patients carrying more than 20 lung metastases compared to those with less than 20, as observed in the GSE14378 dataset (Fig. 3A and B). This suggests that elevated FUCA1 expression may attenuate the metastatic potential of ccRCC. To confirm this hypothesis, we conducted both internal and external experiments. Two ccRCC-derived cell lines, 786O and TK-10, were selected for further analysis due to their remarkable expression levels of CTCF and FUCA1 compared to other cell lines (Fig. 3C). A Transwell assay was employed to assess cellular migration and invasion in ccRCC using 786O and TK-10 cell lines, which revealed a significant reduction in migratory and invasive cells upon ectopic FUCA1 expression compared to the control (Fig. 3D). The results of the wound healing assays demonstrated lower closure percentages in 786O cells overexpressing FUCA1, and a similar phenomenon was observed in TK-10 cells (Fig. 3E). Furthermore, to gain further insight into the role of FUCA1 on the infiltrative and migratory phenotype of renal clear cell carcinoma, we designed transwell experiments using two tumour cell lines, 786O and TK10, to investigate the effects of FUCA1 knockdown and FUCA1 overexpression and the interaction between the two on ccRCC. The results showed that knockdown of FUCA1 significantly increased cell invasion and migration ability, while overexpression of FUCA1 attenuated the invasion and migration ability of tumour cells. Interestingly, knockdown of FUCA1 in cells overexpressing FUCA1 restored the invasive and migratory abilities of cells (Fig. 3F). Subsequently, we orthotopically implanted renca cell lines harbouring firefly luciferase vectors into the kidney capsules of mice for tumorigenesis. Four male mice were transplanted with normal renca cell lines, while another four were transplanted with cells overexpressing FUCA1. The final tumour volume was monitored using a bioluminescent imaging system and we observed that the total flux of tumours with FUCA1 overexpression was significantly lower than that in the control group (Fig. 3G). Taken together, these findings provide compelling evidence supporting the inhibitory role of FUCA1 on ccRCC cell invasion and migration.

Fig. 3.

FUCA1 affects migration and invasion of ccRCC cells. (A) The correlation between FUCA1 mRNA expression and number of pulmonary metastases in ccRCC patients from GSE14378 dataset. (B) Expression of FUCA1 mRNA between ccRCC patients with less pulmonary metastases (No.<20) and those with more pulmonary metastases (No.≥20). (C) Western blot was used to measure the proteins level of FUCA1 in normal renal cell line(HK-2) and RCC cell lines (caki1,caki2,A498, 786O, TK10). (D) Transwell migration and invasion assay of FUCA1-overexpressing cells at 100× magnification. (E) Wound healing assays of FUCA1-overexpressing cells at 100×magnification. (F) Transwell migration of 786O and TK10 cells overexpressing FUCA1 and knocking down FUCA1 under 100x magnification. (G) Mouse Renal orthotopic tumor models established using FUCA1 overexpression of Renca lines. *P < 0.05,**P < 0.01,***P < 0.001

CTCF functions as a transcriptional regulator of FUCA1 to augment its expression

Through conducting GSEA analysis on differential genes between high and low expression groups of FUCA1 in TCGA, we serendipitously identified a significant enrichment of these genes in the CTCF pathway (Fig. 4A), a pivotal regulatory factor governing gene transcription. Intrigued by this finding, we endeavoured to unravel the intricate relationship between CTCF and FUCA1. Correlation analysis revealed a positive association between the expressions of FUCA1 and CTCF (Fig. 4B). Immunohistochemistry further demonstrated synchronous changes in the expression levels of both FUCA1 and CTCF, while western blotting experiments indicated that knockdown of CTCF through siRNA transfection led to a corresponding decrease in FUCA1 expression in both two ccRCC cell lines (Fig. 4C and D). To investigate potential binding sites for CTCF within homo sapiens, we consulted the JASPAR database (Fig. 4E). In order to verify whether CTCF could bind to the promoter region of FUCA1, luciferase assays were designed using two reporter plasmids: one containing the wild-type promoter region and the other with a mutant version. The sequences for both WT&MUT promoters are provided in Table S2. Additionally, an expression plasmid for CTCF along with a control vector were constructed, alongside an internal reference plasmid expressing rellina luciferase. Binding of CTCF to the promoter region would enhance firefly luciferase gene expression, resulting in an increase in the firefly/rellina ratio value. Our results demonstrated that this value was significantly higher in the 786O-WT + CTCF group compared to the 786O-MUT + CTCF group (Fig. 4E), suggesting that the wild-type promoter region can indeed bind with improved affinity by CTCF. This conclusion was further validated through CHIP-qPCR assay (Fig. 4F).

Fig. 4.

FUCA1 is the CTCF target gene. (A) Differential expression gene analysis of high and low FUCA1 group. (B) Differentially expressed genes were enriched in CTCF pathway through GSEA analysis. (C) Immunohistochemical results of correlation between CTCF and FUCA1. (D) FUCA1 expression were assessed by Western blot after CTCF knockdowned in 786O and TK10 cells. (E) JASPAR (http://jaspar.genereg.net/) shows CTCF binding sites with FUCA1 promoter regions and Luciferase assay showed that CTCF binds to the wild type FUCA1 promoter. (F) The enrichment of CTCF on FUCA1 promoter was detected by ChIP qpcr. ***P < 0.001,****P < 0.0001

The associations between CTCF and clinicopathological variables as well as survival outcomes in ccRCC patients

As previously mentioned, CTCF has the ability to bind to specific fragments of the FUCA1 promoter and positively regulate the expression of FUCA1. Interestingly, the role of CTCF in ccRCC remains unclear. A significant upregulation of CTCF expression was observed in patients with earlier pathologic stages and lower histological grades compared to those in advanced stages, as evidenced by analysis of patient data obtained from the TCGA database (Fig. S1, S2A-D). Similar to FUCA1, Kaplan-Meier curves also demonstrated that high CTCF expression was associated with improved OS (HR = 0.52, P < 0.001) and PFI (HR = 0.57, P < 0.001) (Fig. S2E-F). Furthermore, a multivariate COX regression analysis incorporating CTCF and other clinicopathological variables revealed that lower expression levels of CTCF effectively predicted shorter PFI (Fig. S2H), while its significance for assessing overall survival remained uncertain (Fig. S2G). To investigate whether these two genes have a synergistic effect on predicting prognosis in ccRCC, we examined the interaction between FUCA1 and CTCF by categorising patients into four groups based on their gene expressions. Notably, patients classified as “HH” with high expressions of both FUCA1 and CTCF displayed superior OS and PFI compared to other groups (Fig. S2I-J). These findings suggest that elevated expression of CTCF may serve as a protective factor for ccRCC similar to FUCA1.

Revealing the regulatory role of FUCA1 in modulating the autophagy pathway

In order to elucidate the underlying framework of FUCA1 affecting ccRCC phenotypes, further in-depth research was conducted. The preliminary GSEA analysis revealed that differentially overexpressed genes in the high FUCA1 groups were significantly enriched in the autophagy pathway (Fig. 5A). Therefore, we specifically investigated this important pathway that maintains cellular homeostasis and its correlation with FUCA1 in ccRCC. Initially, we sought to investigate the impact of FUCA1 deficiency on the autophagy process by depleting FUCA1 expression in 786O and TK10 cells using three siRNAs. Western blot analysis revealed that the absence of FUCA1 led to a general increase in the autophagosome markers LC3II compared to normal ccRCC cells, suggesting that loss of FUCA1 may contribute to autophagosome generation (Fig. 5B). However, considering that cellular autophagy is a dynamic procedure and the formation of autophagosomes is just an intermediate step in this process, followed by their fusion with lysosomes to form autolysosomes, it is insufficient to solely rely on the number of autophagosomes as an indicator for overall levels of autophagy. Therefore, in order to better assess the effect of FUCA1 on autophagosomes within the context of autophagic flux, two groups of 786O cells were established: 786O-NC (no downregulation or upregulation of FUCA1) and 786O-FUCA1 (overexpression of FUCA1), and incubated both cell lines with EBSS to induce autophagy while monitoring changes in LC3II expression as a measure for alterations in autophagosome levels throughout different stages. Western blot analysis revealed that the levels ofLC3II were consistently lower in the 786O-FUCA1 lines compared to the 786O-NC lines during all stages of induced-autophagy (Fig. 5C). Immunofluorescence analysis revealed a notable reduction in the puncta luminosity of LC3 in 786O and TK10 cells with upregulated FUCA1 expression, whereas an increase was observed in cells with downregulated FUCA1 expression. This provided a more intuitive demonstration of the content of autophagosomes in cells with different levels of FUCA1 expression(Fig. 5D). Subsequently, the mRFP-GFP-LC3 dual fluorescence system was used to detect autophagy, which revealed that the knockdown of Fuca1 inhibited autophagosomal and autophagolysosomal fusion in both the 786O and TK10 cell lines. Conversely, overexpression of Fuca1 was observed to promote autophagosomal and autophagolysosomal fusion (Fig. 5E). Furthermore, electron microscopy provided detailed evidence showing that the upregulation of FUCA1 resulted in a reduction in the accumulation of autophagosomes compared to control cells when incubated with EBSS (Fig. 5F). Interestingly, there was no significant difference observed between the 786O cells overexpressing FUCA1 and the control group regarding the increase of autolysosomes induced by EBSS compared to full medium culture conditions (Fig. 5G). These findings collectively indicate that alterations in FUCA1 can disrupt the normal process of autophagy in cells. However, further investigations are required to elucidate the specific mechanism by which FUCA1 modulates autophagy. To address this question, we employed CQ as an inhibitor targeting the fusion process between autophagosomes and lysosomes to antagonize cellular autophagy [33]. When CQ takes effect and completely blocks the fusion process, any increment in autophagosome formation resulting from FUCA1 overexpression would suggest that it impacts their formation. Conversely, if there is no significant change observed in autophagosome levels, it indicates that FUCA1 affects the fusion stage between autophagosomes and lysosomes. Our results showed that after treating 786O and TK10 cell lines with CQ for 4 h, upregulation of FUCA1 did not noticeably reduce LC3II density compared to control group where contrary outcome was presented (Fig. 5H). Therefore, we propose that overexpression of FUCA1 may lead to a reduction in the number of autophagosomes by accelerating their fusion stage during the process known as autophagy flux.

Fig. 5.

FUCA1 regulates autophagy. (A) GSEA analysis revealed that differentially overexpressed genes in high FUCA1 groups were significantly enriched in the autophagy pathway. (B) Following the knockdown of CTCF in 786O and TK10 wild-type and their overexpressing FUCA1 cell lines, the presence of LC3 was detected by Western blotting. (C) The 7860 cell line overexpressing FUCA1 and the control group were incubated in EBSS forthe indicated time period. LC3B was detected by Western blot analysis of cell lysates. (D) After 4 h of EBSS starvation, LC3 and Lamp2 changes in 786O and TK10 cell lines overexpressing and knocking down FUCA1 were observed by confocal microscopy. The nucleus was stained with DAPI. Representative images were shown. (E) GFP-mRFP-LC3 lentiviruses were transfected in the 786O and TK10 cell lines, which had been knocked down and overexpressed FUCA1. Confocal microscope images were taken after a four-hour starvation treatment. In the merged images, yellow spots indicate autophagosomes and red spots indicate autophagic lysosomes. (F) The control group and the 786O cell line overexpressing FUCA1 were cultured in EBSS for 4 h. Electron microscope image showing autophagosome (red arrow). (G) The control group and 7860 cell lines overexpressing FUCA1 were cultured in full medium and EBSS for 4 h. Electron microscope image showing autophagosome (red arrow). (H) The 786O and TK10 cell lines overexpressing FUCA1 were treated with 10 µM CQ for 4 h. Cell lysates were detected by Western blotting. *P < 0.05

CTCF rescues the impact of FUCA1 loss on autophagy and behaviour in ccRCC cells

We have determined that CTCF, acting as a transcription factor, can bind to the promoter region of FUCA1 and enhance its expression. However, further experimental validation is required to ascertain whether CTCF can reverse autophagy dysfunction or the heightened infiltration and metastasis characteristics observed in ccRCC due to FUCA1 deficiency. Immunoblot analysis revealed a significant increase of LC3 in 786O and TK10 cells with CTCF interference by siRNAs, indicating a parallel effect between CTCF and FUCA1 on autophagy regulation (Fig. 6A). Confocal microscopy analysis demonstrated that 786O and TK10 cells with downregulated FUCA1 through siRNA exhibited higher LC3 luminosity compared to those without downregulation after EBSS incubation for 4 h; however, transfection of exogenous CTCF into si-FUCA1 786O and TK10 cells resulted in diminished LC3 luminosity once again (Fig. 6B). To investigate the interaction between CTCF and FUCA1 in ccRCC migration, we categorized each of the 786O and TK10 cell populations into four groups based on the presence or absence of exogenous CTCF and knockdown of FUCA1: CTCF⁺/si-FUCA1⁺, CTCF⁺/si-FUCA1⁻, CTCF⁻/si-FUCA1⁺, and CTCF⁻/si-FUCA1⁻. The CTCF⁻/si-FUCA1⁻ group was used as a control. We then conducted transwell and wound healing assays to assess cell migration. In the transwell assay, we observed a higher number of migrated cells in the CTCF-/si-FUCA1 + group compared to control, while the CTCF⁺/si-FUCA1⁻ group showed a lower number of migrated cells. Notably, there was no significant difference in cell migration between the CTCF⁺/si-FUCA1⁺ group and the control (Fig. 6C). Wound healing analysis revealed that the CTCF⁺/si-FUCA1⁻ group had a significantly reduced wound closure after 24 h compared to the control, while the CTCF⁻/si-FUCA1⁺ group showed significantly increased wound closure. Additionally, transfection of cells with siRNA-mediated FUCA1 knockdown with a plasmid containing exogenous CTCF resulted in minimal differences compared to the control (Fig. 6D). These findings suggest that both CTCF and FUCA1 play a role in regulating autophagy in ccRCC. Specifically, the overexpression of CTCF may compensate for functional decline caused by decreased expression of FUCA1 in ccRCC.

Fig. 6.

CTCF regulates autophagy and invasion and metastasis of renal clear cell cancer cells. (A) Following the knockdown of CTCF in 786O and TK10 wild-type and their overexpressing FUCA1 cell lines, the presence of LC3 was detected by Western blotting. (B) The changes of LC3B and Lamp2 in 7860O and TK10 cell lines overexpressing CTCF and FUCA1 knocking down were observed by confocal microscopy after 4 h of EBSS starvation. The nuclei were stained with DAPI. (C) transwell detected changes in migration and invasion ability of 786O and TK10 cell lines that overexpressed CTCF and knocked down FUCA1. (D) Wound healing tests of 786O and TK10 cell line overexpressing CTCF and FUCA1 knockdown under 100x magnification

Blocking autophagy reverses the role of FUCA1 in ccRCC

Western blot analysis was used to verify the changes in autophagy levels and Fuca1 content in 786O and TK10 cell lines following a 4-hour chloroquine treatment. The results demonstrated that there was no significant alteration in the level of Fuca1 in cells after the blockade of autophagy, which supports the hypothesis that FUCA1 may act as an upstream regulator of autophagy regulation (Fig. 7A). Subsequently, chloroquine was introduced to TK10 and 786O cell lines genetically engineered to overexpress Fuca1, with the aim of inhibiting autophagy. The invasive and metastatic abilities of the cells were then observed and it was found that the effects of Fuca1 were reversed after autophagy had been inhibited (Fig. 7B-C).

Fig. 7.

Blocking autophagy reverses the role of FUCA1 in RCC.(A)The 786O and TK10 cell lines were treated with 10 µM CQ for 4 h. Cell lysates were detected by Western blotting.(B-C)Transwell migration and invasion assays were conducted on 786O and TK10 wild-type and their overexpressed FUCA1 cell lines, treated with 10 µM CQ for 24 h

The serum AFU serves as a reliable prognostic indicator for patients with ccRCC

The protein-protein interaction network identified several hub proteins that exhibited a strong correlation with FUCA1, including FUCA2, the encoding gene of serum AFU (Fig. 8A and B). Moreover, the nomogram analysis indicated that patients with high expression of FUCA2 had a more definite and longer probability of survival (Fig. 8C). Additionally, we collected clinical information from patients diagnosed with ccRCC at Qilu Hospital and investigated the association between serum AFU levels and patient survival as well as other factors. Our findings demonstrated that patients in early stages had higher AFU levels and those with elevated AFU levels generally experienced better survival outcomes (Fig. 8D and E). Furthermore, multi-cox regression analysis showed that AFU could independently predict PFI in sufferers but lacked sufficient predictive power for OS (Fig. 8F and G). Consistent with these findings, the nomogram model also suggested that higher AFU levels were associated with an increased probability of exceeding survival expectations, aligning closely with the results for FUCA2 (Fig. 8H).

Fig. 8.

The effect of FUCA2 expression and serum AFU level on clinical outcome of ccRCC patients. (A) Protein–protein interaction network analysis showed FUCA1 that interacts with FUCA2 and other proteins. (B) The scatterplot showing Spearman correlation between FUCA1 and FUCA2 expression based on TCGA ccRCC cohort. (C) Nomogram model for predicting survival rates of ccRCC patients at 3,5, and 10years. (D) Serum AFU level in different subgroups of ccRCC petients from Qilu hospital(left panel, N stage; right panel, Pathologic stage); (E) The Kaplan-Meier curves of OS(left panel) and PFS (right panel) stratified by different serum AFU level in ccRCC patients from Qilu hospital. (F) Multivariate Cox regression analysis of serum AFU level for OS in ccRCC patients from Qilu hospital. (G) Multivariate Cox regression analysis of serum AFU level for PFS in ccRCC patients from Qilu hospital. (H) Low expression of FUCA1/2 gene and serum AFU level indicates poor prognosis of ccRCC patients. ccRCC, clear cell renal cell carcinoma; FUCA2, α-L-fucosidase2; AFU, α-L-fucosidase; OS, overall survival; PFS, Progression free survival; HR, hazard ratio. *P < 0.05

Discussion

Our research has identified FUCA1 as a potential tumour suppressor that can effectively inhibit the invasion and migration of ccRCC cells by targeting the autophagy pathway. Furthermore, we have discovered that its functional deficiency can be rescued by CTCF, thereby highlighting a novel therapeutic target for advanced ccRCC patients. FUCA1 is one of the main genes encoding alpha-L-fucosidase, and its products are mainly found in tissues [12]. Previous studies have extensively investigated the role of FUCA1 in various cancer types, but there is limited evidence regarding its involvement in ccRCC [34]. A synthesis of existing literature revealed that FUCA1 plays a dual role in progression of tumour and various malignancies such as prostate cancer, colorectal tumours, breast cancer, and neuroblastoma. It is frequently downregulated and has been observed to exert inhibitory effects on tumour development [12, 35, 36].Conversely, upregulated FUCA1 can predict poor prognosis in esophageal squamous cell carcinoma and glioma [15, 16]. At present, the partial mechanisms by which FUCA1 regulates tumour phenotype have been elucidated and it has been identified as a novel target gene of P53, which induces programmed cell death or apoptosis for dampening the growth of cancer cells [11, 37]. Moreover, FUCA1 has been demonstrated to reduce the activation of the epidermal growth factor receptor via disassembling α1,6-fucosylation of EGFR as well as inhibiting Akt phosphorylation. This subsequently restricts EGFR downstream signalling, a common tumorigenesis-dependent pathway [11, 12]. Some studies have explored the correlation between FUCA1 and tumour immune microenvironment. In ccRCC, FUCA1 has been shown to promote M2 macrophage infiltration, which facilitates immune evasion of tumours and indirectly maintains tumour progression [38]. In this study, we identified several proteins that interact closely with FUCA1, with FUCA2 being the most prominent one. Previous literature has consistently reported a correlation between FUCA2 and poor survival outcomes in various cancers including breast cancer, liver cancer, and lung cancer [39]. Conversely, our investigation revealed that overexpression of FUCA2 was associated with a favourable prognosis, and the serum AFU encoded by FUCA2 was validated as a robust prognostic predictor for ccRCC.

To elucidate the precise impact of FUCA1 on the nature of ccRCC, our GSEA analysis revealed a close association between FUCA1 and CTCF as well as autophagy pathways. CHIP-qPCR and luciferase assays demonstrated that CTCF functions as a transcription factor that not only activates FUCA1 transcription but also mitigates the effects caused by FUCA1 downregulation. These findings suggest that FUCA1 may be regulated by CTCF in ccRCC. CTCF was identified as a conserved transcription factor for maintaining three-dimensional genome organization in normal cells, and its disruption was closely linked to tumorigenesis [40]. CTCF played a paradoxical role in different cancers and its regulatory mechanism exhibited a wide range of sophistication. In endometrial cancer, CTCF was identified as a robust tumour suppressor gene since its haploinsufficiency led to the locus-specific alterations in gene expression including downregulation of tumour-suppressor genes and upregulation of estrogen-sensitive genes [41]. Peculiarly, overexpression of CTCF increased the proliferative activity and prevented cancer cells from programmed cell death in breast cancer [42]. Moreover, overexpression of CTCF was also detected in hepatocellular carcinoma and this was associated with shorter disease-free survival of patients. Conversely, the suppression of CTCF resulted in a reduction in cell proliferation, motility and invasiveness in HCC cell lines, which was due to a decrease in the levels of telomerase reverse transcriptase (TERT), shelterin complex member telomerase repeat-binding factor 1 and forkhead box protein M1 (FOXM1) resulting from CTCF deficiency [43]. In addition to alterations in CTCF expression level, structural variations in the protein itself were also observed in cancers, particularly in zinc finger domains (ZFs) [44]. Abnormality in ZFs of CTCF would directly influence CTCF binding affinity and stability to DNA [45]. The transcription factor CTCF exhibits dual functionality, acting as both an activator to upregulate target gene expression and as an inhibitor to downregulate target gene expression. Our study revealed that FUCA1 is a novel downstream gene positively regulated by CTCF in ccRCC.

Cell autophagy is an adaptable process that enables cells to respond to a range of cellular stress, including protein and organelle damage and redox imbalance. It plays a critical role in maintaining cell integrity and homeostasis by realizing nutritional reuse. A substantial body of evidence has demonstrated that autophagy is closely associated with various cancer types. However, the dual role of autophagy both in cancer progression and inhibition remains a topic of debate [24, 46]. In our study, we elucidated the pivotal role of FUCA1 in promoting autophagy in ccRCC cells. FUCA1 enhances the fusion of autophagosomes and lysosomes, thereby facilitating cargo degradation and ultimately inducing cell death in ccRCC. Previous research has suggested that P53 gene, an upstream regulator of FUCA1, can undergo autophagic suppression as it is sequestered and degraded within autophagosomes in ccRCC [47]. From this perspective, it appeared that the induction of cell death autophagy may result in a reduction in P53 density and further stimulation of tumour progression. Conversely, FUCA1 has been demonstrated to promote the fusion of autophagosomes and lysosomes, thereby facilitating the degradation of substances contained within autophagosomes. This may result in the release of P53, which could then resume its role. It is becoming increasingly evident from the latest research that the facilitation of autophagy flux has an anti-tumour effect in ccRCC. It has been reported that TRAF2, which acts as an oncogene in ccRCC, can be depleted in order to suppress the proliferation and progression of ccRCC, depending on the autophagy inactivation-induced polarization of M2 macrophages [48]. Furthermore, another investigation has revealed that the obesity-associated protein FTO also governs phenotypic traits of ccRCC through the modulation of the autophagy pathway, and inhibition of FTO impairs ccRCC growth and metastasis by augmenting autophagy flux [49].

Significantly, our study has elucidated the novel role of FUCA1 in ccRCC and has provided initial insights into its upstream and downstream regulatory pathways through the utilization of multiple advanced methodologies. This discovery opens up new possibilities for identifying potential therapeutic targets for ccRCC. However, certain limitations were inevitably encountered in this project, one of which was that we primarily focused on investigating the activation of autophagy-induced cell death by FUCA1 as a suppressor of ccRCC development, rather than extensively exploring how cellular autophagy influences phenotypes of ccRCC. Addressing this aspect is crucial and will be prioritized in our future investigations.

Conclusion

Our study provides novel insights into the role of FUCA1 in ccRCC, revealing a marked reduction in FUCA1 expression in ccRCC tissues compared to normal tissues. We establish FUCA1’s pivotal function as an anti-tumour factor, as it significantly inhibits tumour cell migration, and invasion. Our research identifies CTCF as a key activator of FUCA1 expression, functioning as a transcription factor by binding to the promoter region. Notably, CTCF also demonstrates inhibitory effects on ccRCC growth. Furthermore, our findings underscore FUCA1’s involvement in regulating autophagy within ccRCC cells. Alterations in FUCA1 expression disrupt the autophagic process, particularly affecting the fusion between autophagosomes and lysosomes. Additionally, FUCA1 interacts closely with FUCA2, and their combined product, AFU, shows potential as a prognostic biomarker for patients with ccRCC (Fig. 9).

Fig. 9.

Working models propose that CTCF governs autophagy in ccRCC through FUCA1, thereby exerting an impact on patient prognosis

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

CTCF

CCCTC-binding factor

CHIP

Chromatin immunoprecipitation

ccRCC

Clear cell renal cell carcinoma

EBSS

Earle’s balanced salt solution

AFU

α-L-fucosidase

FUCA1

α-L-fucosidase1

FUCA2

α-L-fucosidase2

IHC

Immunohistochemical

LC3

Microtubule-associated protein 1 light chain3

Lamp2

Lysosomal associated membrane protein 2

MUT

Mutation

OS

Overall survival

PFI

Progress free interval

PFS

Progress free survival

DFS

Disease free survival

TCGA

The Cancer Genome Atlas

WT

Wildtype

Author contributions

JK.L and YD.F designed this study; S.Z, JJ.S and QZ.C conducted experiments; JK.L, S.Z, JJ.S, QZ.C, S.P and NZ.Z collected and analyzed data; S.Z, JJ.S, QZ.C, S.P, NZ.Z, YD.F, NW.Y, and JK.L wrote the manuscript. All authors approved the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant Number 82102999).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This study was approved by Ethics Committee of the Shandong University Qilu Hospital(Ethics Number: KYLL-2022(ZM)-477). The patients provided their written informed consent to participate in this study.

Competing interests

The authors declare no competing interests.