Inhibition of SLC25A10 promotes cellular senescence and impedes hepatocellular carcinoma progression

Introduction

Hepatocellular carcinoma (HCC) is the predominant subtype of primary liver cancer, accounting for 75–85% of cases (1). Globally, liver cancer ranks as the sixth most diagnosed malignancy and third leading cause of cancer-related mortality, with HCC patients facing a 5-year survival rate below 18% due to frequent tumor recurrence and aggressive metastatic dissemination (2). Current first-line therapies-including surgical resection, liver transplantation, and radiofrequency ablation-demonstrate efficacy predominantly in early-stage disease. However, over 70% of patients present with advanced or unresectable tumors at initial diagnosis, limiting therapeutic options to systemic regimens with suboptimal response rates (3). Persistent challenges such as intrinsic chemoresistance and acquired therapeutic escape mechanisms further diminish clinical outcomes, necessitating a deeper mechanistic understanding of HCC pathogenesis to guide innovative treatment development.

In recent years, the induction of cellular senescence in cancer cells has emerged as a promising anti-tumor strategy to halt the uncontrolled proliferation of malignant hepatocytes (4). Cellular senescence, characterized by irreversible cell-cycle arrest, can be initiated through two primary mechanisms: replicative senescence mediated by telomere attrition (5), and premature senescence triggered by diverse stressors including genotoxic agents, oncogenic activation, or mitochondrial damage (6). Notably in HCC, senescent cells exert tumor-suppressive effects through the senescence-associated secretory phenotype (SASP). SASP-derived cytokines mediate dual therapeutic effects by upregulating cyclin-dependent kinase inhibitor 1A (CDKN1A, or p21) and cyclin-dependent kinase inhibitor 2A (CDKN2A, or p16) to enforce cell-cycle arrest, while simultaneously facilitating immune-mediated clearance of malignant cells (7,8). These mechanistic insights have prompted clinical exploration of pro-senescence therapies as adjuvants to conventional treatments, offering a novel paradigm for personalized HCC management with enhanced therapeutic outcomes.

As a member of the mitochondrial solute carrier family 25 (SLC25), the largest group of transmembrane transporters in humans (9), SLC25 member 10 (SLC25A10) plays critical roles in cellular metabolism through substrate shuttling across the mitochondrial inner membrane (10). This carrier protein specifically mediates the electroneutral exchange of dicarboxylates (malate/succinate) with inorganic phosphate, sulfate, or thiosulfate, thereby establishing metabolic crosstalk between mitochondrial and cytosolic compartments (11). In hepatocytes, SLC25A10 supports de novo lipogenesis by supplying acetyl-CoA precursors and maintains metabolic flexibility through dual regulation of redox homeostasis [via nicotinamide adenine dinucleotide phosphate (NADPH) production] and glucose utilization (12,13). Clinically significant upregulation of SLC25A10 has been documented across various malignancies, including colorectal carcinoma, high-grade osteosarcoma, epithelial ovarian cancer, and non-small cell lung cancer, positioning it as a potential therapeutic target (14-16). However, the mechanistic role of SLC25A10 in HCC pathogenesis remains unclear.

In this study, we first systematically profiled SLC25A10 expression patterns in clinical in HCC specimens and cells lines. Through targeted knockdown of SLC25A10, we systematically examined its roles in suppressing cellular proliferation and inducing senescence-associated phenotypes. Mechanistic dissection employing pathway-specific inhibitors ultimately revealed that SLC25A10 orchestrates HCC progression via high mobility group box 1 (HMGB1)-dependent modulation of the SASP. These findings collectively position SLC25A10 as a pivotal metabolic nexus coordinating senescence evasion and malignant growth, providing a conceptual framework for developing senescence-focused therapeutic interventions against refractory HCC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2319/rc).

Methods

ICGC and GAO et al. dataset acquisition

Transcriptional profiles of SLC25A10 in the liver hepatocellular carcinoma (LIHC) cohort were retrieved from two independent publicly accessible genomic repositories: (I) the International Cancer Genome Consortium (ICGC; https://www.icgc-argo.org/) and (II) the HCC multi-omics database established by GAO et al. (https://ngdc.cncb.ac.cn/) (17).

Clinical human liver specimens

Fresh-frozen primary HCC tumor tissues and matched histologically normal adjacent liver tissues were obtained from eight HCC patients undergoing curative surgical operation at the Hepatobiliary Surgery Department of Affiliated Hospital of Nantong University. This study complied with the ethical principles of the Declaration of Helsinki and its subsequent amendments and received institutional review board approved from the Affiliated Hospital of Nantong University (ethical approval No. 2020-L093). All participants provided written informed consent prior to tissue collection.

Histological and immunohistochemical (IHC) staining

Human liver tissues and murine subcutaneous tumors specimens were fixed in 4% paraformaldehyde (PFA), processed through graded ethanol dehydration series, paraffin-embedded, and sectioned at 5 µm thickness. Tissue sections were deparaffinized in xylene and rehydrated through a descending ethanol gradient. Histopathological evaluation was performed using standard hematoxylin and eosin (H&E) staining protocols.

For IHC analysis, antigen retrieval was achieved through microwave-mediated heating in sodium citrate buffer [pondus hydrogenii (pH) 6.0]. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide treatment for 15 min. Sections were immunostained using the following primary antibodies: SLC25A10 (1:100 dilution, Cat#12086-1-AP, Proteintech, Wuhan, China), anti-KI67 (1:200, Cat#GB121141-100, Servicebio Technology, Wuhan, China), anti-PCNA (1:800, Cat#GB12010-100, Servicebio Technology, Wuhan, China), and anti-HMGB1 (1:200, Cat#ab79823, Abcam, UK), with overnight incubation at 4 ℃. After three washes in phosphate buffered saline (PBS), sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 90 min at room temperature. Diaminobenzidine chromogenic substrate was applied for signal development, followed by hematoxylin counterstaining. Digital imaging was performed using an OLYMPUS VS200, and quantitative analysis was conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from tissues using TRIzol reagent (TIANGEN BIOTECH, Beijing, China) following the manufacturer’s protocol. RNA purity and concentration were quantified using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA) with absorbance ratios (A260/A280) between 1.8 and 2.0, indicating optimal quality. First-strand complementary DNA (cDNA) synthesis was performed with 1 µg total RNA using the HiScript III RT SuperMix (R323-01, Vazyme Biotech, Nanjing, China) under the following conditions: 42 ℃ for 2 min [genomic DNA (gDNA) wiper reaction], followed by 50 ℃ for 15 min and 85 ℃ for 5 s.

Quantitative PCR (qPCR) amplification was carried out on a QuantStudio5 Real-Time PCR system (Thermo Fisher Scientific, USA) using SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). Gene expression quantification was performed using the comparative 2^−ΔΔCT method, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) endogenous control. Primer sequences (synthesized by Tsingke Biotechnology, Beijing, China) were as follows: SLC25A10 forward 5’-CAGGAGACTGTTCTCGGGTG-3’ and reverse 5’-GTCACCAGCGGCTGCAATA-3’, GAPDH forward 5’-AATGGGCAGCCGTTAGGAAA-3’ and reverse 5’-GCCCAATACGACCAAATCAGAG-3’.

Western blot (WB) analysis

Cellular and tissue protein lysates were prepared using RIPA Lysis Buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing 1× protease/phosphatase inhibitor cocktail (P1045, Beyotime, Shanghai, China). Lysates were centrifuged at 12,000 rpm for 10 min at 4 ℃ to collect supernatants. Protein concentrations were determined via bicinchoninic acid (BCA) assay (23225, Thermo Fisher Scientific, Waltham, USA). Samples (30 µg/lane) were denatured in 5× SDS-PAGE Loading Buffer (Biosharp, Hefei, China) at 95 ℃ for 10 min, then resolved on 10% SDS-polyacrylamide gels (EpiZyme, Shanghai, China) with pre-stained protein markers (26616, Thermo Fisher, Waltham, USA). Electrophoretically separated proteins were transferred onto 0.45 µm PVDF membranes (LC2005, Vazyme Biotech, Nanjing, China) using a semi-dry transfer system (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in Tris-Buffered Saline with Tween-20 (TBST) (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 2 h at room temperature, followed by incubation with primary antibodies diluted in blocking buffer: Anti-SLC25A10 (1:1,000, #12086-1-AP, Proteintech, Wuhan, China), Anti-CDK4 (1:1,000, #D9G3E, Cell Signaling Technology, Danvers, MA, USA), Anti-Cyclin D1 (1:1,000, #92G2, Cell Signaling Technology, USA), Anti-CDKN2A (1:1,000, #ab108349, Abcam, Cambridge, UK), and Anti-GAPDH (1:5,000, #60004-1-Ig, Proteintech, Wuhan, China). After overnight incubation at 4 ℃ with gentle agitation, membranes were washed 3 times for 10 min each in TBST and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies: Goat anti-Rabbit IgG (1:5,000, #111-035-003, Jackson ImmunoResearch, West Grove, PA, USA), Goat anti-Mouse IgG (1:5,000, #115-035-003, Jackson ImmunoResearch, Lancaster, USA). Chemiluminescent signals were developed using Enhanced Chemiluminescence (ECL) Prime Kit (WBKLS0100, Millipore, Boston, USA) and captured on a ChemiDoc XRS+ Chemiluminescence Apparatus (Bio-Rad, California, USA). Densitometric analysis was performed using Image Lab 6.1 software (Bio-Rad) with GAPDH as a loading control. Quantitative data normalization was conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell culture and treatment

Human cell lines (SK-Hep1, HepG2, Hep3B, Huh7, HCC-LM3, MHCC-97L and MHCC-97H) were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS, Pricella, Wuhan, China) and 1% penicillin-streptomycin (PS2101; NCM Biotech, Suzhou, China) at 37 ℃ in a humidified 5% CO₂ incubator (Thermo Scientific™ Heracell™ 150i). For stable gene knockdown, SK-Hep1 and Hep3B cells were seeded in 6-well plates at 2×10⁵ cells/well and allowed to adhere for 24 h. At 60–70% confluence, cells were transfected with 2.5 µg of either SLC25A10-targeting short hairpin RNA (sh-SLC25A10) plasmid or scramble control short hairpin RNA (shRNA) (sh-control) using lipofectamine 3000 transfection kit (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. Following 48 h of transfection, selection was performed with 2 µg/mL puromycin (A1113803; Gibco™) for 10 days to establish HCC cell lines with stabilized knockdown of SLC25A10. The stable knockdown efficiency was validated through qRT-PCR and WB analysis as described in Sections 2.4 and 2.5, respectively.

Cell proliferation assays

CCK-8 assay

Cells were seeded in 96-well plates at 2×10³ cells/well with six technical replicates per group. After 24–72h incubation under standard culture conditions, 10 µL CCK-8 reagent (CK04; Dojindo Laboratories, Kumamoto, Japan) was added to each well containing 100 µL serum-free medium. Following 2 h incubation at 37 ℃ protected from light, absorbance at 450 nm was measured using an automatic microplate reader (Thermo Fisher Scientific, Waltham, USA).

Colony formation assay

Cells were plated in 6-well plates (1×10³ cells/well) and maintained for 14 days with medium refreshed every 3 days. Colonies were fixed with 4% PFA for 15 min, stained with 0.5% crystal violet (C0775; Sigma-Aldrich, Germany) in methanol for 30 min, then washed with PBS until clear background achieved. Air-dried plates were scanned using an Epson Perfection V800 scanner. Colonies (>50 cells/colony) were quantified using ImageJ software.

5-ethynyl-2'-deoxyuridine (EdU) incorporation assay

Cells grown on 24-well chamber slides (1×10⁴ cells/well) were pulsed with 10 µM EdU (Beyotime Biotechnology, Shanghai, China) for 2 h. Following fixation with 4% PFA, click chemistry reaction was performed using Alexa Fluor^® 488-conjugated azide (C0078S; Beyotime, Shanghai, China) according to the manufacturer’s protocol. Nuclei were counterstained with 5 µg/mL Hoechst 33342 (Beyotime Biotechnology, Shanghai, China) for 10 min. Fluorescent images were acquired using a Leica THUNDER Imager DMi8 with 20×/0.75NA objective under consistent exposure settings. The EdU-positive cell proportion was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Senescence-associated β-galactosidase (SA-β-Gal) assay

Cellular senescence was assessed using the SA-β-Gal Activity Assay Kit (C0602; Beyotime Biotechnology, Shanghai, China) with protocol modifications. Brief, cells were seeded in 6-well plates at 5×10⁴ cells/well and cultured until 80% confluence. After PBS washing, cells were fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS (pH 6.0) for 30 min at room temperature. The SA-β-Gal staining solution containing 1 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) in dimethylformamide, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂ was applied to completely cover the fixed cells. Staining proceeded for 16 h at 37 ℃ in a dry incubator without CO₂. Senescent cells were identified by development of perinuclear blue precipitates using an Olympus IX83 inverted microscope equipped with DP80 CCD camera (Olympus Corporation, Tokyo, Japan). Five random fields per well were captured at 200× magnification (20× objective/10× ocular). Quantitative analysis was performed using ImageJ with the Color Threshold plugin, setting thresholds at hue: 200–255, saturation: 50–255, brightness: 0–255 for blue signal detection. Results were expressed as percentage of SA-β-gal-positive cells relative to total cells counterstained with Nuclear Fast Red (ab146284; Abcam, Cambs, UK).

Cell cycle analysis

Cells were harvested at 80% confluence using 0.25% trypsin-EDTA (25200056; Gibco™, California, USA) and washed twice with ice-cold PBS (pH 7.4). Single-cell suspensions were fixed in 70% pre-chilled ethanol (−20 ℃) for 18 h at 4 ℃. Following fixation, cells were centrifuged at 300 ×g for 5 min at 4 ℃ and permeabilized with 0.1% Triton X-100 in PBS for 15 min on ice. Cell cycle profiling was performed using the cell cycle detection kit (CA1510; Solarbio Science & Technology, Beijing, China). Cells were resuspended in 500 µL staining solution containing 50 µg/mL propidium iodide (PI), 100 µg/mL RNase A, and 0.1% sodium citrate. After 30 min incubation at 37 ℃ protected from light, samples were analyzed on a Beckman Coulter CytoFLEX LX flow cytometer (Brea, CA, USA) equipped with a 488 nm laser and a 610/20 nm emission filter. A minimum of 10,000 single-cell events were acquired per sample at 300–500 events/s. Doublet discrimination was performed using pulse geometry gating (FSC-H vs. FSC-A). Cell cycle phase distribution (G0/G1, S, G2/M) was quantified using FlowJo software (BD Biosciences) with Dean-Jett-Fox algorithm. Data normalization was performed using exponentially growing asynchronous cells as reference controls.

Subcutaneous tumorigenesis assay

Male BALB/c athymic nude mice (6–8 weeks old) were obtained from the specific pathogen-free (SPF) Laboratory Animal Center of Nantong University and housed under controlled environmental conditions (temperature: 22±1 ℃, 12-hour light/dark cycle) with free access to autoclaved food and water. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Nantong University (approval No. S20200315-009), with all procedures performed in strict accordance with institutional animal welfare guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. A protocol was prepared before the study without registration. For tumor xenograft establishment, Hep3B cells (1×10⁶ cells/mouse) with or without SCL25A0 knockdown were resuspended in 200 µL sterile PBS and subcutaneously inoculated into the flanks of nude mice. The animals were randomly divided into two groups (n=5 per group). Tumor implantation was successfully achieved in all animals (success rate: 100%), with no unexpected mortality or exclusion throughout the study period. Tumor growth was monitored daily and measured twice weekly using calipers. Tumor volume was calculated as (length × width²)/2. At the experimental endpoint (day 21 post-inoculation), mice were euthanized by CO₂ asphyxiation followed by cervical dislocation. Tumors were surgically excised, weighed, and processed for histopathological analysis H&E staining and IHC evaluation of target protein expression.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 software (version 9.0; GraphPad Software, San Diego, CA, USA). Experimental data from a minimum of three independent biological replicates are presented as means ± standard error of the mean (SEM). Intergroup comparisons were assessed using a two-tailed Student’s t-test, with statistical significance defined as *P<0.05, **P<0.01, and ***P<0.001.

Results

Upregulation of SLC25A10 in HCC

To investigate the role and molecular mechanisms of SLC25A10 in the pathogenesis and progression of HCC, our initial analysis of the ICGC database and Gao et al. dataset revealed significantly elevated transcription levels of SLC25A10 in HCC liver tissues than normal liver tissues (Figure 1A). To validate these findings, we obtained paired liver tumor and adjacent normal samples from eight HCC patients undergoing curative hepatectomy. Multimodal assessment through IHC, qRT-PCR, and WB analyses uniformly demonstrated upregulated SLC25A10 expression at both messenger RNA (mRNA) and protein levels in tumor tissues (Figure 1B-1D). Subsequently, we detected the expression level of SLC25A10 protein in seven HCC cell lines (SK-Hep1, HepG2, Hep3B, Huh7, HCC-LM3, MHCC-97L and MHCC-97H) for subsequent functional validation. The results showed that SLC25A10 was highly expressed in SK-Hep1, HepG2 and Hep3B cells (Figure 1E). The grayscale quantification results of the WB analyses presented in Figure 1D,1E are shown in Figure S1.

Figure 1 Expression profile of SLC25A10 in HCC tissues and cell lines. (A) Transcriptional expression of SLC25A10 in HCC specimens from the ICGC database and GAO et al. cohort. (B) IHC technique was used to investigate the differential immunohistochemical staining intensity of SLC25A10 protein between HCC tissues and paired adjacent non-tumor tissues. (C) qRT-PCR assessment of SLC25A10 mRNA level in matched HCC tissues and peri-tumoral hepatic tissues. (D) WB quantification of SLC25A10 protein abundance in HCC specimens compared with adjacent normal controls. (E) Differential SLC25A10 protein expression patterns in different cell lines, as demonstrated by western blot analysis. HCC, hepatocellular carcinoma; ICGC, International Cancer Genome Consortium; IHC, immunohistochemistry; mRNA, messenger RNA; N, normal; qRT-PCR, quantitative real-time polymerase chain reaction; T, tumor; WB, western blot.

Inhibition of SLC25A10 suppresses cells proliferation

To investigate the role of SLC25A10 on cellular biological behaviors, we performed SLC25A10 knockdown in SK-Hep1 and Hep3B cells (selected based on their relatively high endogenous SLC25A10 expression levels) using distinct shRNA constructs (sh-SLC25A10-1 and sh-SLC25A10-2). Successful gene silencing was confirmed at both transcriptional and protein levels by qRT-PCR and WB, respectively (Figure 2A,2B). Functional assessments revealed that SLC25A10 knockdown significantly impaired malignant phenotypes: CCK-8 assay demonstrated a marked reduction in cell viability in both cell lines (Figure 2C). Colony forming capacity was substantially attenuated in knockdown groups compared to controls (Figure 2D). EdU incorporation assays (quantifying proliferating cells) showed decreased positivity rates, further corroborating the anti-proliferation effect of SLC25A10 inhibition (Figure 2E).

Figure 2 Knockdown of SLC25A10 inhibited cell proliferation capacity. (A,B) SK-Hep1 and Hep3B cells were transfected with siRNA-1 and siRNA-2 targeting SLC25A10, followed by qRT-PCR (A) and WB (B) analyses to evaluate the knockdown efficiency of SLC25A10 at both mRNA and protein levels. ***, P<0.001. (C-E) Functional characterization of proliferation dynamics using CCK-8 viability assays (C), colony formation analysis (D) and EdU incorporation assays (E) in SK-Hep1 and Hep3B cells transfected with shRNA constructs targeting SLC25A10 (sh-SLC25A10-1 and sh-SLC25A10-2) versus non-targeting shRNA controls; (D) cells were stained with crystal violet; (E) cells were pulsed with EdU and Hoechst 33342 (20×). EdU, 5-ethynyl-2'-deoxyuridine; mRNA, messenger RNA; OD, optical density value; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short hairpin RNA; siRNA, small interfering RNA; WB, western blot.

SLC25A10 knockdown induces cellular senescence and cell cycle arrest

Given that cellular senescence is characterized by suppressed proliferation capacity and contributes to tumor suppression (18), we first evaluated senescence-associated phenotypes using SA-β-galactosidase (SA-β-Gal) staining. Quantitative analysis revealed significantly increased SA-β-Gal-positive cells in both SK-Hep1 and Hep3B lines following SLC25A10 knockdown, indicating senescence induction (Figure 3A). Consistent with the known association between senescence and cell cycle arrest, flow cytometry analysis demonstrated G1 phase accumulation in SLC25A10-deficient cells (Figure 3B,3C). To mechanistically link these observations, we examined key cell cycle regulators using WB. The results showed that downregulation of G1/S transition promoters (CDK4 and Cyclin D1) and upregulation of cell cycle inhibitor CDKN2A (p16) in SK-Hep1 and Hep3B cells with SLC25A10 knockdown (Figure 3D). The grayscale quantification results of the WB analyses presented in Figure 3D was shown in Figure S2. These coordinated molecular changes substantiate that SLC25A10 knockdown impede cell cycle progression through senescence-mediated mechanisms, providing a plausible explanation for the observed anti-proliferative effects.

Figure 3 SLC25A10 suppression induces cellular senescence and cell cycle dysregulation. (A) SA-β-Gal staining of senescent cells in SK-Hep1 and Hep3B cells with or without SLC25A10 knockdown. (B) Flow cytometric evaluation of cell cycle phase distribution using PI staining in SLC25A10 knockdown cell lines. Black line: represents the histogram of the experimentally measured DNA content distribution (raw data), reflecting the actual distribution of cells across different DNA content levels in the sample. Red Line: indicates the theoretical curve generated by a mathematical model (e.g., the Dean-Jett-Fox model), which is used to classify cell cycle phases (G1, S, and G2/M). The comparison between the black raw data and the red fitted curve allows evaluation of the model’s accuracy. (C) Quantification analysis of cell cycle phase proportions following SLC25A10 knockdown. (D) Immunoblot analysis of cell cycle regulatory proteins (CDK4, Cyclin D1, and CDKN2A) in SK-Hep1 and Hep3B cells transfected with or without SLC25A10 shRNA. PI, propidium iodide; shRNA, short hairpin RNA.

SLC25A10 knockdown attenuates tumor growth in vivo

To validate the anti-tumor efficacy of SLC25A10 knockdown, we established subcutaneous xenograft model by implanting Hep3B cells stably expressing sh-SLC25A10-2 or control shRNA into nude mice. Based on longitudinal measurements of subcutaneous tumor volume in both experimental groups, quantitative analysis revealed that SLC25A10 knockdown significantly suppressed tumor progression (Figure 4A). Histopathological and molecular analyses further corroborated these findings: H&E staining revealed reduced pathological karyokinesis, and IHC quantification showed downregulation of proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) in SLC25A10-knockdown tumor tissues (Figure 4B). HMGB1 has been established as a canonical component of the SASP, serving dual roles as a paracrine stimulator of SASP signaling and a canonical biomarker of cellular senescence (19). Thus, in this study, we also detected the expression of HMGB1 protein using IHC staining in tumor tissues, and found that HMGB1 was intensified in tumor tissues with SLC25A10 inhibition (Figure 4B). These in vivo data align with our in vitro observations, demonstrating that SLC25A10 silencing exerts anti-cancer effects through proliferation inhibition and senescence induction.

Figure 4 SLC25A10 knockdown suppressed tumor growth in vivo. (A) Representative macroscopic morphology of subcutaneous xenografts derived from Hep3B cells transduced with sh-SLC25A10-2 versus non-targeting shRNA control plasmids (left). Quantification of tumor volume upon SLC25A10 inhibition (right). *, P<0.05. (B) H&E staining, IHC staining of tumor proliferation markers (KI67 and PCNA), and HMGB1 of tumor tissues. H&E, hematoxylin and eosin; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen; shRNA, short hairpin RNA.

Inhibition of HMGB1 expression partially restores the cellular biological behavior changes induced by SLC25A10 knockdown

To delineate the senescence-dependent mechanism underlying SLC25A10-mediated tumor suppression, we pharmacologically targeted HMGB1 using NecroX-7 (10 µM, 24 h) in SLC25A10-depleted HCC cells (sh-SLC25A10-2) and controls (sh-control). Comprehensive functional analyses revealed that HMGB1 inhibition counteracted the anti-cancer effects of SLC25A10 silencing through multiple pathways: proliferative recovery (Figure 5A-5C), senescence attenuation (Figure 6A), and alleviation of G1 phase arrest (Figure 6B,6C). Molecular profiling further demonstrated that NecroX-7 stimulation could reverse SLC25A10 knockdown-induced downregulation of cell cycle accelerators (CDK4 and Cyclin D1) and attenuate upregulation of senescence effector CDKN2A (Figure 6D). The grayscale quantification results of the WB analyses presented in Figure 6D are shown in Figure S3. These coordinated results establish that SLC25A10 depletion exerts tumor-suppressive effects primarily in a senescence-dependent manner.

Figure 5 HMGB1 inhibition rescues SLC25A10 knockdown-mediated suppression of proliferative capacity. Multi-parametric proliferation analysis via CCK-8 viability assays (A), colony formation assays (B), and EdU incorporation assays (C) in SK-Hep1 and Hep3B cells transfected with control plasmids and sh-SLC25A10-2 plasmids with or without pharmacological HMGB1 inhibition (NecroX-7, 10 µM); (B) cells were stained with crystal violet; (C) cells were pulsed with EdU and Hoechst 33342. EdU, 5-ethynyl-2'-deoxyuridine; OD, optical density value.

Figure 6 HMGB1 blockade attenuates SLC25A10 silencing-induced senescence and cell cycle dysregulation. (A) SA-β-Gal activity quantification in SK-Hep1 and Hep3B cells transfected with control plasmids and sh-SLC25A10-2 plasmids with or without NecroX-7. (B,C) Flow cytometric cell cycle profiling with propidium iodide staining and phase distribution quantification in experimental cohorts. In (B), black line represents the histogram of the experimentally measured DNA content distribution (raw data), reflecting the actual distribution of cells across different DNA content levels in the sample; red line indicates the theoretical curve generated by a mathematical model (e.g., the Dean-Jett-Fox model), which is used to classify cell cycle phases (G1, S, and G2/M). The comparison between the black raw data and the red fitted curve allows evaluation of the model’s accuracy. (D) WB analysis of cell cycle regulatory proteins (CDK4, Cyclin D1, CDKN2A) across combinatorial treatment groups. WB, western blot.

Discussion

Despite recent advances in therapeutic strategies for HCC, clinical outcomes remain suboptimal. HCC frequently develops long before clinical detection, with most patients presenting at intermediate-to-advanced stages characterized by advanced hepatic fibrosis and cirrhosis (20,21). This diagnostic delay results in disease progression to unresectable status or metastatic dissemination, posing significant therapeutic (22). These clinical realities emphasize the urgent need for identifying novel molecular targets for pharmacological intervention.

The mitochondrial carrier SLC25A10 plays a pivotal role in maintaining metabolic homeostasis through its critical function in dicarboxylate transport (23). Pathological dysregulation of SLC25A10 expression disrupts mitochondrial bioenergetics, precipitating metabolic reprogramming and oxidative stress accumulation (24). Beyond redox regulation, this transporter actively modulates tricarboxylic acid (TCA) cycle stability and facilitates lipogenesis by shuttling essential substrates for fatty acid biosynthesis (25). Mounting clinical evidence demonstrated elevated SLC25A10 expression across diverse malignant tumor tissues. In non-small cell lung carcinoma and high-grade serous ovarian cancer, elevated SLC25A10 levels correlate with accelerated tumor proliferation, chemoresistance development, and unfavorable survival outcomes (14,26). Nevertheless, the precise role and expression pattern of SLC25A10 in HCC pathogenesis remains incompletely characterized. In this study, through comprehensive analysis of the ICGC database and the dataset reported by Gao et al. (17), we have identified a significant upregulation in SLC25A10 transcriptional levels within HCC liver tissues compared to normal hepatic counterparts. Subsequent validation through IHC analysis, qRT-PCR, and WB assays consistently demonstrated tumor-specific elevation of SLC25A10 expression at both transcriptional and translational levels. These findings suggest that SLC25A10 may play a critical role in the process of HCC development and progression.

Senescence serves as a robust tumor-suppressive mechanism in oncogenesis. As a hallmark of mammalian aging, cellular senescence exerts dual influences by functioning as both a physiological process and a pathogenic driver of age-associated pathologies, including carcinogenesis (27). During hepatocarcinogenesis, senescence induction establishes stable proliferation arrest in neoplastic cells, thereby exposing their inherent vulnerabilities, while concurrently mobilizing immune-mediated elimination of pre-malignant cells through a coordinated “one-two punch” mechanism (28). This secondary phase features SASP-mediated activation of immunosurveillance mechanisms that enable selective clearance of senescent cancer cells, effectively counteracting malignant progression (29). Notably, p53 restoration-triggered senescence has demonstrated preventive efficacy against HCC development through SASP-dependent immune clearance pathways (30). Given that conventional anticancer modalities—including radiotherapy, chemotherapeutic agents, and molecular-targeted therapies—exhibit intrinsic senescence-inducing capacities (31,32), adjunctive pro-senescence therapies may synergistically enhance therapeutic efficacy by amplifying tumor cells debilitation and impeding HCC progression. Notably, the biological implications of SLC25A10 in modulating cellular senescence and their interplay in HCC pathogenesis remain unreported. In this study, we employed small interfering RNA (siRNA)-mediated knockdown of SLC25A10 to systematically investigate its regulatory effects on both cellular proliferation and senescence. Our experimental findings demonstrated that knockdown of SLC25A10 could suppress cell proliferation, induce senescence-associated β-galactosidase activity, and trigger G1 phase arrest by downregulating CDK4/Cyclin D1 and upregulating CDKN2A. Interestingly, we also found that SLC25A10 silencing reduced tumor growth and decreased KI67/PCNA expression, while enhancing HMGB1, a SASP marker, suggesting a critical role of SLC25A10 in maintaining HCC cell homeostasis through senescence-associated pathways.

Senescent cells exhibit unique features such as irreversible cell-cycle arrest, resistance to apoptosis, and SASP (33). Extensive research has established that SASP represents a complex secretome comprising proinflammatory cytokines, chemokines, and extracellular matrix-remodeling proteases. This phenotype not only serves as a biomarker of cellular aging but also actively contributes to age-related pathophysiology through sustained promotion of chronic low-grade inflammation and progressive tissue dysfunction (33). The HMGB1 protein, a highly conserved non-histone chromatin-associated protein belonging to the HMG superfamily (34), serves as a critical alarmin in inflammatory responses. HMGB1 exhibits dual secretion mechanisms: active release by immunocompetent cells such as activated monocytes and macrophages through non-classical secretory pathways, and passive leakage from necrotic or membrane-compromised cells. Upon extracellular release, HMGB1 functions as a damage-associated molecular pattern (DAMP) that binds pattern recognition receptors—particularly the receptor for advanced glycation end products (RAGE)—to initiate potent proinflammatory signaling cascades (35-37). Notably in senescent cells, HMGB1 undergoes subcellular redistribution from its nuclear localization to cytoplasmic accumulation, followed by active secretion. This extracellular HMGB1 subsequently amplifies inflammatory responses through activation of NF-κB signaling via engagement of toll-like receptors (TLRs), establishing an autocrine/paracrine feedback loop that perpetuates SASP-mediated tissue inflammation (38).

Emerging studies have established the oncogenic centrality of HMGB1 in HCC, where it orchestrates tumor promotion through distinct molecular cascades. Mechanistically, HMGB1 has been shown to drive tumor progression via Krüppel-like factor 7 (KLF7)-mediated upregulation of TLR4 and PTK8 signaling pathways (39), while extracellular HMGB1 exacerbates metastatic potential through miR-21 dysregulation that amplifies CD44 expression (40). To elucidate the senescence-dependent tumor-suppressive mechanism of SLC25A10, we pharmacologically inhibited HMGB1 using NecroX-7 in SLC25A10-depleted HCC cells. Intriguingly, HMGB1 blockade substantially counteracted the anti-tumor effects of SLC25A10 silencing, manifesting as restored proliferative capacity, attenuated senescence-associated-β-gal activity, and alleviated G1 phase arrest. Mechanistically, NecroX-7 treatment reversed SLC25A10 knockdown-induced suppression of cell cycle accelerators—CDK4 and Cyclin D1—while diminishing the upregulation of senescence effector CDKN2A. These coordinated findings establish that SLC25A10 deficiency exerts tumor suppression primarily through HMGB1-dependent senescence activation, positioning the SLC25A10-HMGB1 axis as a critical regulator of cellular senescence in HCC pathogenesis. While our data reveal the criticality of SLC25A10 in HCC progression, it must be emphasized that these findings do not directly support its clinical targetability. Future studies comparing SLC25A10 dependency between malignant and normal hepatocytes are essential to evaluate therapeutic feasibility.

Conclusions

SLC25A10 serves as a critical regulator in HCC cellular proliferation and senescence, demonstrating its potential as a strategic target for anti-cancer therapies and highlighting the therapeutic implications of its inhibition in oncology.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2319/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2319/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2319/prf

Funding: This study was supported by the China Postdoctoral Science Foundation (No. 2022M711719), Chinese Foundation for Hepatitis Prevention and Control (No. TQGB20210029), Chinese Foundation for Hepatitis Prevention and Control of the WBN Research Foundation (No. 2024029), Health Commission of Nantong (No. QA2021002), the National Natural Science Foundation of China (No. 82070624), and the Natural Science Foundation of Jiangsu Province (No. BK20210841).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2319/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments and was approved by the ethics committee of the Affiliated Hospital of Nantong University (ethical approval No. 2020-L093). Informed consent was taken from all the patients. Animal experiments were performed under a project license (approval No. S20200315-009) granted by the ethics committee of the Affiliated Hospital of Nantong University and in strict accordance with institutional animal welfare guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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