A total of six male C57BL/6J mice (age, 8-10 weeks; weight, 20-25 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were performed according to international guidelines and approved by the Animal Care and Use Committee of Tianjin Medical University (approval no. TMUaMEC2024050; Tianjin, China). Animals were maintained under specific-pathogen-free conditions (21-23°C; humidity, 60-65%; light/dark cycle, 12:12 h; free access to food/water). A diethylnitrosamine (DEN)-induced liver cancer mouse model was established as previously described (29). Briefly, 2-week-old C57BL/6J mice (weight, 8-12 g) were intraperitoneally administered 25 mg/kg body weight DEN (cat. no. W610685; Energy Chemical) (Fig. S1A). When they reached 4 weeks of age, mice were treated with weekly intraperitoneal injection of CCl₄ (5 ml/kg body weight, at a concentration of 10% in olive oil) for a total of 20 times. Mice were monitored daily for signs of distress and weighed three times/week. Abdominal distension was assessed by palpation twice/week. Behavioral parameters, including feeding efficiency and locomotor activity, were recorded daily. At 24 weeks, all mice were euthanized via inhalational exposure to CO₂ (40% chamber volume/min) in a sealed chamber, followed by confirmation of death through absence of cardiac and respiratory function for 5 min. The humane endpoints were defined as follows: i) Tumor volume >2×10³ mm³; ii) tumor diameter >20 mm; iii) rapid or continuous body weight loss >20% within 72 h; iv) tumor ulceration or necrosis leading to skin rupture or persistent exudation for >48 h and v) abdominal distension or ascites burden >10% of body weight, exhibiting an appearance analogous to that of a pregnant mouse compared with age-matched control mice. Liver tissue samples were collected following euthanasia. Cancer and adjacent paracancerous tissues were dissected, with the normal adjacent tissues sampled at a distance of ≥0.5 cm from the tumor margin to ensure clear distinction. All tissue samples were immediately snap-frozen in liquid nitrogen (−196°C) for subsequent analyses.

Hep3B, HepG2, Huh7, Hepa1-6 and 293T cells were obtained from the American Type Culture Collection. The cells were cultured in DMEM (cat. no. MA0212; Dalian Meilun Biology Technology Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; cat. no. MN012103; Mengma (Tianjin) Biotechnology Co., Ltd.) and 1% antibiotics at 37°C under a 5% CO₂ atmosphere. A total of 20 μg lentiCRISPRv2 (cat. no. 52961; Addgene, Inc.) served as the core plasmid for constructing knockout-SSBP1 (for both mouse and human sources), and H128 pLenti-EF1α-EGFP-3FLAG-PGK-Puro (HeYuan Biotechnology Co., Ltd.) for SSBP1 overexpression. Additionally, packaging plasmids (5 μg pMD2.G and 15 μg psPAX2, second-generation lentiviral packaging plasmids) were purchased from Addgene, Inc. The plasmids [sg-SSBP1 (sg, single-guide RNA), 5′-CACCGCAAGTGACTACCTGTAATAC-3′; sg-Ssbp1 (sg, single-guide RNA), 5′-CACCGGATAGTGAAGTATACCAAAT-3′; SSBP1-Flag, forward, 5′-CCGGAATTCATGTTTCGAAGACCTGTATTAC-3′ and reverse, 5′-CCTGGATCCTTCCTCTTTGCTGTCTG-3′] or control plasmids, along with packaging plasmids, were co-transfected into 293T cells (5×10⁵ cells/well) of a 6 well plate using polyethyleneimine (cat. no. 408727; Sigma-Aldrich). The transfection was performed according to the manufacturer's protocol at 37°C for 6-8 h. medium was replaced with DMEM containing 10% FBS (cat. no. MN012103; Mengma (Tianjin) Biotechnology Co., Ltd.). Following incubation at 37°C for 24 and 48 h, viral particles were collected and filtered through a 0.45 μm filter. After filtration, centrifugation was performed at 4°C for 2 h at 60,000 × g. At one day before infection, Hep3B, HepG2, Huh7, Hepa1-6 cells were passaged into 6 well plates, and infection was performed when the cell confluency reached 70%. Before infection, fresh complete medium was added, 10-20 μl viral solution was introduced, the plate was shaken to mix evenly and then incubated at 37°C. After 8 h, the medium was replaced with fresh complete medium. At 48 h later, puromycin (2 μg/ml) was added for selection. Following viral infection, Hep3B, HepG2, Huh7 and Hepa1-6 cells were maintained in medium containing 2 μg/ml puromycin (cat. no. P8230; Beijing Solarbio Science & Technology Co., Ltd.) to establish stable cell lines with SSBP1 knockout and SSBP1 overexpression. The successful establishment of the aforementioned cell lines was verified by western blotting.

Sorafenib (cat. no. S1040; Selleck Chemicals) was dissolved in DMSO to a final concentration of 10 mM/ml. Hep3B cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and treated with 8 μM sorafenib for 12 h at 37°C.

The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) data-sets were retrieved from the National Cancer Institute (NCI) Genomic Data Commons (30) (portal.gdc.cancer.gov/projects/TCGA-LIHC, accession no. phs000178), while the mitochondrial localization gene set was sourced from MitoCarta3.0 (31) (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). Differentially expressed genes (DEGs), were identified using R language (32) (version 3.6.2; http://www.r-project.org/) with the criteria of |log2 fold change (log2FC)|>1.0 (corresponding to a more than 2-fold absolute change in expression) and adjusted P-value <0.05 (to control for false discovery rate in multiple testing), and analyzed with the Metascape online tool (33) (Metascape 3.5; https://metascape.org/gp/index.html#/main/step1). GSE14520 (34) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14520), GSE101685 (35) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101685), GSE151412 (36) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE151412) and GSE62813 (37) (ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62813) datasets were downloaded from National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) profiles (38) (https://www.ncbi.nlm.nih.gov/geo/), and the relative mRNA expression levels of SSBP1 and POLG (DNA polymerase gamma) within these cohorts were analyzed. The expression patterns of SSBP1 across TCGA in cancer were analyzed using the TIMER2.0 (39) (http://timer.cistrome.org/) and UALCAN (40) (https://ualcan.path.uab.edu/) analysis tools. The effects of SSBP1 on the survival of patients with liver cancer and those undergoing treatment with sorafenib were evaluated using the Kaplan Meier Plotter online survival analysis tool (41) (https://kmplot.com/analysis/).

Total RNA was extracted from tumor tissue or cells using the TransZol Up RNA isolation reagent (cat. no. ET111-01-V2; TransGen Biotech Co., Ltd.) and reverse-transcribed into cDNA with the TransScriptII First Strand cDNA Synthesis SuperMix (cat. no. 11120ES60; Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacturer's instructions. qPCR analysis was performed using the SYBR Green PCR Mix (cat. no. 11200ES; Shanghai Yeasen Biotechnology Co., Ltd.) and targeted gene-specific primers (Table SI). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 10 sec, 60°C for 10 sec, and 60°C for 30 sec. The relative mRNA expression levels were calculated using the ΔΔCq method (42), while β-actin served as an internal control. All primers were designed by NCBI and were synthesized by Genewiz, Inc.

To measure the copy number of mtDNA, genomic DNA extracted from liver cancer tissue or cells was quantified using qPCR. The primer sequences used are presented in Table SII.

Western blot analysis was performed as previously described (43). Briefly, total protein was extracted from tumor tissue and cells using RIPA lysis buffer (cat. no. WB3100; NCM Biotech; www.). The protein concentration was determined using a BCA kit (cat. no. CW0014S; Jiangsu CoWin Biotech Co., Ltd.) and equal amounts of protein (50 μg/lane) were separated by 10% SDS-PAGE, followed by electrotransferring onto a PVDF membrane (MilliporeSigma). Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight. Following washing with PBST (containing 0.1% Tween-20) three times (5 min each), the membrane was incubated with the corresponding Peroxidase AffiniPure™ Goat Anti-Mouse (cat. no. 115-035-003) and anti-Rabbit IgG (H+L; cat. no. 111-035-003; both 1:10,000; both Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies at room temperature for 1.5 h. The membrane was washed with PBST three times and the immunoreactive signals were detected using the ECL Western Blotting Detection Reagent (cat. no. CW0049M; Jiangsu CoWin Biotech Co., Ltd.). The protein bands were scanned using the MiniChemi 610 chemiluminescence imaging system (SinSage Technology, Co., Ltd.) and relative intensity was semi-quantified by ImageJ software (ImageJ 2.0; National Institutes of Health). β-actin served as an endogenous control.

Cell viability was assessed using MTT reagent (cat. no. M8180; Beijing Solarbio Science & Technology Co., Ltd.), as previously described (44). Briefly, liver cancer cells were plated into 96 well plates at a density of 3×10³ cells/well and incubated either in the presence or absence of sorafenib (8 μM). Following incubation at 37°C for 24, 48, and 72 h, respectively the culture medium was replaced with 100 μl MTT reagent (0.5 mg/ml), followed by incubation for 4 h at 37°C. Then, the purple formazan was dissolved in dimethyl sulfoxide (DMSO). Absorbance at a wavelength of 490 nm was measured using a microplate reader (45).

Liver cancer cells were seeded into 6-well plates at a density of 1×10³ cells/well in the presence or absence of sorafenib (2.5 μM) at 37°C for ~2 weeks. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet solution (cat. no. G1064; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min, both at room temperature. The colonies (>50 cells) were counted using ImageJ software (ImageJ 2.0; National Institutes of Health) and images were captured under a light microscope (magnification, ×40).

The migration of liver cancer cells were assessed using 24 well Transwell chambers. The upper and lower culture compartments were separated by polycarbonate membranes with 8 μm pores (Corning, Inc.). The bottom chamber was filled with DMEM (cat. no. MA0212; Dalian Meilun Biology Technology Co., Ltd.) supplemented with 20% FBS (cat. no. MN012103; Mengma (Tianjin) Biotechnology Co., Ltd.) as a chemoattractant. A total of 5×10⁴ cells in serum-free DMEM (were seeded into the upper chamber and incubated at 37°C for 48 h in a humidified incubator with 5% CO₂. The cells that migrated to the underside of the membrane were stained with 0.1% crystal violet solution (cat. no. G1064; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature and images were captured under a light microscope (magnification, ×40).

Liver cancer cells (Hep3B and Hepa1-6 cells) were cultured in a 6 well plate until ~100% confluence and a scratch was made. Following washing with PBS to remove detached cells, cells were then cultured under serum-free conditions for 24 h. Images were captured under a light microscope at 0 and 24 h to record the changes in wound width (magnification, ×40). Wound distance was expressed as ratio to the initial distance at 0 h.

Mitochondrial morphology was evaluated using the MitoTracker^®-Red kit (cat. no. C1035; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, liver cancer cells cultured in cell chambers at 37°C were treated in the presence or absence of sorafenib (8 μM, 12 h at 37°C). Following washing with PBS, cells were incubated in serum-free DMEM (cat. no. MA0212; Dalian Meilun Biology Technology Co., Ltd.) supplemented with Mito-Tracker Red CMXRos (1:1,000) for 30 min at 37°C. After washing with PBS, 1 ml serum-free medium was added to the cell chamber. A total of ≥8 randomly selected fields/sample were observed under a laser confocal scanning microscope (LSM-800; Carl Zeiss AG).

Following treatment with sorafenib (8 μM, 12 h at 37°C), cells were harvested and centrifuged at 300 × g for 5 min at room temperature. Following washing with PBS three times, cells were incubated with the MitoTracker^®-Red probe for 30 min at 37°C. Cells were washed three times with PBS and analyzed using a 579 nm channel flow cytometer (BD FACSVerse; BD Biosciences). Data analysis was performed using FlowJo software (version 10.8.1; Tree Star, Inc.).

ROS levels were measured using a DCFH-DA assay (cat. no. S0035S; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Liver cancer cells were seeded into 6 well plates at a density of 1×10⁵ cells per well and treated in the presence or absence of sorafenib (8 μM) at 37°C for 12 h. Cells were washed with PBS three times and the fluorescence intensity was measured using a microplate reader, as previously described (44). A total of 1×10⁴ cells/sample were analyzed on a BD FACSVerse flow cytometer (BD Biosciences) using an argon-ion laser (15 mW) and an incident beam at 488 nm. Data analysis was performed using FlowJo software (version 10.8.1; Tree Star, Inc.).

Mitochondrial membrane potential (MMP) was measured using a JC-1 assay kit (cat. no. C2003S; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Hep3B cells were collected following centrifugation at 600 × g for 4 min at room temperature and washed twice with PBS. Cells were incubated with the JC-1 dye in the dark at 37°C for 20 min. Following gently washing with PBS twice, the cells were analyzed using a BD FACSVerse flow cytometer (BD Biosciences) to detect aggregates and monomers. Data analysis was performed using FlowJo software (version 10.8.1; Tree Star, Inc.). MMP was calculated as the ratio of the fluorescence intensity of aggregates to that of monomers.

Following centrifugation at 600 × g for 5 min at room temperature, 5×10⁵ Hep3B cells were collected and washed twice with PBS. The cells were re-suspended in PBS and stained with propidium iodide and Annexin (cat. no. C1067S; Beyotime Institute of Biotechnology) in the dark for 20 min. Cells were washed with PBS and 1×10⁴ cells/sample were analyzed on a BD FACSVerse flow cytometer (BD Biosciences) equipped with a 15 mW argon-ion laser and an incident beam at 488 nm. Data analysis was performed using FlowJo software (version 10.8.1; Tree Star, Inc.). Cell death was calculated as the percentage of cells positive for propidium iodide fluorescence (46).

C11-BODIPY^581/591 (5 μM; cat. no. D3861; Thermo Fisher Scientific, Inc.) was used to measure lipid ROS levels. Hep3B cells seeded into glass-bottom cell culture dishes at a density of 2×10⁵ cells/ml, were treated with sorafenib (8 μM, 12 h at 37°C) and incubated with C11-BODIPY^581/591 for 30 min at room temperature. Following washing with PBS three times, ≥50 cells/group were observed under a confocal microscope (Carl Zeiss AG) and the fluorescence intensity was measured using ImageJ software (ImageJ 2.0; National Institutes of Health). FITC/PE ratio was used to determine lipid oxidation, and the mean ratio of each group was normalized to that of the control.

All statistical analyses were performed with SPSS software (version 26.0; IBM Corp.). All data are expressed as the mean ± SD, and all experiments were repeated at least three times. Unpaired two-tailed Student's t-test was used for comparisons between two groups when data passed normality and equal variance test; otherwise, the Mann-Whitney test was used. For data that were normally distributed containing >2 groups, one-way ANOVA was used followed by Tukey's comparison post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Among all mitochondrial-localized genes, 225 DEGs were identified between liver cancer and normal tissue, including 85 up and 170 downregulated genes (Fig. 1A). To determine the main functions of DEGs, the Metascape online tool was utilized. The analysis revealed that the DEGs were primarily enriched in 'mitochondrion organization' (Fig. 1B). Among 17 genes involved in mitochondrial biogenesis, eight genes, namely ALAS1, SIRT3, TFAM, TFB2M, SSBP1, CYCS, POLRMT and POLG, were significantly associated with liver cancer phenotypes within the TCGA-LIHC datasets (data not shown). A DEN-induced liver cancer mouse model was established and RT-qPCR showed that only SSBP1 and POLG2 were upregulated, while ALAS1 was significantly downregulated in tumors cells (Fig. 1C). However, in the GEO datasets, the expression of SSBP1, but not POLG, was markedly increased in liver cancer tissues compared with adjacent non-cancerous tissue (Fig. 1D). SSBP1 was upregulated in numerous types of cancer tissues compared with normal tissue (Fig. S1B and C), thus suggesting that SSBP1 could serve a key role in cancer, particularly in liver cancer (Fig. 1E). Kaplan-Meier survival analysis revealed that high SSBP1 expression was associated with poor prognosis in liver cancer (Fig. 1F). Collectively, SSBP1 may be a potential biomarker for the diagnosis and prognosis of liver cancer.

To investigate the effect of SSBP1 on the growth and tumorigenesis of liver cancer, SSBP1 was knocked out using independent lentiviral sgRNA. Western blot analysis demonstrated that SSBP1 was successfully knocked out in the liver cancer cell lines (Figs. 2A and S2A, G and J). MTT assay was employed to monitor cell proliferation at 24 h intervals. Proliferation capacity of SSBP1-knockout Hep3B cells was notably decreased (Fig. 2B). Similarly, the number of colonies formed by SSBP1-knockout Hep3B cells was significantly decreased compared with control cells, consistent with the reduced proliferation capacity observed in the MTT assay (Fig. 2C and D). To explore the effect of SSBP1 on liver cancer cell migration, a Transwell assay was performed. The loss of SSBP1 markedly decreased the migration of Hep3B cells compared with the control group (Fig. 2E). Additionally, wound healing assay was utilized to assess the migration ability of SSBP1-knockout Hep3B cells. SSBP1 knockout significantly impaired the motility of Hep3B cells compared with the control (Fig. 2F). The same results were observed in Hepa1-6 (Fig. S2B-F), HepG2 (Fig. S2H and I) and Huh7 cells (Fig. S2K and L).

As previously described, SSBP1 upregulation is associated with most types of cancers (28). Therefore, the role of SSBP1 in promoting liver cancer growth was investigated. Flag-tagged SSBP1 was overexpressed in liver cancer cell lines using a lentiviral system and SSBP1 overexpression was validated using western blot analysis (Figs. 3A and S3A). Compared with the control, the proliferative capacity of SSBP1-overexpressing Hep3B cells was significantly enhanced (Fig. 3B). SSBP1 overexpression promoted the colony formation capacity of Hep3B cells (Fig. 3C and D). Transwell migration and wound healing assays demonstrated that SSBP1 overexpression enhanced the migration potential of liver cancer cells (Fig. 3E and F). The same results were also obtained in HepG2 cells (Fig. S3B and C).

SSBP1 is a key mitochondrial protein; its abnormal regulation affects mtDNA content, eventually leading to alterations in mitochondrial function (27). mtDNA copy number was elevated in liver cancer tissue (Fig. 4A). Concurrently, SSBP1 knockout in liver cancer cells decreased the number of mitochondria while increasing their migration towards the nucleus (Fig. 4B and C). Additionally, the mtDNA copy number was decreased (Fig. 4D). Western blot analysis indicated that SSBP1 knockout diminished the expression of mitochondria-related proteins (Fig. 4E). As mitochondria serve as the primary source of cellular ROS generation (47), the accumulation of mitochondrial ROS in Hep3B cells was evaluated. A significant increase in mitochondrial ROS accumulation and a marked decrease in MMP were observed in SSBP1-deficient cells (Fig. 4F-I). These results suggested that SSBP1 knockout promoted mitochondrial damage in liver cancer cells.

As aforementioned, the relative cell viability was significantly decreased by SSBP1 deficiency. Therefore, the present study aimed to investigate whether, in addition to impaired proliferation, cell death also occurred. Flow cytometry showed that SSBP1 deletion significantly increased the percentage of dead cells compared with control cells (Fig. 5A-B). Among the various types of cell death, ferroptosis is involved in the genesis and progression of liver cancer, with mitochondria, where SSBP1 is located and which are crucial energy-supplying organelles, serving a key role in this process (27-29,44,48). Therefore, C11-BODIPY fluorescence staining was employed to determine the extent of ferroptosis, and the results revealed an increase in lipid peroxidation in SSBP1-knockout Hep3B cells, confirming enhanced ferroptosis (Fig. 5C-F). Consistently, quantitative analysis showed that antioxidant genes (e.g., GPX4, SLC7A11) were downregulated at both protein and mRNA levels in SSBP1-deficient cells (Fig. 5G and H), demonstrating that SSBP1 regulates liver cancer progression via modulating ferroptosis. The same results were also observed in HepG2 (Fig. S4A and B) and Hepa1-6 cells (Fig. S4C).

Based on the dual role of sorafenib as both a tumor suppressor and inducer of ferroptosis in liver cancer (8), the present study explored the effect of SSBP1 on sorafenib treatment. GEO datasets showed that when liver cancer cells were treated with sorafenib, the expression of SSBP1 notably decreased (Fig. S5A), whereas SSBP1 was upregulated in sorafenib-resistant cells compared with sorafenib-sensitive cells (Fig. S5B). In addition, high SSBP1 expression was associated with poor prognosis in sorafenib-treated liver cancer (Fig. S5C). Therefore, Hep3B cells were treated with sorafenib, and western blot analysis showed that SSBP1 was markedly downregulated in these cells (Fig. 6A). SSBP1 knockout enhanced sorafenib-induced inhibition of clonogenicity and growth inhibition (Fig. 6B-D). Subsequently, the effect of SSBP1 deficiency on sorafenib treatment in liver cancer was assessed. Following SSBP1 knockout, the mtDNA copy number and mitochondrial mass of liver cancer cells were markedly decreased following treatment with sorafenib (Fig. 6E-G). In addition, MitoTracker staining indicated that mitochondrial fragmentation was enhanced and mitochondria migrated closer to the nuclear periphery in SSBP1-knockout liver cancer cells (Fig. 6E). Furthermore, treatment of SSBP1-knockout liver cancer cells with sorafenib enhanced intracellular ROS levels (Fig. 6H and I) and decreased MMP compared with the control group (Fig. 6J and K). Lipid peroxidation levels were markedly elevated, and the protein and mRNA expression of ferroptosis-associated genes was reduced in Hep3B cells treated with sorafenib (Fig. 6A and L-N). In summary, SSBP1 deficiency enhanced the sensitivity of liver cancer cells to sorafenib and may serve as a novel target for overcoming sorafenib resistance (Fig. 7).