GOT1 inhibits hepatocellular carcinoma progression by regulating SLC25A5-dependent mitochondrial apoptosis

Abstract

Hepatocellular carcinoma (HCC), the most common subtype of primary liver cancer, is characterized by a poor prognosis, with most patients diagnosed at advanced stages that are unresectable and resistant to targeted therapies. This underscores the critical need for novel therapeutic targets. In this study, we investigated the role of glutamate-oxaloacetate transaminase 1 (GOT1) in HCC progression and evaluated its therapeutic potential. GOT1 expression was markedly downregulated in HCC tissues compared with adjacent non-tumor tissues, and higher GOT1 expression correlated with improved patient survival. Functional experiments revealed that overexpression of GOT1 suppressed HCC cell proliferation and tumor growth while promoting apoptosis both in vitro and in vivo. Mechanistically, integrated transcriptome sequencing and mass spectrometry identified solute carrier family 25 member 5 (SLC25A5) as a GOT1-interacting partner. GOT1-induced oxidative stress, mitochondrial membrane depolarization, and activation of the apoptotic cascade were mediated through upregulation of SLC25A5; Conversely, SLC25A5 knockdown rescued these phenotypic effects. Importantly, adeno-associated virus-mediated delivery of GOT1 (AAV-TBG-GOT1) significantly inhibited tumor growth in preclinical HCC models, underscoring its translational relevance. Collectively, these results establish GOT1 as a tumor suppressor in HCC that acts via SLC25A5-dependent mitochondrial apoptosis and propose GOT1 as a promising prognostic indicator and therapeutic target for HCC.

Introduction

Liver cancer ranks as the sixth most prevalent malignancy globally and stands as the third leading cause of cancer-related deaths, with hepatocellular carcinoma (HCC) representing the predominant subtype [1] and having a poor prognosis. Liver resection is the main treatment for early HCC [2]. However, most patients are diagnosed with mid-stage or advanced HCC and cannot undergo resection [3]. Despite the availability of targeted agents, such as sorafenib, regorafenib, and lenvatinib, the therapeutic efficacy in HCC remains unsatisfactory, with low objective response rates [4–6]. Therefore, there is an urgent need to develop novel treatment strategies for aggressive HCC. Glutamic oxaloacetic transaminase 1 (GOT1) is a key isozyme of aminotransferases in humans, along with Glutamic oxaloacetic transaminase 2 (GOT2). Both enzymes play crucial roles in amino acid metabolism by catalyzing transfer reactions. It is well understood that GOT1 functions as a metabolic enzyme, enhancing the NADP + /NADPH ratio and preserving cellular redox balance [7]. However, its non-metabolic functions remain underexplored. For example, GOT1 has been found to constrain T(H)17 cell differentiation [8]. Recent research has focused on the dysregulation of GOT1 in tumors, such as pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast invasive carcinoma (BRCA), where high expression of GOT1 promotes tumor proliferation, invasion, and glutamine metabolism [9–12]. However, the role of GOT1 in HCC progression remains poorly understood. Our previous study confirmed that knockdown of GOT2 promotes HCC proliferation and metastasis in vitro and in vivo [13], suggesting a distinct role of GOT in HCC compared to other cancers. Therefore, these findings highlight the need to further investigate the function of GOT1 in HCC. Rapid tumor growth leads to increased oxygen consumption, leading to hypoxia, a hallmark of solid tumors [14, 15]. Hypoxia-induced reactive oxygen species (ROS) generation, primarily derived from mitochondria [16, 17]. Oxidative stress activates the mitochondrial permeability transition pore (MPTP) [18], where solute carrier family 25 member 5 (SLC25A5) modulates mitochondrial matrix small molecule concentrations, influencing MPTP opening [19]. MPTP opening precipitates cellular bioenergetic depletion and apoptosis, antagonistic to cell proliferation [18].

In this study, we observed significant downregulation of GOT1 in HCC tissues, with its expression level positively correlating with patient prognosis. We further demonstrated that GOT1 overexpression reduces mitochondrial membrane potential via upregulation of SLC25A5, thereby promoting apoptosis and inhibiting proliferation in HCC cells. These results identify GOT1 as a promising prognostic biomarker and therapeutic target for HCC.

Materials and methods

Reagents and antibodies

GOT1 and Vinculin antibodies were purchased from Wuhan Proteintech Biotechnology Co., Ltd., SLC25A5, CMYC and FLAG antibodies were purchased from Wuhan ABclonal Biotechnology Co., Ltd., cell counting kit-8 (CCK-8) and MG132 were purchased from MedChemExpress Biotechnology Co., Ltd., USA, EDU cell proliferation detection kit, reactive oxygen detection kit, mitochondrial membrane potential detection kit, GSH and GSSG detection kit, NADP + /NADPH detection kit were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., and cell apoptosis kit and cell cycle kit were purchased from Hangzhou Lianke Biotechnology Co., Ltd. Protein A/G magnetic beads for immunoprecipitation were obtained from Selleck Co., Ltd.

Clinical samples and animal studies

This study adhered to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from patients diagnosed with HCC who underwent partial hepatectomy at the Department of Hepatobiliary Surgery, Nanjing Drum Tower Hospital. Consent was obtained for the use of tumor and adjacent non-tumor tissues for research purposes. The experimental protocol involving human subjects was approved by the Ethics Committee of Nanjing Drum Tower Hospital.

Five-week-old BALB/c male nude mice and 5-week-old C57BL/6 J male mice were procured from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The animals were housed in a specific pathogen-free facility at the Laboratory Animal Research Center under controlled environmental conditions (temperature: 22 °C ± 2 °C, humidity: 60 ± 10%, light/dark cycle: 12/12 h). All animal care procedures and experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. The study received approval from the Institutional Animal Care and Use Committee of the Laboratory Animal Center at Drum Tower Hospital, China.C57BL/6 J mice (6–8 weeks old, male) were used to establish the HCC model. Tumorigenesis was induced by hydrodynamic tail vein injection of plasmid DNA containing AKT (15 μg), NRAS (15 μg), and Sleeping Beauty transposase (SB, 3 μg). One week after injection, mice were randomly assigned into two groups: control vector group (AAV-CON) and experimental group (AAV-GOT1). Each mouse received a single intravenous injection of AAV-CON or AAV-GOT1 (2*10¹¹ viral particles, via tail vein). Six weeks after treatment, all mice were sacrificed, and liver tissues were collected for histological, immunohistochemical, and molecular analyses to evaluate the effect of GOT1 on HCC development.

Lentivirus infection

Lentiviral plasmids designed for GOT1 overexpression with a flag tag and short hairpin RNA (shRNA) sequences targeting GOT1 were constructed by Beijing Qingke Biotechnology Co., Ltd. These constructs were transiently transfected into 293 T cells alongside auxiliary plasmids. Subsequently, the resulting supernatant was harvested and meticulously preserved at -80°C. This supernatant, enriched with lentivirus particles carrying either overexpression or knockdown elements, was then employed to infect HCCLM3 and Hep3B cell lines. Following infection, puromycin served as a selective agent to isolate successfully infected cells.

Cell proliferation and migration analysis

All cell lines were purchased from Servicebio (Wuhan, China) and were authenticated by the supplier as qualified for experimental use before the experiments. Cell viability was assessed using the cell counting kit-8 (CCK-8) proliferation assay in accordance with the manufacturer’s instructions. For clone formation analysis, an appropriate number of cells were seeded into six-well plates and cultured in a complete medium supplemented with 30% fetal bovine serum (FBS) for two weeks. Subsequently, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, stained with 0.1% crystal violet, and visualized using a digital camera. The proliferation rate was further evaluated using the 5-ethynyl-2-deoxyuridine (EDU) proliferation detection kit. Cells were labeled with an EDU fluorescent probe and the expression of fluorescence was detected by Leica fluorescence inverted microscopy. Migration capacity was assessed utilizing the Transwell assay. Cells were seeded into the cell culture chamber and inserted into a 24-well plate, with 200 μL of serum-free medium added to the top chamber and 600 μL of complete medium added to the bottom chamber. After 24 h of incubation, the chamber was fixed with 4% PFA for 30 minutes, stained with 0.1% crystal violet for 20 min, and observed under a microscope for imaging.

Measurement of GSH/GSSG and NADP + /NADPH ratios, ROS, and mitochondrial membrane potential

The GSH/GSSG ratio detection kit and NADP + /NADPH ratio detection kit were employed to conduct the assays and calculations in accordance with the manufacturer’s instructions. Following the manufacturer’s protocols, the DCFH-DA probe and JC-1 probe were incubated with the cells, respectively, and then the intracellular ROS accumulation and changes in mitochondrial membrane potential were observed under the microscope.

Western blot analysis

Proteins were extracted using RIPA lysis buffer, separated by electrophoresis, and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. Block the PVDF membrane using 5% skim milk at room temperature for 1 h. The membranes were then incubated overnight at 4 °C with specific primary antibodies. After washing the membrane three times with TBST, incubate with the secondary antibody for 1 h. Protein expression was detected using an enhanced chemiluminescent reagent.

Real-time fluorescence quantitative qRT-PCR

Total RNA was extracted using the Trizol method, followed by reverse transcription. Quantitative real-time PCR was conducted on a real-time fluorescence quantitative PCR system utilizing ChamQ Universal SYBR qPCR premix, with calculation performed using the 2^-ΔΔCT method. The primer sequences used were as follows:

β-actin:

Forward: 5’-CTACGTCGCCCTGGACTTCGAGC-3’

Reverse 5’-GATGGAGCCGCCGATCCACACGG-3’

GOT1:

Forward: 5’-GCTGACCGGATTCTGACCAT-3’

Reverse 5’-TCAACCTGCTTGGGGTTCAA-3’

SLC25A5:

Forward: 5’-TTATAGACTGCGTGGTCCGTA-3’

Reverse: 5’-GGCGAAGTTAAGAGCCTGGG-3’

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM) unless otherwise specified. All datasets were confirmed to meet the assumptions of normality and homogeneity of variances prior to conducting parametric tests. Comparisons between two groups were conducted using an unpaired, two-tailed Student’s t-test. Comparisons involving three or more groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test, as specified in the figure legends. p-value < 0.05 was considered statistically significant. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with “ns” indicating nonsignificant differences.

Ubiquitination assay

To assess the effect of GOT1 on SLC25A5 ubiquitination, ubiquitination assays were performed in 293 T cells. Cells were seeded in 10-cm dishes and transiently transfected with the following plasmid combinations: (1) HA-UB alone, (2) CMYC-SLC25A5 alone, (3) HA-UB plus CMYC-SLC25A5, and (4) HA-UB plus CMYC-SLC25A5 plus FLAG-GOT1, using a standard lipofection reagent according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were treated with the proteasome inhibitor MG132 (final concentration 10 μM) for 6 h before harvest.

Cells were then lysed in ice-cold immunoprecipitation buffer supplemented with protease inhibitors. Equal amounts of protein were incubated with anti-CMYC magnetic beads at 4 °C with gentle rotation. After extensive washing, immunocomplexes were eluted by boiling in SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting. Ubiquitinated SLC25A5 was detected by anti-HA immunoblotting, whereas anti-CMYC immunoblotting was used to verify efficient immunoprecipitation of CMYC-SLC25A5.

Result

The downregulation of GOT1 is associated with a poor prognosis in HCC

The TCGA database showed the expression patterns of GOT1 in tumor tissues (T) and adjacent non-tumor tissues (N) across various cancer types (Fig. 1A). GOT1 was upregulated in BRCA, Pancreatic adenocarcinoma (PAAD), Prostate adenocarcinoma (PRAD), and lung squamous cell carcinoma (LUSC), whereas it was downregulated in acute myeloid leukemia (LAML) and Liver HCC (LIHC). Interestingly, the overall survival (OS) of the high GOT1 expression group in LIHC surpassed that of the low expression group (Fig. 1B), suggesting a unique role of GOT1 in LIHC compared to other malignancies. In addition to TCGA-LIHC, we assessed GOT1 expression across multiple independent public HCC cohorts, in which GOT1 was consistently downregulated in tumor tissues compared with matched non-tumor liver (Fig. 1C). To further investigate GOT1 expression in HCC patients, samples from ten pairs of adjacent non-tumor (N) and tumor (T) tissues were analyzed for GOT1 expression at both protein and mRNA levels (Fig. 1D, E and Supplement Fig. S1). Densitometric quantification of Western blots from paired HCC and adjacent non-tumorous liver tissues demonstrated that, overall, GOT1 protein expression was lower in tumor than in corresponding non-tumor liver. Later, immunohistochemistry (IHC) staining of T and N tissues (Fig. 1F) confirmed this finding, showing a significant decrease in GOT1 value in tumor tissue. Furthermore, three mouse models of HCC (AKT + CMYC; AKT + NRAS; CCL4) were utilized to validate the decreased expression of GOT1 in HCC (Fig. 1G).

Fig. 1. Downregulation of GOT1 predicts a poor prognosis in HCC.

A Pan-cancer analysis of GOT1 expression in cancers from the TCGA database. B Low expression of GOT1 predicts worse OS based on the TCGA database. C The expression of GOT1 in multiple independent public HCC datasets (GSE14520, GSE25097, GSE39791, GSE45267, GSE45436, GSE36411) D,E Western blot and RT-qPCR analysis of GOT1 expression in adjacent non-tumor tissues and tumor tissues from ten pairs of clinical HCC samples. F Representative IHC images of GOT1 in human HCC adjacent non-tumor tissues and tumor tissues (10×). G Western blot analysis of GOT1 expression in normal liver tissue and cancer tissue from three mouse models of HCC. Scale bar,100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns nonsignificant. N non-tumor tissues, T tumor tissues).

GOT1 overexpression inhibits HCC progression in vitro

GOT1 is downregulated and shows a positive correlation with the prognosis of HCC, indicating its potential role as a tumor suppressor in HCC. To characterize baseline GOT1 expression, we first examined its protein levels across different cell lines, including 293 T, LO2, Hep3B, HepG2, C3A, HCCLM3, MHCC97L, MHCC97H, SMCC-7721 and Huh7 (Fig. 2A). Hep3B and HCCLM3, characterized by lower GOT1 expression, were chosen to establish lentivirus-mediated GOT1 overexpression cell lines (lvcon and lvGOT1) (Fig. 2B). The results of the CCK-8, Colony Formation and EDU assay showed that the overexpression of GOT1 significantly inhibited the proliferation of HCC cells (Fig. 2C–E). However, wound-healing (scratch) and transwell migration assays indicated that overexpression of GOT1 did not affect the migratory capacity of HCC cells (Fig. 2F).

Fig. 2. GOT1 overexpression inhibits HCC progression in vitro.

A Western blot and RT-qPCR analysis of GOT1 protein and mRNA expression in a range of cells. B Western blotting and RT-qPCR verified the efficiency of infection of HCCLM3 and Hep3B cells with GOT1 overexpressing lentivirus. C–E CCK-8 (Fig. 2C), colony formation (Fig. 2D) and EDU (Fig. 2E) assays the effect of GOT1 overexpression on the proliferation of HCCLM3 and Hep3B. F Wound healing assay and transwell assay to detect the effect of GOT2 overexpression on the migration ability of HCCLM3 and Hep3B. Scale bar, 25 or 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

GOT1 overexpression inhibits HCC progression in vivo

In vivo experiment, HCCLM3 cells transfected with lvcon and lvGOT1 were injected into nude mice (n = 5) to establish a subcutaneous tumor model. Notably, mice in the lvGOT1 group exhibited significantly slower tumor growth rates and reduced tumor weights compared to the control group (Fig. 3A–D). Histological examination revealed that tumors in the lvGOT1 group exhibited fewer intravascular tumor thrombi, indicating reduced vascular invasion compared with the control group, and Immunohistochemistry indicated a reduced positive rate of the proliferation marker Ki67 in the lvGOT1 group (Fig. 3E). In the NRAS + AKT-induced orthotopic HCC model, exogenous plasmid-mediated expression of GOT1 led to a significantly lower number of tumors and hepatosomatic index compared to the control group (Fig. 3F, G). Immunohistochemistry analysis of both groups revealed normal nuclear cytoplasmic morphology and reduced Ki67 expression in the GOT1 overexpression group, indicating decreased proliferative capacity. Furthermore, Masson muscle fiber staining showed less liver fibrosis compared to the control group (Fig. 3H). Therefore, GOT1 is specifically under expressed in HCC and its overexpression inhibits the progression of HCC.

Fig. 3. GOT1 overexpression inhibits HCC progression in vivo.

A A model of subcutaneous tumor in nude mice was constructed using stable transfection lvcon and IvGOT1 cells. Representative images of tumors are shown (n = 5 mice/group). B,C Volume (Fig. 3B) and weight (Fig. 3C) of subcutaneous tumors in nude mice in the lvcon and lvGOT1 groups. D Growth curves of subcutaneous tumors in nude mice in the lvcon and lvGOT1 groups. E Representative images of HE, GOT1 and Ki-67 of lvcon and lvGOT1 groups. F Representative image of overexpression of GOT1 inhibiting the progression of AKT + NRAS HCC model. G Liver-body ratio of Control group and GOT1 group of the AKT + NRAS HCC model. H Representative IHC images of HE, GOT1, Ki-67, and Masson of Control group and GOT1 group of the AKT + NRAS HCC model. Scale bar, 0.1 or 0.2 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

In conclusion, our results suggest that overexpression of GOT1 inhibits the proliferation of HCC both in vivo and in vitro, potentially attenuating its malignant progression.

Overexpression of GOT1 induces apoptosis in HCC cells

In vitro and in vivo experiments consistently showed that overexpression of GOT1 inhibits the proliferation of HCC. To investigate the underlying mechanisms, we performed RNA-seq analysis on the lvcon and lvGOT1 groups of HCCLM3 cells (Supplement Table. S1). KEGG enrichment analysis identified the HIF-1 signaling pathway as enriched in GOT1-overexpressing cells, although it did not reach the multiple-testing–corrected significance threshold (q value > 0.05) (Fig. 4A). HIF-1 signaling is critically involved in cellular responses to hypoxia and is known to promote ROS generation. Subsequently, we quantified the levels of ROS after overexpressing GOT1 using fluorescence microscopy and flow cytometry, which revealed a significant increase in ROS levels in GOT1-overexpressing cells (Fig. 4B, C). Although GOT1 has been reported to sustain redox homeostasis by generating NADPH, we observed a decreased GSH/GSSG ratio and an increased NADP⁺/NADPH ratio in lv-GOT1-treated HCCLM3 and Hep3B cells (Fig. 4D, E), indicating disrupted redox balance. Furthermore, GO analysis of RNA-seq data emphasized transition metal ion binding as the most relevant biological signaling pathway influenced by GOT1 overexpression (Fig. 4F), which correlated with a reduction in mitochondrial membrane potential (Fig. 4G), a hallmark of early apoptosis. Flow cytometric analysis demonstrated a significantly higher apoptosis rate in the GOT1 overexpression group (Fig. 4H). Overall, these findings suggest that overexpression of GOT1 leads to oxidative stress, reduced mitochondrial membrane potential, and ultimately triggers apoptosis in HCC cells.

Fig. 4. GOT1 induces HCC cells apoptosis.

A KEGG pathways enriched in lvcon group and lvGOT1 group of HCCLM3. B ROS content in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells. C Flow cytometry was utilized to assess ROS changes in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells. D GSH/GSSG ratio in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells. E NADP⁺/NADPH ratio in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells. F GO pathways enriched in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells. G JC-1 probe was employed to investigate alterations in mitochondrial membrane potential after GOT1 overexpression in HCCLM3 and Hep3B cells. H Cell apoptosis in lvcon and lvGOT1 group of HCCLM3 and Hep3B cells were detected by flow cytometry. Scale bar,100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns nonsignificant.

GOT1 inhibits HCC cell proliferation through SLC25 A5

Our studies have demonstrated that the overexpression of GOT1 induces apoptosis and suppresses proliferation in HCC cells. To investigate the molecular mechanism involved, we conducted immunoprecipitation (IP) by combining GOT1 antibody with magnetic beads (Fig. 5A). The effectiveness of IP was confirmed through silver staining (Fig. 5B). Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze the GOT1-interacting proteins (Fig. 5C). SLC25A5 was identified as a high-confidence binding partner based on interaction scores and functional relevance. The physical association between GOT1 and SLC25A5 was further confirmed by co-immunoprecipitation (Co-IP) in HCCLM3 and Hep3B cells (Fig. 5D). Moreover, immunofluorescence staining revealed substantial colocalization of GOT1 and SLC25A5 in both cell lines (Fig. 5E). Additionally, GOT1 positively regulated the protein and mRNA expression levels of SLC25A5 (Fig. 5F, G). Consistent with this, ubiquitination assays revealed that GOT1 attenuated the ubiquitination of SLC25A5, thereby potentially contributing to its increased protein stability (Supplementary Fig. S2). Knockdown of SLC25A5 reversed the anti-proliferative effect induced by GOT1 overexpression (Fig. 5H–J) and concurrently restored mitochondrial membrane potential (Fig. 5K), indicating that SLC25A5 is a downstream factor of GOT1. In conclusion, these results suggest that GOT1 inhibits HCC cell proliferation through upregulation of SLC25A5, and that silencing SLC25A5 reverses the suppressive effects of GOT1 on cellular proliferation.

Fig. 5. GOT1 inhibits HCC cell proliferation through SLC25A5.

A IP assay of GOT1 overexpression in 293 T cells. B the results of silver staining of GOT1 overexpression in 293 T cells. C the top five target proteins bound to GOT1 were identified by LC-MS mass spectrometry, including SLC25A5. D Co-IP verifies the combination of GOT1 and SLC25A5. E Diagram showing the co-localization results of GOT1 and SLC25A5. F Changes in SLC25A5 protein expression when GOT1 is overexpressed or knocked down. G RT-qPCR analysis of changes in SLC25A5 mRNA expression when GOT1 was overexpressed or knocked down. H Western blot verifies the knockdown efficiency of siSLC25A5. I Colony formation assay detected changes in the proliferation ability of liver cancer cells after siSLC25A5 treatment in lvGOT1 cells, accompanied by a quantitative bar graph. J CCK8 assay evaluating changes in the proliferation ability of liver cancer cells following lvGOT1 and siSLC25A5 treatment. K Utilization of the JC-1 probe to demonstrate changes in mitochondrial membrane potential following lvGOT1 and siSLC25A5 treatment. Scale bar, 20 or 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns nonsignificant.

Therapeutic delivery of GOT1 suppresses HCC progression in vivo

To evaluate the therapeutic potential of GOT1 invivo, we constructed an AAV-TBG-GOT1 vector (AAV-GOT1) and a corresponding AAV-TBG-Control vector (AAV-CON). Each NRAS + AKT-induced orthotopic HCC model mouse was intravenously injected with 2*10¹¹ virus particles and euthanized after six weeks (Fig. 6A). Macroscopic examination of the livers revealed a marked reduction in total tumor burden in the AAV-GOT1 group compared to the AAV-CON group (Fig. 6B). The liver-body ratio was also lower in the AAV-GOT1 group compared to the AAV-CON group (Fig. 6C). Blood biochemistry analysis indicated no significant adverse effects of AAV-GOT1 on hepatic or renal function (Fig. 6D). Consistent with the above results, histopathological analysis further verified the suppressive effect of AAV-GOT1 on HCC tumor growth in vivo, as reflected by immunohistochemical staining and corresponding H-score quantification of GOT1, Ki-67, and SLC25A5 (Fig. 6E). In summary, these results demonstrate that AAV-mediated delivery of GOT1 effectively inhibits HCC progression in vivo without detectable organ toxicity, supporting its potential as a gene therapy strategy for HCC.

Fig. 6. AAV-GOT1inhibits HCC progression in vivo.

A Schematic representation of the experimental timeline for evaluating the effect of AAV-GOT1 treatment on HCC progression in vivo. B Representative images of mouse livers after 6 weeks of AAV-CON or AAV-GOT1 treatment. C Liver-body weight ratio of mice in the AAV-CON or AAV-GOT1 groups. D Serum biochemical indicators related to liver and kidney function. E Representative images of HE staining, and immunohistochemical staining for GOT1, Ki-67, and SLC25A5 of AAV-CON or AAV-GOT1 group. Scale bar, 0.1 mm. Quantitative analysis of immunohistochemical H-scores for GOT1, Ki67, and SLC25A5 in the indicated groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns nonsignificant.

Discussion

Research on the role of GOT1 in various solid tumors has gained significant attention [10, 20–22]. However, investigations into its correlation with HCC remain limited. Given the high expression of GOT1 in the liver and its distinct pattern compared to other cancers, such as BRCA, CRC, and PDAC (Fig. 1A), we explored its mechanism in HCC development. Tissue analysis of HCC patients revealed a notably lower expression of GOT1 in tumor tissues compared to adjacent non-tumor tissues. Survival analysis from public databases indicated that low GOT1 expression correlates with poorer prognosis in HCC patients.

GOT1 has emerged as a potential therapeutic target across multiple cancers [23]. In PDAC, CRC, and breast cancer, GOT1 acts as a pro-tumorigenic metabolic hub that strengthens the malate–aspartate shuttle, preserves cytosolic redox balance, and supports NAD(P)H-dependent anabolic metabolism, ultimately driving tumor cell proliferation and resistance to oxidative stress [24]. In contrast to its pro-tumorigenic role in other malignancies, our data suggest that in HCC, GOT1 exerts a tumor-suppressive effect. GOT1 is highly active in hepatocytes, where it sustains the malate–aspartate shuttle and integrates carbon–nitrogen metabolism. The diagnostic sensitivity of AST/ALT elevations as indicators of hepatocellular injury underscores the liver’s intrinsic reliance on transamination [25]. Accordingly, in HCC, GOT1 is likely to preferentially drive mitochondrial oxidative stress and apoptosis. Functionally, GOT1 overexpression in HCC cells markedly increases intracellular ROS and induces oxidative stress, while concomitantly weakening the capacity of the GSH redox cycle to maintain redox homeostasis, as evidenced by a decreased GSH/GSSG ratio, thereby suppressing HCC cell proliferation and promoting cell death [26]. Mechanistically, we further demonstrate that GOT1 upregulates the mitochondrial ADP/ATP carrier SLC25A5 and enhances its protein stability by attenuating its ubiquitination, thereby increasing mitochondrial ROS production, promoting dissipation of the mitochondrial membrane potential and opening of the MPTP, and ultimately augmenting mitochondrial apoptosis in HCC cells. However, the precise molecular mechanisms through which GOT1 governs the ubiquitination of SLC25A5 remain to be fully delineated and are currently under active mechanistic investigation. Moreover, the increased NADP + /NADPH ratio post-GOT1 overexpression suggests a potential mechanism for overcoming ROS-induced cell death [27]. This finding is consistent with the concept of a ROS “threshold,” in which moderate levels of ROS promote cellular adaptation and survival, whereas excessive ROS disrupt redox homeostasis and trigger apoptosis [28].Unlike in PDAC or CRC, where GOT1 primarily supports redox equilibrium, in HCC it appears to push cells beyond this adaptive threshold, highlighting a tissue-specific divergence. However, the precise molecular basis underlying this difference remains to be fully elucidated.

SLC25A5, situated in the inner mitochondrial membrane, facilitates ADP/ATP exchange [29]. ATP generation by mitochondria fuels tumor metastasis [30]. SLC25A5 influences mitochondrial uncoupling and MPTP activity, key in various pathologies and organ damage [18]. Our study revealed that GOT1 overexpression positively regulated SLC25A5, leading to MPTP opening without a significant impact on HCC cell migration. Although SLC25A5 is non-essential for MPTP, it modulates its permeability transition [31]. Increased ROS and decreased membrane potential induced by GOT1 overexpression serve as second messengers for MPTP opening, influencing cell apoptosis [32]. Given that ROS also regulates autophagy and mitophagy via AMPK–mTOR and PINK1/Parkin pathways, it is plausible that GOT1-induced ROS not only triggers apoptosis but may also engage mitophagy. It remains to be determined whether this response protects cells through the removal of damaged mitochondria or instead promotes cell death.

In conclusion, our animal experiments demonstrate that GOT1 may serve as a potential therapeutic target for HCC, as it upregulates SLC25A5 to promote MPTP opening, disrupt mitochondrial function, induce apoptosis, and thereby suppress HCC cell proliferation.

Author contributions

WWZ, CXC, and YKZ contributed equally to this work. WWZ, CXC, and YKZ were responsible for designing the study, performing experiments, analyzing data, and drafting the manuscript. YJL, ZWL, and TCL assisted with in vitro and in vivo experiments, data collection, and analysis. YZL contributed to study design, supervised data interpretation, and provided critical revisions to the manuscript. DCY conceived and supervised the project, acquired funding, and critically revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (no. 82173129, 82302942, 82103384, 82372834), the China Postdoctoral Science Foundation (no. 2023M731622), and the Start-up Funding for Talent Introduction of Drum Tower Hospital (RC2023-032).

Data availability

All data supporting the findings of this study are available within the paper and its supplementary information. Additional data are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Nanjing Drum Tower Hospital (approval No. 2023-LCYJ-PY-32). All animal experiments were approved by the Animal Ethics Committee of Nanjing Drum Tower Hospital (approval No. 2023AE01093) and complied with relevant institutional and national guidelines.

Supplementary information

The online version contains supplementary material available at 10.1038/s41388-026-03769-x.