ABSTRACT
-
Background/Aims
Acetyl coenzyme A (acetyl-CoA) is one of the most essential metabolites in cell metabolism but its function and concentration in hepatocellular carcinoma (HCC) remain elusive and controversial.
-
Methods
A comprehensive analysis of acetyl-CoA levels and acetyl-CoA synthetase 2 (ACSS2) expression across a range of samples, including patient specimens from both hepatitis B virus (HBV) positive and HBV negative HCC individuals, HBV-transgenic mouse HCC models, and multiple cell lines. Furthermore, to evaluate the functional significance of ACSS2 in HBV-related HCC, we implemented both genetic and pharmacological inhibition strategies targeting ACSS2. Molecular mechanism and mitophagy assessment were revealed by cleavage under target and tagmentation sequencing, RNA sequencing, bioinformatic analyses, transmission electron microscopy and JC-1 staining.
-
Results
Our study revealed a distinct metabolic signature of HBV-related HCC, marked by elevated acetyl-CoA, which was driven by ACSS2. ACSS2 was upregulated by the carbohydrate response element-binding protein in HBV-related HCC. Furthermore, ACSS2 improved tumor cell proliferation, an effect that was dependent on its enzymatic activity. Mechanistically, ACSS2-induced acetyl-CoA accumulation activated voltage-dependent anion channels 1 transcription through increased H3K27ac occupancy, which subsequently promoted mitophagy and HBV-related HCC tumorigenesis. Notably, targeting ACSS2 by depletion or inhibition with a catalytic inhibitor significantly suppressed tumor growth.
-
Conclusions
These findings not only illustrate the interplay between metabolic reprogramming, epigenetic modification, and tumorigenesis in the context of HBV infection, but also highlight ACSS2 as a novel metabolic vulnerability in HBV-related HCC. Therefore, targeting ACSS2 could be a novel strategy against HBV-related HCC.
-
Keywords: Liver cancer; Hepatitis B virus; Acetyl coenzyme A; Acetylation; Mitophagy
Study Highlights
• ACSS2 is elevated in HBV-related HCC, driving acetyl-CoA accumulation and tumor growth.
• ACSS2 increases H3K27ac occupancy on the VDAC1 promoter to enhance mitophagy.
• ACSS2 exhibits as a promising therapeutic target for HBV-related hepatocellular carcinoma.
Graphical Abstract
INTRODUCTION
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide [
1]. Hepatitis B virus (HBV) is currently recognized as a predominant etiological factor in the pathogenesis of HCC, accounting for a significant proportion of cases both globally (~50%) and particularly in China (~80%) [
1,
2]. HBV-related HCC is a proliferation-driven malignancy [
3-
5]. Compared with other types of HCC, HBV-related HCC is characterized by poor prognosis owing to its early onset and accelerated progression [
2,
6]. Therefore, understanding the aggressive nature of HBV-related HCC is crucial for developing targeted and personalized treatment approaches for this cohort of patients.
Accumulating evidence has defined cancers, including HCC, as metabolic disorders, in which numerous metabolic pathways are rewired to fulfill tumor proliferation and progression [
7]. For instance, altered metabolites, such as elevated lactate or acetate levels, act not only as an alternative resource for the biosynthetic requirements of cancer cells but also as regulators that control the expression of downstream oncogenes [
8-
11]. By hijacking host metabolism, HBV infection can largely affect various metabolic pathways in host cells, including enhanced glycolysis, disordered lipid metabolism, and upregulated hexosamine biosynthesis pathway [
12-
14]. Therefore, the metabolic landscape of HBV-related HCC is extremely intricate and distinct due to the interplay between viral replication and carcinogenesis. However, the precise roles and mechanisms by which key enzymes or metabolic intermediates contribute to HBV-related HCC progression remain unclear.
As a central metabolic intermediate, acetyl coenzyme A (acetyl-CoA) is indispensable as fuel for the tricarboxylic acid (TCA) cycle, a key substrate for fatty acid biosynthesis, and the sole donor of acetyl groups for acetylation [
15]. Considering the multifunctional role of acetyl-CoA in cellular physiology, it is not surprising that it also plays an important role in cancer. Typically, elevated acetyl-CoA levels in various malignancies support the growth and proliferation of tumor cells [
16-
18]. For example, KRAS-driven upregulation of acetyl-CoA promotes tumorigenesis in the pancreas by fueling lipid synthesis, and targeting acetyl-CoA-dependent processes exerts anti-cancer effects [
16]. Nonetheless, the reported levels of acetyl-CoA have been discordant in HCC. One European study found a dramatic decrease in acetyl-CoA in HCC compared with paired normal tissue, whereas an Asian study reported the opposite relationship [
19,
20]. Notably, the latter Asian study, which predominantly included patients with HBV-related HCC (>85%), differed from the former study, which primarily involved patients without HBV infection. However, the influence of HBV infection on acetyl-CoA levels in HCC and its underlying mechanisms remain unclear.
In this study, acetyl-CoA levels are found significantly elevated in HBV-positive HCC, compared to HBV-negative HCC. Our study also reveals that acetyl-CoA synthetase 2 (ACSS2) is upregulated upon HBV infection and appears to be a key determinant of acetyl-CoA levels in HBV-related HCC. ACSS2 plays a key role in the ACSS family, crucial for lipid synthesis and protein acetylation by producing acetyl-CoA [
21]. It’s highly expressed in the liver and heart, with broad cellular distribution, primarily affecting acetylation [
21,
22]. ACSS1, on the other hand, is mainly found in brain and muscles, located in mitochondria, and involved in acetate oxidation [
22]. ACSS3, distributed in mitochondria with propionate as its substrate, has unclear enzymatic activity and disease mechanisms [
21,
23]. Therefore, ACSS2 is particularly important for acetyl-CoA production among ACSS family, especially in the liver. In this study, ACSS2 promotes the tumor proliferation via upregulating voltage-dependent anion channels 1 (VDAC1)-mediated mitophagy. More importantly, our study suggests that ACSS2 could be a novel prognostic biomarker and a promising therapeutic target for HBV-related HCC.
MATERIALS AND METHODS
Animal models
HBV-transgenic (HBV-Tg) mice (6–8-week-old, male C57 BL/6 mice) were provided by Prof. Ning-shao Xia (Xiamen University, China). To induce HCC, 2-week-old HBV-Tg mice were administered an initial intraperitoneal injection of diethylnitrosamine (DEN; 50 mg/kg), followed by administration of CCl
4 (2 mL/kg, twice a week for 12 weeks) from 4 weeks of age. Subsequently, the mice were divided into four groups. Two groups were intravenously injected with pSECC-sg
Acss2 or pSECC-sg
Control (
Supplementary Table 1) when the mice were 6–8 weeks old. The other two groups were treated with either an intraperitoneal injection of 25 mg/kg ACSS2 inhibitor or vehicle control once every 2 d for 12 weeks. All HBV-Tg mice were euthanized at 28 weeks of age for subsequent assessments.
In the context of xenograft implantation models, 6–8-week-old male NU/NU mice were purchased from Beijing Vital River Laboratory and divided into six groups. MHCC-97H cells (1×10
6) suspended in 50% volume of 50-μl Matrigel were implanted into the left liver lobe of each mouse (
Supplementary Table 2). Six weeks after implantation, all mice were euthanized and the tumor tissues were harvested for subsequent analysis.
Both HBV-positive HCC and HBV-negative HCC samples and paired, adjacent normal liver tissues were obtained from the Second Affiliated Hospital of Chongqing Medical University. Written informed consent was obtained from all patients involved in this study. The diagnosis of HBV-related HCC was based on the previous HBV infection history and was verified by laboratory examinations, including HBsAg and HBV DNA.
Ethical
All procedures performed in this study were approved by the Institutional Review Board of Chongqing Medical University (reference number: 2024063). All animal experiments were meticulously conducted in compliance with ethical guidelines and protocols sanctioned by the Institutional Animal Care and Use Committee at Chongqing Medical University (approval no: IACUC-CQMU-2023-07014).
RESULTS
Acetyl-CoA levels were elevated in HBV-related HCC
To investigate whether HBV infection was related to the differences in acetyl-CoA levels in HCC, we first compared acetyl-CoA concentrations between HBV-positive and HBV-negative HCC tissues using patient samples. Surprisingly, acetyl-CoA levels were significantly higher in tumor tissues from HBV-positive patients with HCC than those in adjacent non-tumor tissues, a pattern that was not observed in HBV-negative patients with HCC (
Fig. 1A). This trend was consistently confirmed in HCC mouse models, where acetyl-CoA levels were significantly elevated in HBV-Tg mice subjected to DEN/carbon tetrachloride (CCl
4) treatment relative to those in C57 BL/6 wild-type mice (
Fig. 1B). An increase in acetyl-CoA levels was also observed in HBV-infected hepatoma cells (Huh7-AdHBV1.3 and HBVinfected HepG2-NTCP), stable HBV-expression HepAD38, and liver tissue of HBV-Tg mice, compared with their non-HBV counterparts (
Supplementary Fig. 1A,
1B). We subsequently employed Gene Set Enrichment Analysis (GSEA) to investigate the metabolic differences between HBV-positive and HBV-negative patients with HCC, revealing significant alterations in the acetyl-CoA metabolic pathway (
Fig. 1C). Notably, we further compared the enrichment of the acetyl-CoA metabolic pathway between HBV-related HCC and other HCC, including HCV-related and metabolic dysfunction-associated steatotic liver disease (MASLD)-related HCC. Our analysis revealed significant differences in the acetyl-CoA metabolic pathway enrichment among these distinct etiologies of HCC (
Supplementary Fig. 1C). Taken together, these data indicate that HBV infection is responsible for elevated acetyl-CoA.
Subsequently, we investigated the alterations in mRNA levels of various enzymes related to the acetyl-CoA metabolic pathway (
Fig. 1D) before and after HBV infection, using NTCP and Huh7 cell lines. Our results demonstrated that the mRNA and protein levels of ACSS2 were significantly upregulated following HBV infection (
Fig. 1E and
Supplementary Fig. 1D–
1F). Through the analysis of data from the GEO (Gene Expression Omnibus) database, we further explored the expression patterns of ACSS2 across different types of liver cancer. Our findings indicate that ACSS2 expression is significantly elevated and is among the most prominently regulated genes within the acetyl-CoA gene set in HBV-related HCC when compared to HCV-related HCC and other types such as MASLD-related HCC (
Fig. 1F). Moreover, we examined ACSS2 expression in mouse HCC tissues. Significantly higher mRNA and protein levels of ACSS2 were detected in the DEN/CCl4-induced HBV-Tg mouse model than in those in the C57 BL/6 model (
Fig. 1G–
1I). More importantly, ACSS2 expression was markedly elevated in tumor tissues of HBV-positive patients with HCC compared with that in paired non-tumor tissues, a trend that was less pronounced in the HBV-negative HCC cohort (
Fig. 1J,
1K). Similar results were obtained when comparing ACSS2 expression between hepatoma cells with HBV replication and hepatoma cells without HBV replication, as well as liver tissues from C57 BL/6 and HBV-Tg mice (
Supplementary Fig. 1E–
1H).
As ACSS2 is the key enzyme in acetyl-CoA generation in cancer cells, we investigated whether the increased level of acetyl-CoA in HBV-related HCC was dependent on differences in ACSS2 expression. Following the knockdown of ACSS2 in HepAD38 and Huh7-AdHBV1.3 cells using shRNA, acetyl-CoA levels were significantly decreased (
Supplementary Fig. 1I,
1J). Additionally, according to The Cancer Genome Atlas (TCGA)-LIHC database, patients with high ACSS2 expression had a poor prognosis (
Fig. 1L), suggesting that ACSS2 may play a role in the progression of HBV-related HCC.
To gain insight into the mechanism by which both ACSS2 mRNA and protein levels increase after HBV infec-tion, the
ACSS2 promoter was cloned into a luciferase reporter plasmid and it was found that HBV infection significantly enhanced
ACSS2 promoter activity (
Fig. 2A and
Supplementary Fig. 2A). Subsequently, potential transcription factors that could mediate
ACSS2 expression in two HBV-related HCC cohorts from TCGA and GSE121248 were screened. Among the three candidates (carbohydrate response element-binding protein [ChREBP], SREBP, and SP1) [
9,
24], only ChREBP was positively correlated with ACSS2 expression (r>0.3;
Fig. 2B and
Supplementary Fig. 2B). Interestingly, a substantial elevation in ChREBP mRNA and protein levels was observed upon HBV infection (
Fig. 2C–
2H and
Supplementary Fig. 2C–
2I).
Subsequently, it was investigated whether ChREBP was involved in the transcriptional regulation of ACSS2. Notably, ChREBP inhibition by small interfering RNAs (siRNAs) led to a marked decrease in ACSS2 expression in HepAD38 and Huh7 cells, with a more pronounced effect observed in the presence of HBV infection (
Fig. 2I and
Supplementary Fig. 2J). Furthermore, chromatin immunoprecipitation (ChIP) assay verified that HBV infection augmented the binding affinity of ChREBP to the
ACSS2 promoter region (
Fig. 2J and
Supplementary Fig. 2K). Taken together, these results confirmed the ChREBP-dependent upregulation of
ACSS2 expression in response to HBV infection.
As mentioned previously, ACSS2 expression was correlated with HBV-related HCC prognosis; however, its role in HBV-related HCC progression remains largely unexplored. Considering that the primary function of ACSS2 in cancer cells is to convert acetate to acetyl-CoA, we investigated its enzymatic activity in HBV-related HCC progression. To this end, the concentrations of acetyl-CoA in HBV-expressing or HBV-infected hepatoma cells (HepAD38, Huh7-AdHBV1.3, and PLC/PRF/5-AdHBV1.3) were measured following genetic and pharmacological targeting of ACSS2. Significant decreases in intracellular acetyl-CoA levels were observed following ACSS2 knockdown. More importantly, similar results were obtained after treatment with an ACSS2 inhibitor, which is reported as a specific inhibitor with an IC
50 of 5.5 μM [
25]. Interestingly, acetyl-CoA levels could be restored by the re-expression of ACSS2-WT but not by its catalytically inactive mutant (D552A) (
Fig. 3A,
3B and
Supplementary Fig. 3A,
3B). Overall, these results indicated that targeting ACSS2 enzymatic activity can effectively modulate intracellular acetyl-CoA concentrations in HBV-related HCC.
Intracellular acetyl-CoA levels are essential for cancer progression [
15,
19,
20,
26] and ACSS2 enzymatic activity can effectively modulate acetyl-CoA levels in HBV-related HCC. As anticipated, cell growth and colony formation assays demonstrated that ACSS2 knockdown or pharmacological inhibition substantially inhibited cell proliferation in hepatoma cells, which could be saved by the re-expression of ACSS2-WT but not ACSS2-D552A (
Fig. 3C–
3F and
Supplementary Fig. 3C–
3H). Consistent with prior findings were found in the EdU incorporation assay (
Fig. 3G–
3J and
Supplementary Fig. 3I,
3J). Collectively, these results suggest that ACSS2 promotes HBV-related HCC cell proliferation mainly through its enzymatic activity.
ACSS2, traditionally recognized for its role in lipid metabolism as a source of acetyl-CoA for lipid synthesis, has been increasingly implicated in transcriptional regulation of the liver via regulation of histone acetylation [
27]. Therefore, the potential role of ACSS2 was explored in histone acetylation in HBV-associated HCC. Given the dynamic nature of histone acetylation in response to cellular acetyl-CoA levels, histone acetylation levels were examined in hepatoma cells. HBV infection resulted in a pronounced increase in H3 acetylation (H3ac), but not in H4 acetylation (H4ac) (
Fig. 4A and
Supplementary Fig. 4A). Next, the acetylation levels of specific lysine residues in histone H3, including H3K9 (H3K9ac), H3K27 (H3K27ac), and H3K56 (H3K56ac), were examined in HepAD38 and HepG2-NTCP cells. HBV infection led to a dramatic increase in H3K27ac levels, with less pronounced effects on H3K9ac and H3K56ac levels (
Fig. 4B and
Supplementary Fig. 4B). Consistent with the
in vitro observations, immunoblot and immunohistochemistry (IHC) analyses confirmed a significant increase in H3K27ac levels in liver tissues from HBV-Tg mice and tumor tissues from DEN/CCl
4-induced HBV-Tg mice compared with those in WT mice (
Fig. 4C,
4D and
Supplementary Fig. 4C–
4E).
The relationship between ACSS2 and H3K27ac levels was explored in HBV-associated HCC cells. H3K27 acetylation was dependent on the acetyl-CoA concentration and acetyltransferase activity (
Fig. 4E); thus, we investigated whether the increase in H3K27ac after HBV infection was ACSS2-dependent. By silencing ACSS2 in HepAD38 and Huh7 cells, we found that the lack of ACSS2 expression significantly reduced H3K27ac levels following HBV infection; however, the trend was not obvious without HBV infection (
Fig. 4F). As ACSS2 enzymatic activity is essential for maintaining the acetyl-CoA pool in HBV-related HCC, ACSS2 enzymatic activity was next targeted to determine whether this resulted in H3K27ac changes
in vitro. Accordingly, pharmacological inhibition of ACSS2 enzymatic activity effectively reduced H3K27ac levels in hepatoma cells, and overexpression of ACSS2-WT instead of the enzymatically deficient mutant (ACSS2-D552A) restored H3K27ac levels in ACSS2-knockdown hepatoma cells (
Supplementary Fig. 4F,
4G). In summary, these data highlight the importance of ACSS2 enzymatic activity in maintaining high levels of H3K27ac in HBV-related HCC.
To determine the necessity of ACSS2-mediated alterations in H3K27 acetylation for the proliferative effects of ACSS2, H3K27ac levels were manipulated in hepatoma cells by treating with CTB, an activator of p300 acetyltransferase. Treatment with CTB effectively diminished the suppressive effect of ACSS2 depletion on hepatoma cell proliferation and H3K27ac levels (
Fig. 4G–
4K). Collectively, these data demonstrate that an increase in H3K27ac is necessary for the pro-proliferative effect of ACSS2 in HBV-related HCC.
To determine the potential targets regulated by ACSS2 in HBV-related HCC, a joint analysis of cleavage under target and tagmentation sequencing (CUT & Tag-seq) and RNA sequencing (RNA-seq) was performed in HepAD38 cells (n=3 per group) (
Fig. 5A). Consistent with our previous immunoblot assays, CUT & Tag-seq revealed a decrease in H3K27ac peak enrichment near the transcription start sites after ACSS2 knockdown (
Fig. 5B). The combined analysis revealed that most peaks were located in the promoter region; thus, we screened candidate genes with H3K27ac enrichment in these areas (
Fig. 5C,
5D). Among the 31 candidate genes, six were positively correlated with ACSS2 expression in the HBV-related HCC cohort of TCGA and have been reported to be tumor oncogenes (
Fig. 5D and
Supplementary Fig. 5A). Next, we screened potential downstream targets of ACSS2 using RT-qPCR. Following knockdown of ACSS2, RT-qPCR detected a significant downregulation of VDAC1 mRNA (
Fig. 5E and
Supplementary Fig. 5B). In addition, H3K27ac peaks were validated in the promoter region of
VDAC1, and a marked decrease was observed upon ACSS2 depletion (
Fig. 5F).
In particular, both mRNA and protein levels of VDAC1 were significantly reduced after ACSS2 depletion or ACSS2 inhibitor treatment, and were related to ACSS2 enzymatic activity (
Fig. 5G and
Supplementary Fig. 5C,
5D). Furthermore, restoration of H3K27ac levels by CTB rescued VDAC1 expression after ACSS2 silencing (
Fig. 5H and
Supplementary Fig. 5E). ChIP-qPCR assays revealed enrichment of H3K27ac within the promoter region of
VDAC1. This enrichment was attenuated after ablation or inhibition of ACSS2 and could be effectively rescued after ACSS2-WT re-expression (
Fig. 5I and
Supplementary Fig. 5F). These findings corroborate the hypothesis that ACSS2 activates the transcription of the
VDAC1 gene by increasing H3K27ac occupancy.
To further elucidate the oncogenic mechanism of upregulation of VDAC1 by ACSS2 in HBV-related HCC, we integrated the CUT & Tag-seq and RNA-seq data into GO analyses. Following the knockdown of ACSS2 in HepAD38 cells, GO analyses revealed that differentially expressed genes (DEGs) were mainly enriched in mitochondria-related pathways (
Fig. 6A). Importantly, VDAC1 was involved in all the main enriched clusters. Located on the mitochondrial membrane, VDAC1 is recognized as a mitochondrial gateway that mediates many important cellular processes, including mitophagy and cell proliferation, in different cancer types [
28-
30]. Our results indicate that ACSS2 may be a potent regulator of mitophagy in a VDAC1-dependent manner.
Given the crucial role of VDAC1 in the regulation of mitochondrial membrane potential (MMP) and mitophagy, transmission electron microscopy (TEM) and JC-1 staining were used to examine morphological and functional alterations in mitochondria. Interestingly, the depletion of ACSS2 or VDAC1 or the use of an ACSS2 inhibitor resulted in mitochondrial swelling and a reduction in autophagic vacuoles containing defective mitochondria, as observed in
Figure 6B and
Supplementary Figure 6A. In contrast, reexpression of ACSS2-WT or VDAC1 in shACSS2-cells, rather than the mutant ACSS2-D552A, could restore these phenotypes (
Fig. 6B). Silencing of ACSS2 or VDAC1 and treatment with an ACSS2 inhibitor resulted in a large decline in MMP, as determined by the uptake of JC-1 (
Fig. 6C and
Supplementary Fig. 6B,
6C). In addition, upon ACSS2 depletion, the restoration of expression with VDAC1 or ACSS2-WT reversed the decline in uptake; however, ACSS2-D552A failed to show rescue effects. These results indicate that ACSS2 promotes mitophagy via its catalytic activity in a VDAC1-dependent manner.
Furthermore, ACSS2 depletion, VDAC1 depletion, and ACSS2 inhibitors led to significant decreases in protein levels of mitophagy-related markers, such as Parkin and PINK1. In contrast, the subsequent restoration of VDAC1 or ACSS2 expression by ACSS2-WT in ACSS2-KD cells rescued the expression of PARKIN and PINK1, whereas the re-expression of ACSS2-D552A showed no obvious changes (
Fig. 6D and
Supplementary Fig. 6D,
6E). Consistent with the immunoblot data, targeting ACSS2 or VDAC1 decreased the number of colocalized puncta formed by autophagosomes and mitochondria in HepAD38 and Huh7-HB V1.1 cells, whereas ACSS2-WT and VDAC1 overexpression restored the number of puncta (
Fig. 6E and
Supplementary Fig. 7A,
7B). These results support the hypothesis that ACSS2 promotes VDAC1-mediated mitophagy via its catalytic activity.
Mitophagy is crucial for maintaining cellular homeostasis by eliminating dysfunctional and damaged mitochondria that can potentially disrupt cellular metabolism and induce oxidative stress [
31]. The clearance of dysfunctional mitochondria ensures that tumor cells maintain an efficient energy supply for proliferation [
32-
34]. Taking into account the role of ACSS2 in the enhancement of cell proliferation and mitophagy in HBV-related HCC, we evaluated whether this phenotype was due to upregulation of VDAC1-mediated mitophagy. As expected, cells treated with shACSS2 or shVDAC1 exhibited a large reduction in proliferation and colony formation compared with shControl-treated cells (
Supplementary Fig. 8A,
8B). Similar to overexpression of ACSS2-WT, VDAC1 overexpression restored the proliferation of ACSS2-knockdown cells (
Supplementary Fig. 8A,
8B).
To further investigate whether the tumorigenic function of ACSS2 was related to VDAC1 expression
in vivo, hepatoma cells were injected into the livers of nude mice to establish orthotopically implanted HCC models, and consistent re-sults were obtained in terms of tumor volume, tumor weight, and IHC findings (
Supplementary Fig. 8C–
8F). Moreover, ACSS2-knockdown cells were then transduced with a second shRNA targeting Parkin or shControl. Crucially, subsequent colony formation and growth curve assays revealed that while ACSS2 or VDAC1 overexpression rescued proliferation in shControl cells, this rescue effect was abrogated in Parkin knockdown cells (
Supplementary Fig. 9A–
9E). These findings provide direct causal evidence that mitophagy acts as a critical downstream mediator of ACSS2. Taken together, these findings demonstrate that ACSS2 promotes HBV-related HCC proliferation, largely by upregulating VDAC1-mediated mitophagy.
We measured reactive oxygen species (ROS) levels in cells following ACSS2 knockdown and treatment with the ACSS2 inhibitor. Our findings indicate that both interventions significantly increase intracellular ROS levels, suggesting that ACSS2 inhibition enhances oxidative stress in cancer cells (
Supplementary Fig. 10A–
10D). Furthermore, flow cytometry analysis showed that targeting ACSS2 promotes apoptosis (
Supplementary Fig. 10E,
10F). These findings align well with established roles of mitophagy, which functions to reduce intracellular ROS and inhibit apoptosis [
31-
34]. Thus, ACSS2 inhibition simultaneously disrupts mitochondrial quality control, increases oxidative stress, and tips the balance toward programmed cell death.
Next, DEN/CCl
4-induced HCC models were used in HBV-Tg mice to verify the
in vivo results (
Fig. 7A). We investigated two strategies for targeting ACSS2 in HBV-related HCC: silencing using a lentivirus and inhibition using a smallmolecule inhibitor. The pSECC-sg
Control or pSECC-sg
Acss2 group received an intravenous injection of pSECC lentiviruses, whereas the vehicle or ACSS2i group received an intraperitoneal injection of vehicle or ACSS2i, respectively. Administration of pSECC-sg
Acss2 and ACSS2 inhibitors effectively suppressed tumor progression, as indicated by a smaller tumor mass and fewer tumors in the liver (
Fig. 7B,
7C). Furthermore, acetyl-CoA levels in the tumor decreased considerably after targeting ACSS2 (
Fig. 7C). Importantly, subsequent immunoblotting and IHC confirmed that the levels of H3K27ac, VDAC1, mitophagy-related proteins, such as PARKIN and PINK1, and the proliferation marker PCNA were decreased after depletion or inhibition of ACSS2
in vivo (
Fig. 7D,
7E).
The prognostic value of ACSS2 was evaluated using IHC in a tissue microarray of 86 HBV-related HCC tissues (
Supplementary Table 3). The patients were grouped into a high or low expression cohort of ACSS2 according to their IHC scores (
Fig. 7F). Patients with high expression of ACSS2 had larger tumors, which is consistent with the proliferative effect of ACSS2 on HBV-related HCC (
Fig. 7G). Furthermore, patients with high expression of ACSS2 had significantly worse overall survival, progression-free survival and more advanced stage than those with low expression (
Fig. 7H,
7I and
Supplementary Table 4). Collectively, ACSS2 emerged as a potential prognostic biomarker and novel therapeutic target for HBV-related HCC.
DISCUSSION
Metabolic reprogramming in cancer refers to the distinctive metabolic changes that occur in malignant tumors compared with their benign counterparts [
7]. Recently, numerous studies have revealed that metabolic heterogeneity is present not only between cancerous and normal tissues, but also within tumors of the same oncological category and is highly correlated with drug resistance and patient prognosis [
35,
36]. HCC is one of the most heterogeneous malignancies with different etiologies, including HBV infection, HCV infection, alcohol abuse, and metabolic dysfunction-associated steatotic liver disease [
4]. Consequently, these etiological heterogeneities are responsible for diverse metabolic landscapes, different drug sensitivities, and different prognoses, highlighting the urgent need for precision therapy for HCC [
4,
37]. Our study focuses on HBV-related HCC, the most common and aggressive form of HCC, and observes the specific upregulation of acetyl-CoA exclusively in this subtype. Furthermore, HBV infection enhances acetyl-CoA primarily via ACSS2 and subsequently increases H3K27ac modification so as to promote VDAC1-mediated mitophagy, leading to tumor proliferation. Notably, targeting ACSS2 significantly inhibits the proliferation of HBV-related HCC.
In HCC, the expression levels of ACSS2 demonstrate significant variation [
21]. Sun et al. [
38] reported that ACSS2 expression is downregulated and plays an antimetastatic role in HCC, in contrast to the findings of Comerford et al. [
25], who showed that the loss of ACSS2 suppresses tumor development. This diversity may be attributed to the use of different models, as previous studies exclusively used cell lines under hypoxic conditions, whereas further investigations were conducted using mouse models of spontaneous hepatocarcinogenesis. Moreover, these studies did not investigate ACSS2 expression across HCC etiologies. In this study, we observed that ACSS2 expression was elevated exclusively in HBV-related HCC tissues compared to that in adjacent normal tissues. Another key finding of this study is the identification of ChREBP as a transcriptional activator of ACSS2 in HBV-related HCC. ChREBP, a central regulator of liver metabolic pathways, such as glycolysis and lipid synthesis, is upregulated in HCC and promotes tumor growth via the PI3K/AKT pathway [
39,
40]. Our findings revealed that HBV infection enhanced ChREBP expression in HCC, which in turn upregulated ACSS2, thereby clarifying the mechanism underlying ACSS2 upregulation in HBV-related HCC.
Based on clinical features, HBV-related HCC is the major proliferation class, characterized by rapid growth and enrichment of proliferation pathways, including insulin-like growth factor I and the mechanistic target of rapamycin mTOR [
41,
42]. Here, we revealed the proliferation-driven nature of HBV-related HCC from a novel metabolic perspective. We found that HBV-related HCC showed a dependence on ACSS2 to elevate the acetyl-CoA pool and that targeting ACSS2 activity dramatically inhibited the proliferation of HBV-related HCC
in vitro and
in vivo.
Small molecules targeting ACSS2 demonstrated anticancer efficacy in preclinical models across a spectrum of malignancies, and one such inhibitor, MTB-9655, is currently in the Phase 1 clinical trial for patients with advanced lung and breast cancer [
26,
43-
45]. However, this clinical trial did not include patients with HCC, which may be attributed to the inconsistent expression and role of ACSS2 observed in previous studies [
25,
38]. Our study demonstrated that HBV infection accounts for the observed discrepancies and that inhibition of ACSS2 exerts significant anticancer effects, specifically in HBV-related HCC. This finding provides preliminary preclinical evidence supporting the future clinical application of ACSS2 inhibitors in HBV-related HCC.
Unlike citrate-derived acetyl-CoA, which contributes to lipid synthesis, acetate-derived acetyl-CoA plays an important role in epigenetic and post-translational regulation, particularly in cancer [
19,
26,
46]. Compared with other members of the ACSS family, ACSS2 is the primary enzyme responsible for converting acetate into acetyl-CoA, and it predominantly functions in regulating epigenetics and gene transcription in liver cancer [
15,
19,
21,
26,
46]. Consequently, we investigated the role of ACSS2 in the epigenetic regulation of HBV-related HCC. In this study, we found that ACSS2-mediated accumulation of acetyl-CoA resulted in the most pronounced increase in H3K27 acetylation in HBV-related HCC. This specific increase in H3K27ac may be related to the enzymatic kinetics (K
m) and the selected acetylation substrate of acetyltransferases. Interestingly, KAT3B (p300) is a specific acetyltransferase for H3K27 and has the highest Km value among all acetyltransferases [
47,
48]. Upon HBV infection, the specific elevation of H3K27ac is likely correlated with the high sensitivity of p300 to the elevation of acetyl-CoA mediated by ACSS2.
HBV and other viruses rely on the host cell machinery for energy and replication materials, thus disrupting cell homeostasis and causing mitochondrial damage and oxidative stress, which can lead to cell death [
14,
49-
51]. HBV enhances mitophagy by promoting mitochondrial translocation of Parkin and Drp1, thus supporting the viability of infected cells and facilitating viral replication [
52]. In this study, we identified ACSS2 as a metabolic switch of mitophagy upon HBV infection, demonstrating that it promotes mitophagy by upregulating the key substrate, VDAC1. This study not only elucidated a novel role for ACSS2 in the regulation of mitophagy but also offered new insights into virus-induced mitophagy.
There are also several limitations in this study. Firstly, in this study, extensive experiments have confirmed that ACSS2 can promote the transcription of VDAC1 by increasing the levels of H3K27ac on its promoter loci. However, the overexpression of VDAC1 following ACSS2 knockdown did not fully reverse the inhibition of proliferation. Thus, other downstream targets may also be involved. The precise molecular determinants that direct ACSS2-driven H3K27ac enrichment specifically to the VDAC1 promoter, among thousands of other genomic loci, remain unclear with current technologies. This is a recognized difficulty within the epigenetic research community [
19,
53]. Furthermore, to substantiate the role of ACSS2 in the progression of HBV-related HCC, the employment of ACSS2 liver-specific knockout mice may be essential in future studies. Last, HCC is a highly heterogeneous disease, with other various risk factors such as alcohol consumption and dietary habits contributing to its development and progression. While our study, using public databases and animal models, has confirmed that HBV infection can upregulate ACSS2 expression in HCC, we cannot exclude the possibility that alcohol and high-fat diets may also impact ACSS2 expression.
In summary, our data indicate that ACSS2 is a pivotal enzyme responsible for the specific elevation of acetyl-CoA and H3K27ac levels in HBV-related HCC, which enhances VDAC1-mediated mitophagy and subsequent tumor proliferation. By linking metabolic reprogramming and epigenetic dysfunction in HCC, ACSS2 was identified as a key enzyme and its role was fully revealed in the progression of HBV-related HCC. More importantly, ACSS2 may be a novel prognostic marker and therapeutic target for the treatment and diagnosis of HBV-related HCC.
FOOTNOTES
-
Authors’ contributions
N.T., K.W., A.H., and Y.H. initiated the study concept and experimental design. S.L., Y.Y, X.L. and J. H., led the experimental work, data analysis, and statistical evaluation. S.L., and X.D. contributed to data interpretation, and manuscript drafting. H.L. and X.T. provided assistance with experiments. J. T. and R. Z. conducted immunofluorescence staining. S.L. and J.H. analyzed the RNA-seq and CUT & TAG-seq data. Y.Y. and X.L. acquired electron microscopy images. X.D. and J.H. processed clinical samples and established adenoviruses. S.L. performed bioinformatics analysis of TCGA and GEO database. All co-authors participated in manuscript review and editing.
-
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2023YFC2306800), the China National Natural Science Foundation (Grant no. 82272975, 82273238); Senior Medical Talents Program of Chongqing for Young and Middle-aged, the Kuanren talents program and Joint Project of Pinnacle Disciplinary Group, the Second Affiliated Hospital of Chongqing Medical University; the Chongqing Professional Talents Plan for Innovation and Entrepreneurship Demonstration Team (CQYC202203091047, NT), and the Future Medical Youth Innovation Team of Chongqing Medical University (W0160); Bishan Hospital youth talent project; Chongqing medical youth top-notch talent project (No. YXQN202476); Bishan Open Research Fund Program of the Key Laboratory of Molecular Biology for Infectious Diseases, CQMU, (No.202402), Excellent young talent project, Bishan hospital of Chongqing Medical University, Research and Innovation Team Program, Bishan Hospital of Chongqing Medical University (No. BYKY-CX2024008), young project of science and technology research program of Chongqing Education Commission (KJQN202500458) and Natural Science Foundation of Chongqing (Grant No.CSTB2025NSCQ-GPX0358). We are grateful that Prof. Tongchuan He (University of Chicago, USA) kindly provided the AdEasy system. We thank Prof. Bing Sun (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China) kindly provided the pLL3.7 vector. We also thank Prof. Ding Xue (Tsinghua University, Beijing, China) for supplying the CRISPR/Cas9 system.
-
Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
Acetyl-CoA and ACSS2 levels were upregulated upon HBV infection. (A) Acetyl-CoA level measured by ELISA kit in hepatoma cells. (B) Acetyl-CoA level in liver tissue from C57 BL/6 (n=6) and HBV-Tg mice (n=6). (C) GSEA enrichment plot using the ‘‘acetyl-CoA metabolic process” in TCGA and GEO datasets. (D) The mRNA levels of enzymes related to acetyl-CoA synthesis, identified by RT-qPCR. (E) Relative mRNA and protein levels of ACSS2 in HBV-uninfected and HBV infected hepatoma cells, identified by immunoblots. Relative mRNA (F) and protein (G) level of ACSS2 in C57 BL/6 (n=6) and HBV-Tg mice (n= 6). (H) Representative images of immunohistochemical (IHC) staining of ACSS2 in the liver tissue from C57 BL/6 and HBV-Tg mice, scale bar=50 μm. (I) Immunoblots verified the knockdown of ACSS2 by shACSS2 in HepAD38 and Huh7-AdHBV1.3. (J) Intracellular acetyl-CoA levels in hepatoma cells measured by ELISA kit. Data are shown as the mean±SD. Statistical analysis was performed using Student’s test (A, B, D, F, J). *P<0.05, **P<0.01, ***P<0.001, and ns for not significant.
Supplementary Figure 2.
HBV infection upregulates ACSS2 via ChREBP elevation. (A) Huh7 cells were co-transfected with the ACSS2 promoter luciferase reporter and treated as described, and the luciferase activity was monitored in the panel (n=3, per group). (B) Pearson correlation coefficient analysis of ChREBP mRNA with ACSS2 in patients with HBV-related HCC from TCGA-LICH Cohort (n=118). (C–H) Relative mRNA level of ChREBP and protein expression level of ACSS2 and ChREBP, in vitro and in vivo, identified by RT-qPCR and immunoblots, in HBV-uninfected cells or C57 BL/7 mice were utilized as controls. (I) Representative IHC image of ChREBP expression in DEN/CCl4-induced HCC mice models. Scale bar=50 μm. (J) Relative mRNA level and protein expression of ACSS22 after ChRBEP knockdown by siRNA in both HBV-infected and -uninfected cells. (K) ChIP-qPCR analysis of binding affinity of ChREBP on ACSS2 promoter region in Huh7 cells (n=3). Data are presented as mean±SD. *P<0.05, **P<0.01, ***P<0.001, and ns, not significant.
Supplementary Figure 3.
ACSS2 promotes HBV-related HCC proliferation via its enzymatic activity. (A, B) Relative acetyl-CoA level in HBV-infected hepatoma cells after shACSS2 transfection or ACSS2 inhibitor (ACSS2i) treatment for 24 hours in PLC/RPF/5-AdHBV1.3 cells. ACSS2 knockdown by shACSS2 transfection, and re-expression of ACSS2 by adenovirus Flag-ACSS2 (WT or D552A). Cell proliferations were assessed through cell growth curve in PLC/RPF/5-AdHBV1.3 cells (C, D), Colony formation assay in PLC/RPF/5-AdHBV1.3, HepAD38 and Huh7-AdHBV1.3 cells (E–H) and EdU incorporation assay (I, J) in PLC/RPF/5-AdHBV1.3. Scale bar=50 μm. Data are shown as the mean±SD. Statistical analysis was performed using 1-way ANOVA with Tukey’s test (A, C, E, I) or t-test (B, D, F, J). *P<0.05, **P<0.01, ***P<0.001.
Supplementary Figure 4.
ACSS2 upregulates H3K27ac via enzymatic activity upon HBV infection. (A, B) Immunoblots of histone acetylation markers in HepAD38 with or without tetracycline (Tet) and quantitative analysis were shown (n=3, per group). (C, D) Immunoblots in liver tumors from C57 BL/6 and HBV-Tg mice (n=6, per group) and Alb-Cre mice with or without HBV rcccDNA injection (n=5, per group). (E) IHC of H3K27ac in liver tumor from C57 and HBV-Tg mouse models. Scale bar=50 μm. HepAD38 cells and Huh7-AdHBV1.3 cells were transfected with shACSS2 to deplete ACSS2 and restored by adenovirus ACSS2 (WT or D552A). (F) Immunoblots of H3K27ac after indicated administration in HepAD38 and Huh7-AdHBV1.3. (G) H3K27ac level detected by immunoblots after the treatment of ACSS2i for 24 hours. Statistical analysis was performed using t-test (A, B). *P<0.05, ***P<0.001, and ns for no significance.
Supplementary Figure 5.
ACSS2 promotes VDAC1 expression via H3K27ac occupancy. (A) Pearson correlation coefficient analysis of ACSS2 mRNA with VDAC1 mRNA in patients with HBV-related HCC from TCGA-LICH cohort (n=118). (B) Initial gene screening in Huh7-AdHBV1.3 cells after ACSS2 knockdown, assessed by RT-qPCR (n=3, performed in triplicate). (C) In Huh7-AdHBV1.3 cells, upon ACSS2 genetic manipulation indicated, left panel showed the relative mRNA level of VDAC1, verified by RT-qPCR (n=3, technique duplicates) and right panel showed the protein expression of VDAC1 and indicated genes, assessed by immunoblots. (D) The mRNA and protein level of VDAC1 after the treatment of ACSS2i for 24 hours in HepAD38 and Huh7-AdHBV1.3, shown by immunoblots. (E) The effects of CTB treatment on VDAC1 mRNA level (n=3) and protein expression upon ACSS2 knockdown. (F) Enrichment of H3K27ac on VDAC1 promoter upon ACSS2 genetic interference or ACSS2 inhibitor treatment for 24 hours (n=3, technical duplicates), assessed by ChIP-qPCR in Huh7-AdHBV1.3. Data are shown as the mean±SD. Statistical analysis was performed using t-test (B, D) or 1-way ANOVA with Tukey’s test (C, E, F). *P<0.05, ***P<0.001, and ns for no significance.
Supplementary Figure 6.
ACSS2 promotes VDAC1-mediated mitophagy. ACSS2 and VDAC1 depletion were performed by transfection of shRNA, sgRNA or siRNA as indicated in HepAD38, and overexpression of ACSS2-Flag (WT or D552A) and VDAC1-Flag by transfection of plasmid. (A) Representative TEM images depicted ultrastructure in HepAD38 after the treatment of ACSS2i for 24 hours. Red arrows indicated autophagic vacuoles and yellow arrows showed the swollen cristae. Scale bar=500 nm. (B, C) Representative images (left) and quantitative analysis (right) of mitochondrial membrane potential in HepAD38 cells and Huh7-HBV1.1, determined with JC-1 probe, upon intervention described above. Scale bar=25 μm. (D, E) Immunoblots of PARKIN, PINK1, Bcl-2, and BAX upon treatment mentioned above in HBV-infected hepatoma cells including HepAD38 and Huh7-HBV1.1. Statistical analysis was performed using t-test (B) or one-way ANOVA with Tukey’s test (C). ***P<0.001.
Supplementary Figure 7.
Representative images of localization of mitochondria and autophagosome. (A, B) The localization of mitochondria and autophagosome was determined via immunofluorescence staining, indicating yellow, red for mitotracker, and green for GFP-LC3. Scale bar=25 μm.
Supplementary Figure 8.
ACSS2 facilitates HBV-related HCC proliferation via VDAC1-meidated mitophagy in vitro and in vivo. ACSS2 and VDAC1 depletion was performed by shRNA in HepAD38, Huh7-AdHBV1.3 and MHCC-97H-AdHBV1.3. Overexpression of ACSS2-Flag (WT or D552A) and VDAC1-Flag by transfection of plasmid. Cell proliferation of HepAD38, Huh7-AdHBV1.3 and MHCC-97H-AdH-BV1.3, shown by growth curve (A) and colony formation assay (B). (C) MHCC-97H cells were treated as indicated and injected into the liver of nude mice (n=6 per group). (D) Representative gross appearance of implantation tumors. Tumor volume (E) and liver body weight assessment (F) and representative IHC images in six groups of implantation tumors (Magnification: 40×). Data are represented as mean±SD. One-way ANOVA followed by Tukey’s test, *P<0.05, **P<0.01, ***P<0.001.
Supplementary Figure 9.
ACSS2 facilitates HBV-related HCC proliferation via mitophagy. (A, B) Representative images of colony formation assay in HepAD38 and Huh7-AdHBV1.3 cells with ACSS2 knockdown, followed by indicated treatments. (C, D) The quantification of colony formation in HepAD38 and Huh7-AdHBV1.3, respectively. (E) Cell growth curves of HepAD38 and Huh7-AdHBV1.3 cells treated by indicated method. Data are shown as the mean±SD. Statistical analysis was performed using one-way ANOVA with Tukey’s test; *P<0.05, **P<0.01, ***P<0.001 and ns for no significance.
Supplementary Figure 10.
ACSS2 inhibition elevates reactive oxygen species and induces apoptosis in HBV-related HCC. (A–D) Representative images (left) and quantification (right) of ROS levels in HepAD38 and Huh7-HBV1.1 cells following indicated treatments (scale bar=50 μm). (E–H) Apoptosis analysis by fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry in cells following different treatment (n=3, per group). Data are shown as the mean±SD. Statistical analysis was performed using t-test, ***P<0.001.
Figure 1.HBV-related HCC displays increasing levels of acetyl-CoA and ACSS2. (A) Acetyl-CoA levels measured using ELISA in liver tissue from HBV-negative patients with HCC (n=18) and HBV-positive patients (n=18) with both paired sample of tumor tissue (T) and matched non-tumor tissue (N). (B) Acetyl-CoA levels in liver tumor tissue from the DEN/CCL4 –induced HBV-Tg mice model (n=6) and C57 BL/6 wild-type mice model was used as control (n=6). (C) GSEA enrichment plot using the ‘‘acetyl-CoA metabolic process” in TCGA dataset. (D) Schematic process of acetyl-CoA synthesis. (E) The mRNA levels of enzymes related to acetyl-CoA synthesis, identified by RT-qPCR. (F) The mRNA levels of ACSS2 in different types of HCC from Gene Expression Omnibus datasets. (G–I) Protein and mRNA levels of ACSS2 in DEN/CCL4 -induced C57 mice model (n=6) and HBV-Tg mice model (n=6) identified using immunoblots, RT-qPCR and IHC (magnification: 40×. scale bar = 50 μm). (J) Relative mRNA level of ACSS2 in HCC tissue from HBV-positive patients with HCC (n=18), using HBV-negative patients with HCC as control (n=18), measured using RT-qPCR (performed in triplicate). (K) Quantitative analysis of ACSS2 protein level in patients’ HCC tissue. (L) Kaplan–Meier survival analysis of ACSS2 depicting the overall survival (OS) of patients with HBV-related HCC from the TCGA-LIHC cohort. Significance was determined using the χ2 test, HR for hazard ratio. Data are shown as the mean±standard deviation. Statistical analysis was performed using Student’s t-test (A, B, E–G, J) and paired t-test (K). ACSS2, acetyl-CoA synthetase 2; CoA, coenzyme A; DEN/CCl4, diethylnitrosamine/carbon tetrachloride; GSEA, Gene Set Enrichment Analysis; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription quantitative polymerase chain reaction. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.
Figure 2.HBV infection upregulates ACSS2 via ChREBP elevation. (A) HepG2-NTCP and HepAD38 cells were co-transfected with the ACSS2 promoter luciferase reporter and treated as described, and the luciferase activity was monitored (n=3, per group). (B) Pearson’s correlation coefficient analysis of ChREBP mRNA with ACSS2 in patients with HBV-related HCC from both TCGA-LICH cohort (n=118) and GSE121248 (N=107). (C–G) Relative mRNA level of ChREBP and protein expression level of ACSS2 and ChREBP, in vitro and in vivo, identified using RT-qPCR and immunoblots; cells without HBV replication or C57 BL/6 mice were utilized as controls. (H) IHC image of ChREBP expression in mice liver. Scale bar=50 μm. (I) Relative mRNA level and protein expression of ACSS22 after ChRBEP knockdown by siRNA in HepAD38. (J) ChIP-qPCR analysis of binding affinity of ChREBP on ACSS2 promoter region in HepAD38 cells (n=3). Data are presented as mean±standard deviation. ACSS2, acetyl-CoA synthetase 2; ChIP, chromatin immunoprecipitation; ChREBP, carbohydrate response element-binding protein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; RT-qPCR, reverse transcription quantitative polymerase chain reaction. *P<0.05, ***P<0.001; ns, not significant.
Figure 3.ACSS2 promotes HBV-related HCC proliferation via its enzymatic activity. (A, B) Relative acetyl-CoA level in hepatoma cells after shACSS2 transfection or ACSS2 inhibitor (ACSS2i) treatment for 24 hours. ACSS2 knockdown by shACSS2 transfection and re-expression of ACSS2 by adenovirus Flag-ACSS2 (WT or D552A). Cell proliferation was assessed through cell growth curves (C, D), colony formation assay (E, F) and EdU incorporation assay (G–J) in HepAD38 and Huh7-AdHBV1.3. Scale bar=50 μm. Data are shown as the mean±standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey’s test (A, C, E, F, H) or t-test (B, D, J). ACSS2, acetyl-CoA synthetase 2; CoA, coenzyme A; HBV, hepatitis B virus; HCC, hepatocellular carcinoma. *P<0.05, **P<0.01, ***P<0.001.
Figure 4.H3K27ac level is elevated by ACSS2 and correlates with its pro-proliferative effect upon HBV infection. (A, B) Immunoblots of histone acetylation marker in hepatoma cells and quantitative analysis are shown (n=3). (C) Immunoblots in liver tumors from DEN/CCl4-induced C57 and HBV-Tg mice (n=6, per group). (D) IHC of H3K27ac in liver tumor from C57 BL/6 and HBV-Tg mice models. Scale bar=50 μm. (E) The schematic diagram of the relationships among ACSS2, p300, and H3K27ac levels in the nucleus; ac, histone acetylation. (F) The effects of ACSS2 knockdown on H3K27ac level in hepatoma cells with or without HBV infection, shown by western blots. (G) The effects of CTB treatment for 24 hours on H3K27ac in hepatoma cells after ACSS2 depletion, detected by immunoblots. (H–K) The effects of CTB on cell proliferation in vitro after ACSS2 knockdown, shown by growth curve and colony formation in HepAD38 (H, I) and Huh7-AdHBV1.3 (J, K). Data are shown as the mean±standard deviation. Statistical analysis was performed using t-test (A, B) or one-way ANOVA with Tukey’s test (H–K). ACSS2, acetyl-CoA synthetase 2; CTB, Cholera Toxin B; DEN/CCl4, diethylnitrosamine/carbon tetrachloride; HBV, hepatitis B virus; HBV-Tg, HBV-transgenic; IHC, immunohistochemistry. **P<0.01, ***P<0.001. ns, no significance.
Figure 5.ACSS2 promotes VDAC1 expression via H3K27ac occupancy. (A) Workflow of combination analysis of CUT & TAG-seq and RNA-seq in shControlor shACSS2-transfected HepAD38 cells (n=3, per group). (B) The visualization of the binding density of H3K27ac: the heatmap presents the CUT & Tag-seq tag counts on the different H3K27ac binding peaks in HepAD38 cells between shControl (n=3) and shACSS2 (n=3), ordered by signal strength and black arrow indicates VDAC1 loci. (C) Pie chart showing the distribution of H3K27ac annotated genomic regions in HepAD38. (D) Bioinformatics analysis filtered the potential targeted genes. (E) Relative mRNA levels of initial screening genes in HepAD38 cells assessed using RT-qPCR (n=3, performed in triplicate). (F) A representative image from three duplicates of genome browser tracks of H3K27ac occupancy at the VDAC1 gene locus. (G) Upon ACSS2 genetic manipulation, the left panel shows the relative mRNA level of VDAC1, verified using RT-qPCR (n=3, technique duplicates) and the right panel shows the protein expression of VDAC1 and indicated genes, assessed by immunoblots. (H) The effects of CTB treatment on VDAC1 mRNA level (n=3) and protein expression upon ACSS2 knockdown. (I) ChIP-qPCR analysis of the enrichment of H3K27ac on VDAC1 promoter upon ACSS2 interference or ACSS2 inhibitor treatment for 24 hours (n=3, technical duplicates). Data are shown as the mean±standard deviation. Statistical analysis was performed using t-test (E) or one-way ANOVA with Tukey’s test (G–I). ACSS2, acetyl-CoA synthetase 2; ChIP, chromatin immunoprecipitation; CTB, Cholera Toxin B; CUT, cleavage under target; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Tag-seq, tagmentation sequencing; VDAC1, voltage-dependent anion channels 1. *P<0.05, **P<0.01, ***P<0.001. ns, no significance.
Figure 6.ACSS2 promotes VDAC1-mediated mitophagy. (A) GO analysis of overlapping DEGs identified by CUT & TAG-seq and RNA-seq. ACSS2 and VDAC1 depletion were performed by short hairpin RNA (shRNA), single guide RNA (sgRNA), or siRNA as indicated in HepAD38, and overexpression of ACSS2-Flag (WT or D552A) and VDAC1-Flag by transfection of plasmid. (B) Representative TEM images depicting ultrastructure in HepAD38 upon indicated treatments. Red arrows indicate autophagic vacuoles and yellow arrows show the swollen cristae. Scale bar=500 nm. (C) Representative images (left) and quantitative analysis (right) of mitochondrial membrane potential in HepAD38 cells determined by JC-1 staining upon intervention described above. Scale bar=25 μm. (D) Immunoblots of PARKIN, PINK1, Bcl-2 and BAX upon the indicated treatments. (E) Quantitative analyses of the localization of mitochondria and autophagosomes in HepAD38 and Huh7-HBV1.1 was determined via immunofluorescence staining. Data are shown as the mean±standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey’s test (C&E). ACSS2, acetyl-CoA synthetase 2; CUT, cleavage under target; DEG, differentially expressed gene; siRNA, small interfering RNA; Tag-seq, tagmentation sequencing; TEM, transmission electron microscopy; VDAC1, voltage-dependent anion channels 1. ***P<0.001.
Figure 7.Targeting ACSS2 is a promising diagnostic and therapeutic strategy in HBV-related HCC. (A) Schematic showing experimental procedures of DEN-induced HBV-transgenic (Tg) mice. (B) Gross appearances of liver from HBV-Tg with tumors. (C) Liver nodule numbers, liver body weight, and relative acetyl-CoA levels in HBV-Tg mice (n=6, per group). (D) Immunoblots of indicated protein from HBV-Tg liver tumors from four groups treated as described. (E) Representative images of IHC staining of the indicated proteins in consecutive serial sections of liver from HBV-Tg mice. Scale bar = 50 μm. (F) Representative IHC staining of ACSS2 in an HBV-positive HCC tissue microarray. IHC was calculated according to the percentage of stained cells and intensity. Scale bar=50 μm (G) According to the IHC score, patients were grouped into a low-expression cohort (n=43) and a high-expression cohort (n=43). The mean tumor maximum diameters (TMD) were compared using t-test. *P<0.05 (H, I) Kaplan–Meier analysis of overall survival rate and progression free survival rate in the tissue microarray cohort (n=86), stratified by ACSS2. Data are shown as the mean±standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey’s test (C) or t-test (G). ACSS2, acetyl-CoA synthetase 2; DEN, diethylnitrosamine; HBV, hepatitis B virus; HBV-Tg, HBV-transgenic; HCC, hepatocellular carcinoma; IHC, immunohistochemistry. *P<0.05, ***P<0.001.
Abbreviations
chromatin immunoprecipitation
carbohydrate response element-binding protein
differentially expressed gene
Gene Set Enrichment Analysis
metabolic dysfunction-associated steatotic liver disease
mitochondrial membrane potential
transmission electron microscopy
voltage-dependent anion channels 1
REFERENCES
- 1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-263.
- 2. Lin J, Zhang H, Yu H, Bi X, Zhang W, Yin J, et al. Epidemiological characteristics of primary liver cancer in Mainland China from 2003 to 2020: a representative multicenter study. Front Oncol 2022;12:906778.
- 3. Gao Q, Zhu H, Dong L, Shi W, Chen R, Song Z, et al. Integrated proteogenomic characterization of HBV-related hepatocellular carcinoma. Cell 2019;179:561-577.e522.
- 4. Sia D, Villanueva A, Friedman SL, Llovet JM. Liver cancer cell of origin, molecular class, and effects on patient prognosis. Gastroenterology 2017;152:745-761.
- 5. Hoshida Y, Nijman SM, Kobayashi M, Chan JA, Brunet JP, Chiang DY, et al. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res 2009;69:7385-7392.
- 6. Xue X, Liao W, Xing Y. Comparison of clinical features and outcomes between HBV-related and non-B non-C hepatocellular carcinoma. Infect Agent Cancer 2020;15:11.
- 7. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Discov 2022;12:31-46.
- 8. Gao X, Lin SH, Ren F, Li JT, Chen JJ, Yao CB, et al. Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia. Nat Commun 2016;7:11960.
- 9. Zhao S, Jang C, Liu J, Uehara K, Gilbert M, Izzo L, et al. Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate. Nature 2020;579:586-591.
- 10. Murthy D, Attri KS, Shukla SK, Thakur R, Chaika NV, He C, et al. Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis. Nat Cell Biol 2024;26:613-627.
- 11. Li X, Yang Y, Zhang B, Lin X, Fu X, An Y, et al. Lactate metabolism in human health and disease. Signal Transduct Target Ther 2022;7:305.
- 12. Zhou L, He R, Fang P, Li M, Yu H, Wang Q, et al. Hepatitis B virus rigs the cellular metabolome to avoid innate immune recognition. Nat Commun 2021;12:98.
- 13. Hu J, Gao Q, Yang Y, Xia J, Zhang W, Chen Y, et al. Hexosamine biosynthetic pathway promotes the antiviral activity of SAMHD1 by enhancing O-GlcNAc transferase-mediated protein O-GlcNAcylation. Theranostics 2021;11:805-823.
- 14. Yang F, Yan S, He Y, Wang F, Song S, Guo Y, et al. Expression of hepatitis B virus proteins in transgenic mice alters lipid metabolism and induces oxidative stress in the liver. J Hepatol 2008;48:12-19.
- 15. Guertin DA, Wellen KE. Acetyl-CoA metabolism in cancer. Nat Rev Cancer 2023;23:156-172.
- 16. Carrer A, Trefely S, Zhao S, Campbell SL, Norgard RJ, Schultz KC, et al. Acetyl-CoA metabolism supports multistep pancreatic tumorigenesis. Cancer Discov 2019;9:416-435.
- 17. Xia JK, Qin XQ, Zhang L, Liu SJ, Shi XL, Ren HZ. Roles and regulation of histone acetylation in hepatocellular carcinoma. Front Genet 2022;13:982222.
- 18. Zhang C, Wang XY, Zhang P, He TC, Han JH, Zhang R, et al. Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts. Cell Death Dis 2022;13:57.
- 19. Lu M, Zhu WW, Wang X, Tang JJ, Zhang KL, Yu GY, et al. ACOT12-dependent alteration of acetyl-CoA drives hepatocellular carcinoma metastasis by epigenetic induction of epithelial-mesenchymal transition. Cell Metab 2019;29:886-900.e885.
- 20. Park S, Mossmann D, Chen Q, Wang X, Dazert E, Colombi M, et al. Transcription factors TEAD2 and E2A globally repress acetyl-CoA synthesis to promote tumorigenesis. Mol Cell 2022;82:4246-4261.e4211.
- 21. Ling R, Chen G, Tang X, Liu N, Zhou Y, Chen D. Acetyl-CoA synthetase 2(ACSS2): a review with a focus on metabolism and tumor development. Discov Oncol 2022;13:58.
- 22. Fagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics 2014;13:397-406.
- 23. Yoshimura Y, Araki A, Maruta H, Takahashi Y, Yamashita H. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix. J Biochem 2017;161:279-289.
- 24. Xu H, Luo J, Ma G, Zhang X, Yao D, Li M, et al. Acyl-CoA synthetase short-chain family member 2 (ACSS2) is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells. J Cell Physiol 2018;233:1005-1016.
- 25. Comerford SA, Huang Z, Du X, Wang Y, Cai L, Witkiewicz AK, et al. Acetate dependence of tumors. Cell 2014;159:1591-1602.
- 26. Bacigalupa ZA, Arner EN, Vlach LM, Wolf MM, Brown WA, Krystofiak ES, et al. HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma. J Clin Invest 2024;134.
- 27. Vasudevan NP, Soni DK, Moffett JR, Krishnan JKS, Appu AP, Ghoshal S, et al. Acss2 deletion reveals functional versatility via tissue-specific roles in transcriptional regulation. Int J Mol Sci 2023;24.
- 28. Moras M, Hattab C, Gonzalez-Menendez P, Fader CM, Dussiot M, Larghero J, et al. Human erythroid differentiation requires VDAC1-mediated mitochondrial clearance. Haematologica 2022;107:167-177.
- 29. Geisler S, Holmström KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 2010;12:119-131.
- 30. Arif T, Vasilkovsky L, Refaely Y, Konson A, Shoshan-Barmatz V. Silencing VDAC1 expression by siRNA inhibits cancer cell proliferation and tumor growth in vivo. Mol Ther Nucleic Acids 2014;3:e159.
- 31. Chen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal Transduct Target Ther 2023;8:333.
- 32. Sun B, Ding P, Song Y, Zhou J, Chen X, Peng C, et al. FDX1 downregulation activates mitophagy and the PI3K/AKT signaling pathway to promote hepatocellular carcinoma progression by inducing ROS production. Redox Biol 2024;75:103302.
- 33. Xiong Z, Yang L, Zhang C, Huang W, Zhong W, Yi J, et al. MANF facilitates breast cancer cell survival under glucose-starvation conditions via PRKN-mediated mitophagy regulation. Autophagy 2025;21:80-101.
- 34. Wang S, Cheng H, Li M, Gao D, Wu H, Zhang S, et al. BNIP3-mediated mitophagy boosts the competitive growth of Lenvatinib-resistant cells via energy metabolism reprogramming in HCC. Cell Death Dis 2024;15:484.
- 35. Hensley CT, Faubert B, Yuan Q, Lev-Cohain N, Jin E, Kim J, et al. Metabolic heterogeneity in human lung tumors. Cell 2016;164:681-694.
- 36. Yu TJ, Ma D, Liu YY, Xiao Y, Gong Y, Jiang YZ, et al. Bulk and single-cell transcriptome profiling reveal the metabolic heterogeneity in human breast cancers. Mol Ther 2021;29:2350-2365.
- 37. Yang X, Yang C, Zhang S, Geng H, Zhu AX, Bernards R, et al. Precision treatment in advanced hepatocellular carcinoma. Cancer Cell 2024;42:180-197.
- 38. Sun L, Kong Y, Cao M, Zhou H, Li H, Cui Y, et al. Decreased expression of acetyl-CoA synthase 2 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma. Cancer Sci 2017;108:1338-1346.
- 39. Abdul-Wahed A, Guilmeau S, Postic C. Sweet sixteenth for ChREBP: established roles and future goals. Cell Metab 2017;26:324-341.
- 40. Benichou E, Seffou B, Topçu S, Renoult O, Lenoir V, Planchais J, et al. The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism. Nat Commun 2024;15:1879.
- 41. Villanueva A, Alsinet C, Yanger K, Hoshida Y, Zong Y, Toffanin S, et al. Notch signaling is activated in human hepatocellular carcinoma and induces tumor formation in mice. Gastroenterology 2012;143:1660-1669.e1667.
- 42. Martinez-Quetglas I, Pinyol R, Dauch D, Torrecilla S, Tovar V, Moeini A, et al. IGF2 Is Up-regulated by epigenetic mechanisms in hepatocellular carcinomas and is an actionable oncogene product in experimental models. Gastroenterology 2016;151:1192-1205.
- 43. Miller KD, Pniewski K, Perry CE, Papp SB, Shaffer JD, Velasco-Silva JN, et al. Targeting ACSS2 with a transition-state mimetic inhibits triple-negative breast cancer growth. Cancer Res 2021;81:1252-1264.
- 44. Li Z, Liu H, He J, Wang Z, Yin Z, You G, et al. Acetyl-CoA synthetase 2: a critical linkage in obesity-induced tumorigenesis in myeloma. Cell Metab 2021;33:78-93.e77.
- 45. Perets R, Geva R, McKean M, Goutopoulos A, Erez O, Phadnis M, et al. Phase 1 first-in-human trial of MTB-9655, the first oral inhibitor of ACSS2, in patients with advanced solid tumors. J Clin Oncol 2022;40:e20609-e20609.
- 46. Zhou Z, Ren Y, Yang J, Liu M, Shi X, Luo W, et al. Acetylcoenzyme a synthetase 2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer. Gastroenterology 2022;163:1281-1293.e1281.
- 47. Shvedunova M, Akhtar A. Modulation of cellular processes by histone and non-histone protein acetylation. Nat Rev Mol Cell Biol 2022;23:329-349.
- 48. Jin Q, Yu LR, Wang L, Zhang Z, Kasper LH, Lee JE, et al. Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. Embo j 2011;30:249-262.
- 49. Wu YH, Yang Y, Chen CH, Hsiao CJ, Li TN, Liao KJ, et al. Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2. PLoS Pathog 2021;17:e1008866.
- 50. Xue Q, Liu H, Zhu Z, Yang F, Song Y, Li Z, et al. African swine fever virus regulates host energy and amino acid metabolism to promote viral replication. J Virol 2022;96:e0191921.
- 51. Foo J, Bellot G, Pervaiz S, Alonso S. Mitochondria-mediated oxidative stress during viral infection. Trends Microbiol 2022;30:679-692.
- 52. Kim SJ, Khan M, Quan J, Till A, Subramani S, Siddiqui A. Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. PLoS Pathog 2013;9:e1003722.
- 53. Ding Y, Li N, Dong B, Guo W, Wei H, Chen Q, et al. Chromatin remodeling ATPase BRG1 and PTEN are synthetic lethal in prostate cancer. J Clin Invest 2019;129:759-773.
, Ailong Huang1
