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Abstract Background N4‐acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N‐acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10‐mediated ac4C in HCC progression and provide a promising therapeutic approach. Methods The ac4C levels were evaluated by dot blot and ultra‐performance liquid chromatography‐tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real‐time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10‐ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull‐down, mass spectrometry, and site‐specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10‐ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Results Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high‐throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10‐mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Conclusions Our study identified a novel oncogenic epi‐transcriptome axis involving NAT10‐ac4C/eEF2‐HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

A major obstacle to effective cancer immunotherapy is the tumor immune microenvironment. Natural killer (NK) cell resistance has been suggested as a primary cause of poor prognosis in hepatocellular carcinoma (HCC), which seemingly correlates with CNOT7 overexpression. CNOT7, a cytoplasmic mRNA deadenylase that is highly expressed in HCC, may regulate cytokine transforming growth factor‐β1 (TGF‐β1) secretion by controlling nuclear factor‐κB subunit p65 trafficking. CNOT7 depletion suppresses TGF‐β1 secretion in HCC and promotes interferon‐γ (IFN‐γ) secretion by NK cells, and we previously demonstrated that CNOT7 depletion reversed IFN‐γ resistance in HCC cells. Therefore, we hypothesized that CNOT7 depletion might reverse NK cell resistance by influencing the tumor immune microenvironment of HCC. To test this hypothesis, we examined the correlation between CNOT7, STAT1, TGF‐β1 and IFN‐γ expression with hepatitis B virus‐related cirrhosis and HCC with hepatitis B virus‐related cirrhosis. We found that modulation of CNOT7 expression alters TGF‐β1 secretion in HCC and IFN‐γ secretion in NK cells. We also examined the effects of NK cells in HepG2 cells with CNOT7 knockdown, which showed that NK cell surface CD107a expression is up‐regulated and caspase‐3 expression is significantly enhanced in CNOT7‐deficient HepG2 cells. Overall, our results show that knockdown of CNOT7 expression reverses NK cell resistance in HCC cells. Therefore, CNOT7 depletion has potential as a new adjuvant therapy in immunotherapy for HCC.