Improvement of MASLD and MASH by suppression of hepatic N-acetyltransferase 10.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are characterized by excessive triglyceride accumulation in the liver. However, due to an incomplete understanding of its pathogenesis, more efforts are needed to identify specific and effective treatments. N4-acetylcytidine (ac4C) is a newly discovered RNA modification to regulate mRNA. N-acetyltransferase 10 (NAT10) has not been fully explored in MASLD and MASH. METHODS: The clinical relevance of NAT10 was evaluated based on its expression in various mouse and human models of MASLD and MASH. Acetylated RNA immunoprecipitation sequencing and mRNA stability assays were used to explore the role of NAT10 in regulating ac4C modification and expression of target genes. Genetically engineered mice were employed to investigate the role of NAT10 in MASLD and MASH progression. RESULTS: Hepatic NAT10 expression was significantly increased in multiple mice and humans of MASLD and MASH. Genetic knockout of NAT10 protected mice from diet-induced hepatic steatosis and steatohepatitis, whereas overexpression of NAT10 exacerbated high-fat-diet-induced liver steatosis. Mechanistically, NAT10 binds to Srebp-1c mRNA, promoting its stability and expression, thereby upregulating lipogenic enzymes. Treatment with Remodelin, a NAT10-specific inhibitor, effectively ameliorates liver steatosis and dyslipidemia in a preclinical mouse model. CONCLUSIONS: Our findings indicate that NAT10 could regulate lipid metabolism in MASLD and MASH by stabilizing Srebp-1c mRNA and upregulating lipogenic enzymes. This study highlights the role of NAT10 and RNA acetylation in the pathogenesis of MASLD and MASH. Thus, our findings suggest a promising new therapeutic approach, such as the use of NAT10 inhibitor, for treating metabolic liver disease.

Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation.

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 Review of Research on the Mechanism of Tumor Regulation by N-Acetyltransferase 10.

N-acetyltransferase 10 (NAT10) is an important acetyltransferase that regulates telomerase activity and participates in DNA damage reactions, ribosomal RNA (rRNA) transcriptional activation, cell division, microtubule acetylation, and other important cellular processes. Abnormalities in the expression or distribution of NAT10 result in diseases such as Hutchinson-Gilford progeria syndrome (HGPS) and various tumors, with serious consequences. Remodelin, an inhibitor of NAT10, delays HGPS progression; many studies have been conducted on its role in tumor therapy. A major breakthrough in the study of NAT10 was the discovery of mRNA N4-acetylcytidine (ac4C) modification, which can increase mRNA stability and translation efficiency significantly. In addition, NAT10 modifies the mRNA of ac4C, which is associated with tumor development. Here, we present a review of pertinent studies focusing on NAT10, particularly its role in cancer, to provide researchers with a concise and informative summary of the current state of knowledge about this topic. The conclusions drawn from this review could provide a new direction for tumor treatment.

N-acetyltransferase 10 facilitates tumorigenesis of diffuse large B-cell lymphoma by regulating AMPK/mTOR signalling through N4-acetylcytidine modification of SLC30A9.

BACKGROUND: Accumulating studies suggested that posttranscriptional modifications exert a vital role in the tumorigenesis of diffuse large B-cell lymphoma (DLBCL). N4-acetylcytidine (ac4C) modification, catalyzed by the N-acetyltransferase 10 (NAT10), was a novel type of chemical modification that improves translation efficiency and mRNA stability. METHODS: GEO databases and clinical samples were used to explore the expression and clinical value of NAT10 in DLBCL. CRISPER/Cas9-mediated knockout of NAT10 was performed to determine the biological functions of NAT10 in DLBCL. RNA sequencing, acetylated RNA immunoprecipitation sequencing (acRIP-seq), LC-MS/MS, RNA immunoprecipitation (RIP)-qPCR and RNA stability assays were performed to explore the mechanism by which NAT10 contributed to DLBCL progression. RESULTS: Here, we demonstrated that NAT10-mediated ac4C modification regulated the occurrence and progression of DLBCL. Dysregulated N-acetyltransferases expression was found in DLBCL samples. High expression of NAT10 was associated with poor prognosis of DLBCL patients. Deletion of NAT10 expression inhibited cell proliferation and induced G0/G1 phase arrest. Furthermore, knockout of NAT10 increased the sensitivity of DLBCL cells to ibrutinib. AcRIP-seq identified solute carrier family 30 member 9 (SLC30A9) as a downstream target of NAT10 in DLBCL. NAT10 regulated the mRNA stability of SLC30A9 in an ac4C-dependent manner. Genetic silencing of SLC30A9 suppressed DLBCL cell growth via regulating the activation of AMP-activated protein kinase (AMPK) pathway. CONCLUSION: Collectively, these findings highlighted the essential role of ac4C RNA modification mediated by NAT10 in DLBCL, and provided insights into novel epigenetic-based therapeutic strategies.

Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

N4-acetylcytidine acetylation of neurexin 2 in the spinal dorsal horn regulates hypersensitivity in a rat model of cancer-induced bone pain.

Cancer-induced bone pain (CIBP) significantly impacts the quality of life and survival of patients with advanced cancer. Despite the established role of neurexins in synaptic structure and function, their involvement in sensory processing during injury has not been extensively studied. In this study using a rat model of CIBP, we observed increased neurexin 2 expression in spinal cord neurons. Knockdown of neurexin 2 in the spinal cord reversed CIBP-related behaviors, sensitization of spinal c-Fos neurons, and pain-related negative emotional behaviors. Additionally, increased acetylation of neurexin 2 mRNA was identified in the spinal dorsal horn of CIBP rats. Decreasing the expression of N-acetyltransferase 10 (NAT10) reduced neurexin 2 mRNA acetylation and neurexin 2 expression. In PC12 cells, we confirmed that neurexin 2 mRNA acetylation enhanced its stability, and neurexin 2 expression was regulated by NAT10. Finally, we discovered that the NAT10/ac4C-neurexin 2 axis modulated neuronal synaptogenesis. This study demonstrated that the NAT10/ac4C-mediated posttranscriptional modulation of neurexin 2 expression led to the remodeling of spinal synapses and the development of conscious hypersensitivity in CIBP rats. Therefore, targeting the epigenetic modification of neurexin 2 mRNA ac4C may offer a new therapeutic approach for the treatment of nociceptive hypersensitivity in CIBP.

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

BACKGROUND: Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. METHODS: We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. RESULTS: Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. CONCLUSIONS: Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

tRNA-derived small RNA (tsr007330) regulates myocardial fibrosis after myocardial infarction through NAT10-mediated ac4C acetylation of EGR3 mRNA.

Small non-coding ribonucleic acids (sncRNAs) play an important role in cell regulation and are closely related to the pathogenesis of heart diseases. However, the role and molecular mechanism of transfer RNA-derived small RNAs (tsRNAs) in myocardial fibrosis after myocardial infarction (MI) remain unknown. In this study, we identified and validated sncRNAs (mainly miRNA and tsRNA) associated with myocardial fibrosis after MI through PANDORA sequencing of rat myocardial tissue. As a key enzyme of N4-acetylcytidine (ac4C) acetylation modification, N-acetyltransferase 10 (NAT10) plays an important role in regulating messenger RNA (mRNA) stability and translation efficiency. We found that NAT10 is highly expressed in infarcted myocardial tissue, and the results of acetylated RNA immunoprecipitation sequencing (acRIP-seq) analysis suggest that early growth response 3 (EGR3) may be an important molecule in the pathogenesis of NAT10-mediated myocardial fibrosis. Both in vivo and in vitro experiments have shown that inhibition of NAT10 can reduce the expression of EGR3 and alleviate myocardial fibrosis after MI. tsRNA can participate in gene regulation by inhibiting target genes. The expression of tsr007330 was decreased in myocardial infarction tissue. We found that overexpression of tsr007330 in rat myocardial tissue could antagonize NAT10, improve myocardial function in MI and alleviate myocardial fibrosis. In conclusion, tsRNAs (rno-tsr007330) may regulate the occurrence of myocardial fibrosis by regulating NAT10-mediated EGR3 mRNA acetylation. This study provides new insights into the improvement of myocardial fibrosis after MI by targeting tsRNA therapy.

NAT10-mediated ac4C-modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear-cell renal cell carcinoma by attenuating YWHAE-driven cytoplasmic retention of YAP1.

BACKGROUND: Lymphatic metastasis is one of the most common metastatic routes and indicates a poor prognosis in clear-cell renal cell carcinoma (ccRCC). N-acetyltransferase 10 (NAT10) is known to catalyze N4-acetylcytidine (ac4C) modification of mRNA and participate in many cellular processes. However, its role in the lymphangiogenic process of ccRCC has not been reported. This study aimed to elucidate the role of NAT10 in ccRCC lymphangiogenesis, providing valuable insights into potential therapeutic targets for intervention. METHODS: ac4C modification and NAT10 expression levels in ccRCC were assessed using public databases and clinical samples. Functional investigations involved manipulating NAT10 expression in cellular and mouse models to study its role in ccRCC. Mechanistic insights were gained through a combination of RNA sequencing, mass spectrometry, co-immunoprecipitation, RNA immunoprecipitation, immunofluorescence, and site-specific mutation analyses. RESULTS: We found that ac4C modification and NAT10 expression levels increased in ccRCC. NAT10 promoted tumor progression and lymphangiogenesis of ccRCC by enhancing the nuclear import of Yes1-associated transcriptional regulator (YAP1). Subsequently, we identified ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1 (ANKZF1) as the functional target of NAT10, and its upregulation in ccRCC was caused by NAT10-mediated ac4C modification. Mechanistic analyses demonstrated that ANKZF1 interacted with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) to competitively inhibit cytoplasmic retention of YAP1, leading to transcriptional activation of pro-lymphangiogenic factors. CONCLUSIONS: These results suggested a pro-cancer role of NAT10-mediated acetylation in ccRCC and identified the NAT10/ANKZF1/YAP1 axis as an under-reported pathway involving tumor progression and lymphangiogenesis in ccRCC.

N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

Recent advances in the potential role of RNA N4-acetylcytidine in cancer progression.

N4-acetylcytidine (ac4C) is a highly conserved chemical modification widely found in eukaryotic and prokaryotic RNA, such as tRNA, rRNA, and mRNA. This modification is significantly associated with various human diseases, especially cancer, and its formation depends on the catalytic activity of N-acetyltransferase 10 (NAT10), the only known protein that produces ac4C. This review discusses the detection techniques and regulatory mechanisms of ac4C and summarizes ac4C correlation with tumor occurrence, development, prognosis, and drug therapy. It also comments on a new biomarker for early tumor diagnosis and prognosis prediction and a new target for tumor therapy. Video Abstract.

N-acetyltransferase 10 promotes the progression of oral squamous cell carcinoma through N4-acetylcytidine RNA acetylation of MMP1 mRNA.

The pathogenesis of oral squamous cell carcinoma (OSCC) remains unclear. Therefore, clarifying its pathogenesis and molecular-level development mechanism has become the focus of OSCC research. N-acetyltransferase 10 (NAT10) is a crucial enzyme involved in mRNA acetylation, regulating target gene expression and biological functions of various diseases through mediating N4-acetylcytidine (ac4C) acetylation. However, its role in OSCC progression is not well understood. In this study, we showed that NAT10 was significantly upregulated in OSCC tissues compared to normal oral tissues. Moreover, lentivirus-mediated NAT10 knockdown markedly suppressed cell proliferation, migration, and invasion in two OSCC cell lines (SCC-9 and SCC-15). Interestingly, MMP1 was found to be significantly upregulated in OSCC tissues and was a potential target of NAT10. N-acetyltransferase 10 knockdown significantly reduced both the total and ac4C acetylated levels of MMP1 mRNA and decreased its mRNA stability. Xenograft experiments further confirmed the inhibitory effect of NAT10 knockdown on the tumorigenesis and metastasis ability of OSCC cells and decreased MMP1 expression in vivo. Additionally, NAT10 knockdown impaired the proliferation, migration, and invasion abilities in OSCC cell lines in an MMP1-dependent manner. Our results suggest that NAT10 acts as an oncogene in OSCC, and targeting ac4C acetylation could be a promising therapeutic strategy for OSCC treatment.

Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.

BACKGROUND: N4-acetylcytidine (ac4C), a widespread modification in human mRNAs that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays an important role in promoting mRNA stability and translation. However, the biological functions and regulatory mechanisms of NAT10-mediated ac4C were poorly defined. METHODS: ac4C mRNA modification status and NAT10 expression levels were analyzed in gastric cancer (GC) samples and compared with the corresponding normal tissues. The biological role of NAT10-mediated ac4C and its upstream and downstream regulatory mechanisms were determined in vitro and in vivo. The therapeutic potential of targeting NAT10 in GC was further explored. RESULTS: Here, we demonstrated that both ac4C mRNA modification and its acetyltransferase NAT10 were increased in GC, and increased NAT10 expression was associated with disease progression and poor patient prognosis. Functionally, we found that NAT10 promoted cellular G2/M phase progression, proliferation and tumorigenicity of GC in an ac4C-depedent manner. Mechanistic analyses demonstrated that NAT10 mediated ac4C acetylation of MDM2 transcript and subsequently stabilized MDM2 mRNA, leading to its upregulation and p53 downregulation and thereby facilitating gastric carcinogenesis. In addition, Helicobacter pylori (Hp) infection contributed to NAT10 induction, causing MDM2 overexpression and subsequent p53 degradation. Further investigations revealed that targeting NAT10 with Remodelin showed anti-cancer activity in GC and augmented the anti-tumor activity of MDM2 inhibitors in p53 wild-type GC. CONCLUSIONS: These results suggest the critical role of NAT10-mediated ac4C modification in GC oncogenesis and reveal a previously unrecognized signaling cascade involving the Hp-NAT10-MDM2-p53 axis during GC development.

Regulatory roles of NAT10 in airway epithelial cell function and metabolism in pathological conditions.

N-acetyltransferase 10 (NAT10), a nuclear acetyltransferase and a member of the GNAT family, plays critical roles in RNA stability and translation processes as well as cell proliferation. Little is known about regulatory effects of NAT10 in lung epithelial cell proliferation. We firstly investigated NTA10 mRNA expression in alveolar epithelial types I and II, basal, ciliated, club, and goblet/mucous epithelia from heathy and patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, lung adenocarcinoma, para-tumor tissue, and systemic sclerosis, respectively. We selected A549 cells for representative of alveolar epithelia or H1299 and H460 cells as airway epithelia with different genetic backgrounds and studied dynamic responses of NAT10-down-regulated epithelia to high temperature, lipopolysaccharide, cigarette smoking extract (CSE), drugs, radiation, and phosphoinositide 3-kinase (PI3K) inhibitors at various doses. We also compared transcriptomic profiles between alveolar and airway epithelia, between cells with or without NAT10 down-regulation, between early and late stages, and between challenges. The present study demonstrated that NAT10 expression increased in human lung epithelia and varied among epithelial types, challenges, and diseases. Knockdown of NAT10 altered epithelial mitochondrial functions, dynamic responses to LPS, CSE, or PI3K inhibitors, and transcriptomic phenomes. NAT10 regulates biological phenomes, and behaviors are more complex and are dependent upon multiple signal pathways. Thus, NAT10-associated signal pathways can be a new alternative for understanding the disease and developing new biomarkers and targets.

N-acetyltransferase 10 promotes colon cancer progression by inhibiting ferroptosis through N4-acetylation and stabilization of ferroptosis suppressor protein 1 (FSP1) mRNA.

BACKGROUND: N-acetyltransferase 10 (NAT10) is the only enzyme known to mediate the N4-acetylcytidine (ac4C) modification of mRNA and is crucial for mRNA stability and translation efficiency. However, its role in cancer development and prognosis has not yet been explored. This study aimed to examine the possible role of NAT10 in colon cancer. METHODS: The expression levels of NAT10 were evaluated by immunohistochemical analyses with a colon cancer tissue microarray, and its prognostic value in patients was further analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze NAT10 expression in harvested colon cancer tissues and cell lines. Stable NAT10-knockdown and NAT10-overexpressing colon cancer cell lines were constructed using lentivirus. The biological functions of NAT10 in colon cancer cell lines were analyzed in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell, cell cycle, and ferroptosis assays. Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis and metastasis of colon cancer cells in vivo. Dot blotting, acetylated RNA immunoprecipitation-qPCR, and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in colon cancer progression. RESULTS: NAT10 was upregulated in colon cancer tissues and various colon cancer cell lines. This increased NAT10 expression was associated with shorter patient survival. Knockdown of NAT10 in two colon cancer cell lines (HT-29 and LoVo) impaired the proliferation, migration, invasion, tumor formation and metastasis of these cells, whereas overexpression of NAT10 promoted these abilities. Further analysis revealed that NAT10 exerted a strong effect on the mRNA stability and expression of ferroptosis suppressor protein 1 (FSP1) in HT-29 and LoVo cells. In these cells, FSP1 mRNA was found to be modified by ac4C acetylation, and this epigenetic modification was associated with the inhibition of ferroptosis. CONCLUSIONS: Our study revealed that NAT10 plays a critical role in colon cancer development by affecting FSP1 mRNA stability and ferroptosis, suggesting that NAT10 could be a novel prognostic and therapeutic target in colon cancer.

Inhibition of N-Acetyltransferase 10 Suppresses the Progression of Prostate Cancer through Regulation of DNA Replication.

Cancer suppression through the inhibition of N-acetyltransferase 10 (NAT10) by its specific inhibitor Remodelin has been demonstrated in a variety of human cancers. Here, we report the inhibitory effects of Remodelin on prostate cancer (PCa) cells and the possible associated mechanisms. The prostate cancer cell lines VCaP, LNCaP, PC3, and DU145 were used. The in vitro proliferation, migration, and invasion of cells were measured by a cell proliferation assay, colony formation, wound healing, and Transwell assays, respectively. In vivo tumor growth was analyzed by transplantation into nude mice. The inhibition of NAT10 by Remodelin not only suppressed growth, migration, and invasion in vitro, but also the in vivo cancer growth of prostate cancer cells. The involvement of NAT10 in DNA replication was assessed by EdU labeling, DNA spreading, iPOND, and ChIP-PCR assays. The inhibition of NAT10 by Remodelin slowed DNA replication. NAT10 was detected in the prereplication complex, and it could also bind to DNA replication origins. Furthermore, the interaction between NAT10 and CDC6 was analyzed by Co-IP. The altered expression of NAT10 was measured by immunofluorescence staining and Western blotting. Remodelin markedly reduced the levels of CDC6 and AR. The expression of NAT10 could be altered under either castration or noncastration conditions, and Remodelin still suppressed the growth of in vitro-induced castration-resistant prostate cancers. The analysis of a TCGA database revealed that the overexpression of NAT10, CDC6, and MCM7 in prostate cancers were correlated with the Gleason score and node metastasis. Our data demonstrated that Remodelin, an inhibitor of NAT10, effectively inhibits the growth of prostate cancer cells under either no castration or castration conditions, likely by impairing DNA replication.

[Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell].

Objective: To observe the platinum drugs resistance effect of N-acetyltransferase 10 (NAT10) overexpression in breast cancer cell line and elucidate the underlining mechanisms. Methods: The experiment was divided into wild-type (MCF-7 wild-type cells without any treatment) group, NAT10 overexpression group (H-NAT10 plasmid transfected into MCF-7 cells) and NAT10 knockdown group (SH-NAT10 plasmid transfected into MCF-7 cells). The invasion was detected by Transwell array, the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation. The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined. Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1. Flow cytometry was used to detect the cell apoptosis. Results: Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group, 469.00±40.50 in the NAT10 knockdown group, and 445.00±35.50 in the MCF-7 wild-type cells, and the differences were not statistically significant (P>0.05). In the presence of 10 µmol/L oxaliplatin, the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group, both statistically significant (P<0.05) compared with 219.00±31.50 in wild-type cells. In the presence of 10 µmol/L oxaliplatin, NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells, whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two. Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1, and knockdown of NAT10 expression reduced PARP1 binding to XRCC1. Overexpression of NAT10 enhanced PARP1 binding to LIG3, while knockdown of NAT10 expression decreased PARP1 binding to LIG3. In 10 µmol/L oxaliplatin-treated cells, the γH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells, both statistically significant (P<0.05) compared with 1.00±0.00 in wild-type cells. In 10 µmol/L oxaliplatin treated cells, the apoptosis rate was (6.54±0.68)% in the NAT10 overexpression group and (12.98±2.54)% in the NAT10 knockdown group, both of which were statistically significant (P<0.05) compared with (9.67±0.37)% in wild-type cells. Conclusion: NAT10 overexpression enhances the binding of NAT10 to PARP1 and promotes the acetylation of PARP1, which in turn prolongs the half-life of PARP1, thus enhancing PARP1 recruitment of DNA damage repair related proteins to the damage sites, promoting DNA damage repair and ultimately the survival of breast cancer cells.

Elevation of N-acetyltransferase 10 in hippocampal neurons mediates depression- and anxiety-like behaviors.

Major depressive disorder (MDD) is one of the most debilitating and severe mental diseases globally. Increasing evidence has shown that epigenetics is critical for understanding brain function and brain disorders, including MDD. N-acetyltransferase 10 (NAT10), acting on histones, mRNA and other substrates, has been reported to be involved in epigenetic events, including histone acetylation and mRNA modifications. NAT10 is highly expressed in the brain. However, the potential effects of NAT10 on MDD are still unknown. Here, we exploited chronic mild stress (CMS) to induce anxiety- and depression-like behaviors in mice and found that the expression of NAT10 in the mouse hippocampus was upregulated after CMS treatment. Inhibition of NAT10 by pharmacological methods produced anxiolytic- and antidepressant-like effects. Neuron-specific overexpression of NAT10 in the hippocampus resulted in anxiety- and depression-like behaviors, accompanied by higher SIRT1 protein levels, and lower dendritic spine densities. Overall, it was found that elevation of NAT10 in hippocampal neurons is involved in the occurrence of anxiety- and depression-like behaviors, suggesting that NAT10 could be a potential new target for developing anxiolytics and antidepressants.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability.

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.

Up regulation of NAT10 promotes metastasis of hepatocellular carcinoma cells through epithelial-to-mesenchymal transition.

Dysregulation of N-acetyltransferase 10 (NAT10) is associated with the development of many types of tumors; however, its role in hepatocellular carcinoma (HCC) has not been fully elucidated. Here, we examined the role of NAT10 during epithelial-to-mesenchymal transition (EMT) in HCC and established its role in metastasis. We evaluated expression of NAT10 expression in four HCC cell lines and determined the effects of knockdown by siRNA or treatment with the NAT10 inhibitor, Remodelin. NAT10 was highly expressed in HCC cell lines with a mesenchymal-like phenotype (SNU387 and SNU449). Knockdown or inhibition of NAT10 resulted in diminished cell invasion and migration. Moreover, decreased levels of NAT10 were correlated with increased E-cadherin expression and down regulation of vimentin, both of which are canonical markers of EMT signaling, suggesting that NAT10-promoted metastasis may be mediated by EMT in HCC. Our data suggests that up regulation of NAT10-promoted metastasis of HCC cells may be mediated by EMT.