NAT10-mediated crosstalk between acetylation and PARylation enhances the function of PARP1 promoting primary resistance in esophageal cancer

Abstract

Resistance to platinum-based drugs represents a significant challenge in the clinical treatment of esophageal cancer. Particularly, some esophageal cancer patients exhibit primary resistance to platinum-based drugs. Our study revealed that the level of N-acetyltransferase 10 (NAT10) is inversely correlated with the efficacy of platinum-based drug neoadjuvant therapy. In the presence of oxaliplatin, NAT10 significantly reduced the cytotoxicity of oxaliplatin and enhanced esophageal cancer cells’ invasive, migratory, and clonogenic abilities. Mechanistically, oxaliplatin promoted the binding of NAT10 to PARP1. NAT10 acetylated PARP1 at the K97 site, thereby enhancing its stability. Additionally, acetylated PARP1 increased its PARylation modification, promoting the recruitment of downstream DNA repair proteins. In conclusion, NAT10 contributes to primary resistance by regulating the crosstalk of acetylation and PARylation of PARP1 in esophageal cancer.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06182-5.

Introduction

Esophageal cancer (ESCA) ranks as the sixth leading cause of cancer-related deaths worldwide. Clinically, platinum-based antineoplastic agents, such as oxaliplatin, cisplatin, and carboplatin, are widely used in ESCA treatment. However, resistance to platinum-based drugs remains a critical factor contributing to clinical treatment failure [1, 2]. This resistance can be categorized as Primary or Acquired resistance [3, 4]. Primary resistance profoundly impacts ESCA patients’ responses to platinum-based chemotherapy and contributes to the variability in treatment outcomes following initial platinum-based drug administration [5]. The underlying mechanisms of Primary resistance remain unclear, which poses a significant challenge in treatment.

Acetylation modification is increasingly recognized as a key contributor to cancer chemoresistance [6–9]. NAT10 is a Gcn5-related N-acetyltransferase (GNAT) family member, including an acetyltransferase domain and a lysine-rich C-terminus [10]. It is involved in histone acetylation, telomerase activity regulation, DNA damage response, and cytokinesis [11–13]. Recent studies demonstrate that NAT10 plays diverse roles in cancer development and progression. For instance, NAT10 upregulation is linked to poor prognosis in hepatocellular carcinoma [14–16]. Nevertheless, the relationship between aberrant NAT10 expression and Primary resistance in ESCA is obscure.

This study systematically investigated the role of NAT10 in primary resistance to platinum-based drugs. Through multi-omics approaches, we identified key downstream proteins undergoing NAT10-mediated acetylation and elucidated their contributions to primary resistance. Collectively, these findings provide new insights into the mechanisms underlying primary resistance to platinum-based drug in ESCA.

Materials and methods

Chemicals and reagents

All detailed information for chemical reagents and drugs were provided in Supplementary Tables 2 and 3.

Cell culture

Human esophageal carcinoma cell lines ECA109 and EC9706 were obtained from Xinxiang Medical University. The human embryonic kidney cell line HEK293T was acquired from Zhengzou University. DMEM and RPMI1640 media were purchased from WISENT Biotechnology. The cell lines ECA109, EC9706 and human embryonic kidney cell line HEK293T were cultured in DMEM or RPMI1640 media supplemented with 10% fetal bovine serum plus 1% Penicillin-Streptomycin at 37 °C in 5% CO2 incubator.

Animals

Immune deficient BALB/c nude mice (male, 4 weeks old) were purchased from the GemPharmatech, Nanjing, China. Animal experiments were performed according to the approval of the ethic requirements from Institutional Animal Care and Use Committee at Xinxiang Medical University. the Vector or NAT10 overexpression ECA109 cells were subcutaneously injected into animal (5 × 10⁶ cells per mouse). The body weight of mice was monitored daily. From the 15th days, the length diameter (L) and width diameter (W) of tumors were measured with a vernier caliper. The relative tumor volume was calculated with the formula: V = W² × L × 0.5. Subsequently, the nude mice were treated with oxaliplatin (5 mg/kg/3 days) for further 15 days. On day 21 from infection, the Fluorescence signals of tumors were assessed using PerkinElmer Lumina III instrument. At the end of experiment, tumors were harvested and weighed. Tumor sections were then cut for immunofluorescence analysis.

Human esophageal tumors

Esophageal tumor tissues were obtained from Xinxiang Central Hospital and approved by the Ethics Committee of Xinxiang Central Hospital. A total of 20 cases were subjected to clinicopathological analysis. Patients in this study under therapy with platinum-based drugs and the tumor size was determined using CT scans. According to the different efficacy of neoadjuvant therapy with platinum-based drugs, patients are divided into two groups: those with good efficacy of neoadjuvant therapy and poor efficacy of neoadjuvant therapy. For each tumor specimen, portions reserved for paraffin sectioning were excluded; all remaining tissue was homogenized for subsequent analysis.

Plasmid transfection and lentiviral infection

psPAX2, pMD2G, and other plasmids (Supplementary Table 4) were mixed into Opti-MEM Reduced Serum Medium at the ratio of 3:1:3. The mixture in lipofectamine 2000 reagent were co-transfected into 293T cells and the medium was refreshed after 12 h. Then 48 h later, a supernatant medium with lentiviral particles was collected to target cells. After incubation, the cells were treated with puromycin for 2 weeks.

For transient plasmids transfection, the Plasmids and Lip2000 Transfection Reagent were diluted in serum-free media. Subsequently, they were mixed together for 20 min. The mixture was added into cell cultures. The cells were harvested for transfection efficiency assay after incubation for 48 h.

siRNAs transfection

The siRNAs and corresponding negative control siRNAs (Supplementary Table 5) were purchased from Sangon Biotech, China. Transfection was performed using Lipofectamine 2000 according to the manufacturer’s protocol.

Immunofluorescence staining

Cells were seeded onto poly-L-lysine-coated coverslips and fixed in 4% paraformaldehyde. After blocking with 10% BSA, the coverslips were incubated with primary antibodies (Supplementary Table 6) and subsequent fluorescent secondary antibodies. The fluorescent staining was visualized by fluorescence microscope.

Transwell Matrigel assay

The diluted Matrigel was added to Transwell chambers equipped with 8 μm pore filters. Cells were seeded in chambers and then treated with drugs. Culture media were added into each well and the chambers were placed into 24-well plates. After 36 h incubation, cells were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed.

Flow cytometry analysis

Cells were harvested and washed by PBS, 1–5 × 10⁵ cells were incubated with 5 µL of Annexin V and 5 µL of propidium Iodide in 500 µL Annexin V Binding buffer for 15 min. Flow cytometric analyses were conducted by using Attune NxT Flow Cytometer.

Colony-formation assay

Cells were incubated with a completed culture medium including Oxaliplatin, CBP, or Paclitaxel for 10 days. Colony formation was measured and counted after fixation and stained with crystal violet.

Western blot

Cells were lysed by RIPA buffer and the cell debris was removed by centrifugation. Equal amounts of proteins were subjected to polyacrylamide-SDS gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk, followed by washing with TBST. Membranes were incubated with primary antibodies overnight and then incubated with secondary antibodies. Target proteins were detected using the ECL chemiluminescence solution and quantified with ImageJ software.

Immunoprecipitation

The cells were harvested and lysed as above. A fraction of the cell lysates were subjected to immunoprecipitation using the specialized antibodies (Supplementary Table 6) and bound to protein A/G beads. The whole cell lysates and precipitated samples were subsequently analyzed by western blotting assay.

Real-time quantitative PCR

Total RNA was extracted according to the manufacturer’s instructions. Subsequently, RNA was reverse-transcribed to cDNA for qPCR. The cDNA was used for amplification in a 20 µL SYBR Green PCR system. The relative mRNA expression level was quantified according to the 2^−△△Ct method. Primer sequences used for qPCR were listed in Supplementary Table 7.

Wound healing assay

Cells were seeded into 24-well plates and were scratched with 10 µL pipette tips. The cells were washed to remove the suspended cells and then treated with Oxaliplatin. The cell migration was observed and photographed at 0 h and 24 h using a microscope.

Mass spectrometry (MS) analysis

Mass spectrometry analyses were conducted on a Q Exactive Plus LC/MS system. Peptides were loaded onto a C18 analytical column (75 μm × 150 mm, 2 μm particle size, 120 Å pore size, Acclaim PepMap C18 column, for separation. The mobile phases A (0.1% formic acid) and B (80% ACN, 0.1% formic acid) were used to establish the analysis gradient at a flow rate of 300 nL/min. For DDA mode analysis, each scan cycle included one full-scan mass spectrum (R = 70 K, AGC = 3e6, max IT = 20 ms, scan range = 350–1800 m/z) followed by 15 MS/MS scans (R = 17.5 K, AGC = 2e5, max IT = 100 ms) with HCD collision energy set to 28 and an isolation window for precursor selection of 1.6 Da. A dynamic exclusion of 35 s was applied. The MS raw data were analyzed with Max Quant (version 1.6.6) using the Andromeda search engine. The UniProt Human proteome database was used for spectrum matching with the following parameters: LFQ mode for quantification, variable modifications of Oxidation (M) and Acetyl (Protein N-terminus), fixed modifications of Carbamidomethyl (C), trypsin/P for digestion, and match between runs for identification transfer. The search results were filtered to achieve a 1% false discovery rate (FDR) at both protein and peptide levels.

Molecule docking

We performed five independent AlphaFold3 predictions for each of the two target protein structures, with the number of recycles uniformly set to 10. To select the optimal predicted structures, we established strict screening criteria: The template modeling score for the interface prediction should be ≥ 0.74 to ensure the quality of the predicted protein-protein interaction interface; The overall template modeling score (pTM) should be ≥ 0.74 to guarantee the reliability of the overall folded structure; The ranking score should be > 0.8 to comprehensively evaluate the confidence of the model. Through this strategy, we screened out the qualified structures from a total of ten prediction results for the two target structures, which served as the basis for subsequent studies. When using RosettaDock to dock the predicted protein structures, we generated 10 different conformations for each of the two target complex structures, and selected the conformations with the best Total_Score for analysis.

Statistical analysis

For cell invasion and colony formation assays, cells were counted using ImageJ software. An appropriate cell perimeter size range was defined, and cell counts were determined based on this threshold. For immunofluorescence image analysis, protein levels were quantified using ImageJ’s gray-scale area measurement function. Data were analyzed by GraphPad Prism 8 software and presented as mean ± standard deviation (SD). Statistical significance between the two experimental groups was determined using an unpaired, two-tailed Student’s t test, with significance considered at p < 0.05.

Results

NAT10 overexpression promoted primary resistance to platinum-based drugs in ESCA

Clinical samples from ESCA patients receiving neoadjuvant therapy are ideal for studying the mechanisms of primary resistance. Several hundred esophageal cancer samples were collected from patients who received oxaliplatin or carboplatin as neoadjuvant therapy. The pre- and post-drug treatment imaging data of each tumor were analyzed, and the tumor shrinkage degree (measured by the longest diameter) after neoadjuvant therapy was used as a criterion to evaluate the efficacy of platinum-based drug treatment (Figs. 1A, B; Supplementary Table 1). Proteomic and transcriptomic sequencing analyses were conducted on some of the samples, respectively. The protein Mass spectrometry results indicated that the characteristics of the differential proteins in the two groups of samples were presented in Figs. 1C and D. Moreover, the COG/KOG classification of the differential proteins revealed that a considerable number of differential proteins were classified into “Posttranslational modification, protein turnover, chaperones“(Fig. 1E). Therefore, we investigated the expression differences of genes associated with posttranslational modification. The results showed that the expression level of N-acetyltransferase 10 (NAT10) was markedly elevated in the “poor efficacy of neoadjuvant therapy” group (Fig. 1F). Western blot and immunofluorescence assays further verified the significant upregulation of NAT10 in the “poor efficacy of neoadjuvant therapy” group (Figs. 1G, H). Notably, an inverse correlation between NAT10 levels and tumor shrinkage was observed in ESCA patients who received platinum-based drugs (Fig. 1I), suggesting a strong link between NAT10 and primary resistance.

Fig. 1.

The NAT10 level is inversely correlated with the efficacy of neoadjuvant therapy with platinum based drugs. A Schematic of the selection of clinical ESCA samples for platinum drugs neoadjuvant therapy and multi-omics analysis. B After neoadjuvant therapy with platinum-based drugs, good efficacy group exhibited a significantly greater reduction in maximum tumor diameter compared to poor efficacy group. C Subcellular distribution map of differential proteins between the good efficacy group and poor efficacy group. D Domain characteristics dotplot of differential proteins between the good efficacy group and poor efficacy group. (E) COG/KOG classification of differential proteins between the good efficacy group and poor efficacy group. F The mRNA level of NAT10 in the poor efficacy group was significantly higher than that in the good efficacy group. G The NAT10 protein level in the poor efficacy group was remarkably higher than that in the good efficacy group. H Immunofluorescence and quantification analysis revealed that NAT10 levels in the good efficacy group tumor tissues exhibited significantly lower than those in the poor efficacy group. I The extent of tumor shrinkage was inversely correlated with the levels of NAT10. Data were presented as the mean ± SD and statistical significance were performed using Student’s t-test. **** P < 0.0001

To verify the findings, we proceeded with NAT10 overexpression (OE) and knockdown (KD) in the ESCA cell line using lentiviral vectors (Supplementary Figs. 1A, B). Eca109 cells with NAT10 OE or KD were treated with oxaliplatin, carboplatin, or paclitaxel, and subjected to clonogenicity and invasion assays. The results indicated that, in the presence of oxaliplatin or carboplatin, NAT10 OE significantly increased cellular clonogenicity and invasion capabilities, while NAT10 KD exhibited the opposite phenotypes (Figs. 2A, B). However, under paclitaxel treatment, NAT10 OE had no effect on cellular clonogenicity and invasive potential, suggesting that NAT10 OE-induced chemoresistance is specific to platinum-based drugs (Figs. 2A, B). Moreover, apoptosis and scratch assays demonstrated that NAT10 OE reduced cell apoptosis while promoting cell migration (Figs. 2C, D). Similarly, results from another ESCA cell line, Ec9706, confirmed comparable outcomes with platinum-based drugs (Supplementary Figs. 1C, D).

Fig. 2.

NAT10 attenuates the cytotoxicity of oxaliplatin on ESCA cells. A Colony formation assays showed NAT10 enhanced cell clonogenicity compared with vector cells in the presence of Oxaliplatin (0.2µM, 10 days) and carboplatin (0.5µM, 10 days), but not in the presence of Paclitaxel (0.01µM, 10 days). B Transwell Matrigel Assay indicated NAT10 enhanced cell invasion capability compared with vector cells in the presence of Oxaliplatin (10µM, 36 h), and Carboplatin (10µM, 36 h), but not in the presence of Paclitaxel (0.05µM, 36 h). C Apoptosis assays showed NAT10 decreased cell apoptosis compared with vector cells with oxaliplatin treatment (20µM, 24 h). D Scratch assays showed NAT10 enhanced cell migration with oxaliplatin treatment (10µM, 24 h). E, F Colony formation and Transwell Matrigel assay revealed that NAT10 overexpression or knockdown did not affect cell clonogenic capacity or invasion in Eca109. G, H Colony formation and Transwell Matrigel assays demonstrate that neither overexpression nor knockdown of NAT10 affects the clonogenic capacity or invasion in Ec9706 cells. I Flow cytometry analysis indicated different levels of NAT10 did not affect cell apoptosis under normal cell culture. J NAT10 overexpressing or wild-type ECA109 cells were subcutaneously inoculated into nude mice. Three weeks later, living fluorescent imaging revealed no significant difference in tumor volume between the control and NAT10 OE group. K Four weeks after implantation, tumors were removed and weighed. Data were presented as the mean ± SD and statistical significance were performed using Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Interestingly, in the absence of oxaliplatin, neither NAT10 OE nor KD affected the clonogenic ability of ESCA cells (Figs. 2E, G). Similar results were observed in cell invasion and apoptosis assay results (Figs. 2F– I). Additionally, the vector or NAT10 OE Eca109 cells were subcutaneously injected into immunodeficient BALB/c nude mice. Three weeks later, living fluorescence imaging analysis revealed no significant difference in tumor volume between the two groups (Figs. 2J). Four weeks post-implantation, tumor weights were measured, again showing no significant differences (Fig. 2K). We further investigated whether oxaliplatin treatment would impact the expression level of NAT10. The results demonstrated that oxaliplatin could not influence the protein level and mRNA expression level of NAT10 (Figs. 2L, M). The aforementioned results indicated that NAT10 is the origin of primary resistance to oxaliplatin in ESCA cells rather than a downstream effect. Therefore, further investigation was required to elucidate the role of NAT10 in primary resistance.

Oxaliplatin enhanced the interaction between NAT10 and PARP1

It is well known that NAT10 can catalyze the acetylation modification of numerous crucial proteins, and NAT10 has been reported to acetylate α-tubulin and histones in certain tumors [17, 18]. Therefore, we examined whether NAT10 alters the acetylation level of histone H3 and α-tubulin in response to oxaliplatin treatment. As illustrated in Figs. 3A and B, NAT10 OE didn’t affect the acetylation levels of histone H3 or α-tubulin, indicating there were other mechanisms apart from histone H3 and α-tubulin acetylation.

Fig. 3.

Oxaliplatin enhanced the interaction between NAT10 and PARP1. A, B NAT10 overexpression did not affect the acetylation levels of histone H3 and α-tubulin. C Protein-protein interaction network of top 150 detected proteins and D top 50 hub proteins based on connectivity were identified by using the CytoHubba plugin in Cytoscape software. E Mass spectrometry map of PARP1 peptide. F Predictive proteins interacting with NAT10. G Immunofluorescence analysis revealed oxaliplatin (10µM, 24 h) enhanced the co-localization of NAT10 and PARP1. H ECA109 cells were treated with Oxaliplatin 20 µM for 12 h and immunoprecipitation assay indicated Oxaliplatin treatment promoted the interaction of PARP1 and NAT10 in ECA109. I, J HEK293T cells were treated with Oxaliplatin 20 µM for 12 h and immunoprecipitation assay revealed Oxaliplatin treatment enhanced the interaction of NAT10 and PARP1 in HEK293T cells. Data were presented as the mean ± SD and statistical significance were performed using Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

To reveal the key proteins that are acetylated by NAT10 in primary resistance of ESCA, NAT10-binding proteins were isolated using immunoprecipitation and identified via mass spectrometry. Proteomics and protein interaction network analysis revealed that oxaliplatin treatment induced the specific binding of 249 proteins to NAT10 (Fig. 3C). We used the CytoHubba plugin in Cytoscape software to identify the top 50 proteins based on connectivity, which were designated as hub proteins. Results indicated that poly (ADP-ribose) polymerase 1 (PARP1), a critical DNA damage repair protein, was a key protein binding to NAT10 during oxaliplatin treatment (Fig. 3D). The mass spectrometry peak pattern of PARP1 is shown in Fig. 3E. Additionally, we searched for important proteins interacted with NAT10 from several protein databases (BioGRID https://thebiogrid.org/, Uniport https://www.uniprot.org/, STRING https://cn.string-db.org/, DIP http://dip.doe-mbi.ucla.edu/dip/Main.cgi) and confirmed a close interaction between NAT10 and PARP1 (Fig. 3F; Supplementary Fig. 2A). This interaction was validated through immunofluorescence and immunoprecipitation in Eca109 cells treated with oxaliplatin (Figs. 3G–H). To further confirm the direct interaction between NAT10 and PARP1 under oxaliplatin treatment, NAT10-Flag and PARP1-His plasmids were transfected in HEK293T cells and revealed oxaliplatin enhanced the interaction between NAT10 and PARP1 (Figs. 3I–J).

NAT10 regulated PARP1 stability via acetylation modifications

NAT10 has attracted a great deal of attention due to its acetyltransferase features. To determine whether NAT10 contributes to platinum-based drug resistance through PARP1 acetylation, we examined if PARP1 is acetylated by NAT10 in ESCA cells. IP assays with PARP1 antibody followed by immunoblotting using anti-Ac-K antibody showed that NAT10 OE significantly acetylated PARP1, while NAT10 KD decreased tthe acetylation (Fig. 4A). However, without Oxaliplatin treatment, neither NAT10 OE nor KD had an obvious impact on the acetylation levels of PARP1, highlighting oxaliplatin’s essential role in NAT10-mediated PARP1 acetylation (Fig. 4B). To further validate that NAT10 catalyzes the acetylation of PARP1, cells were treated with Remodelin, a selective inhibitor of NAT10. Treatment with Remodelin significantly decreased PARP1 acetylation levels (Fig. 4C). Additionally, we generated endogenous NAT10-knockout Eca109 cells and reconstituted them with wild-type or catalytically inactive NAT10 variants. Functional analysis demonstrated that NAT10 inactivation caused marked attenuation of PARP1 acetylation (Fig. 4D). These results confirm that NAT10 directly mediates the acetylation modification of PARP1.

Fig. 4.

NAT10 regulated PARP1 stability via acetylation modifications. A ECA109 cells were treated with Oxaliplatin 20 µM for 12 h and immunoprecipitation assay showed NAT10 increased the acetylation level of PARP1. B Without oxaliplatin treatment, either overexpression or knockdown of NAT10 didn’t affect the acetylation level of PARP1. C Treatment with NAT10 inhibitor Remodelin significantly decreased PARP1 acetylation levels. D NAT10 inactivation caused marked attenuation of PARP1 acetylation. E ECA109 cells were treated with CHX (200 µM) for indicated time points and Western blot assay showed that NAT10 prolonged the half-live of PARP1. F ECA109 cells were transfected with HA-ubiquitin and treated with Oxaliplatin 20 µM for 12 h in the presence of MG132 10 µM for 18 h. IP assay showed NAT10 overexpression reduced the ubiquitination levels of PARP1. G Four lysine residues were mutated with non-acetylable arginine in PARP1. H qPCR assay indicated NAT10 expression levels didn’t affect the mRNA levels of PARP1. I Wild-type His-PARP1 and four His-mutants were transfected into SW480 cells and IP assay detected the effect of NAT10 overexpression on the acetylation of PARP1. J Remodelin (0, 10, 20 µM) dose-dependently reduce the acetylation levels of wild-type PARP1 without any effects on the acetylation of K97R PARP1 mutant. K Sequence alignment showed that K97 residue was conserved across indicted species. Data were presented as the mean ± SD and statistical significance were performed using Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Increasing evidence suggests that protein acetylation modifications can alter their stability [19, 20]. Our results showed that NAT10 OE substantially prolonged the half-live of PARP1, whereas NAT10 KD had the opposite effect (Fig. 4E). Considering the association between ubiquitination and the protein half-life, we explored the relationship between NAT10 and ubiquitination of PARP1. Eca109 cells were transfected with HA-ubiquitin and then treated with oxaliplatin in the presence of MG132. The results showed that NAT10 OE significantly decreased PARP1 ubiquitination, while NAT10 KD increased it (Fig. 4F). To assess whether NAT10 regulates PARP1 at the transcriptional level, we performed RT-PCR to measure PARP1 mRNA levels. However, varying NAT10 levels had minimal effects on PARP1 mRNA expression (Fig. 4G). In summary, PARP1 was modulated by NAT10 at the protein level, not at the mRNA level.

NAT10 acetylated PARP1 at K97 residue

Mass spectrometry analysis identified four acetylation sites on PARP1 in ESCA cells. To determine the exact sites of NAT10-catalyzed acetylation modification, the lysine residues at these four sites were mutated to arginine, which cannot be acetylated (Fig. 4H). Subsequently, the mutant plasmids were transfected into 293T cells, and the effects of NAT10 overexpression on each mutant were detected. The results demonstrated that NAT10 overexpression failed to influence the acetylation level of K97R-mutated PARP1 but significantly elevated the acetylation levels of wild-type and other PARP1 mutants (Fig. 4I).

Furthermore, the NAT10 inhibitor Remodelin dose-dependently reduced the acetylation levels of wild-type PARP1, but didn’t affect the acetylation of the K97R mutant (Fig. 4J). These results propose that K97 is the most likely site of NAT10-induced acetylation on PARP1. Moreover, sequence alignment showed that the K97 residue is conserved across species, indicating that acetylation at this residue may have significant molecular biological functions (Fig. 4K).

Acetylated PARP1 promoted DNA damage repair by enhancing its PARylation modification

PARP1 is a nuclear protein involved in a range of biological processes, including DNA damage repair, epigenetic regulation, and apoptosis [20–23].PARP1 binds to DNA damage sites and catalyzes the synthesis of poly (ADP-ribose) chains (PARylation) on itself or other protein substrates, subsequently recruiting other DNA repair proteins to the damage sites for collaborative DNA repair [24, 25]. Herein, we investigated the impact of different acetylation levels of PARP1 on its own PARylation. The results showed that NAT10 OE increased the acetylation level of PARP1, along with enhancing the PARylation level of PARP1 (Fig. 5A). Additionally, the NAT10 inhibitor Remodelin reduced PARP1’s PARylation level by decreasing its acetylation (Fig. 5B). Consistently, NAT10 OE had no effect on the acetylation or PARylation of the mutant PARP1 K97R. These findings suggest a positive correlation between NAT10-mediated acetylation and PARylation (Fig. 5C).

Fig. 5.

Acetylated PARP1 promoted DNA damage repair by enhancing its PARylation modification. A NAT10 overexpression increased the PARylation level of PARP1. B Remodelin (20µM, 18 h) reduced PARP1’s own PARylation level by decreasing its acetylation level. C NAT10 overexpression or knockdown did not affect the PARylation level of K97R mutant PARP1. D The acetylated-mimicking PARP1 promotes the self-interaction of PARP1. E The superimposition of the constructed wild-type PARP1 model with the characterized partial structure (PDB ID: 7KK6). F Total Score of the wild type – wild type PARP1 dimer is -2122.237, while that of the K97 acetylated - K97 acetylated PARP1 dimer is -2197.490. G The Co-Immunoprecipitation experiment demonstrated that acetylation at the K97 enhanced the self-interaction of K97 acetylated PARP1, while it had less interaction with non-acetylated PARP1. H Predictive proteins interacting with PARP1. I, J Cells were treated with Oxaliplatin 20 µM for 12 h and IP assay indicated Oxaliplatin treatment enhanced the binding of XRCC1 or LIG1 to PARP1. K, L Immunofluorescence assay indicated NAT10 overexpression increased the co-localization of XRCC1 or LIG1 to PARP1. M, N the WT-PARP1-His or K97R-PARP1-His plasmids were reintroduced into PARP1 knockdown ECA109 cells and IP assay revealed that K97R mutation abolished the impact of NAT10 knockdown on the interaction between PARP1 and XRCC1 or LIG1

PARP1 initiates its own PARylation modification through the formation of dimers. Based on the above experimental results, we hypothesize that acetylation at the K97 site of PARP1 facilitates dimer formation. Replacing lysine with glutamine which can mimic the acetylation of lysine. Immunoprecipitation experiments confirm that PARP1 K97Q indeed enhances the interaction between PARP1 molecules (Fig. 5D). The results of further molecular docking studies are consistent with the aforementioned experimental results. Specifically, since the structure of the full-length PARP1 protein has not yet been characterized, its wildtype and acetylated at K97 structures were modeled using AlphaFold3 based on the full sequence of PARP1 in UniProt. Figure 5E shows the superimposition of the constructed wildtype PARP1 model with the characterized partial PARP1 structure (PDB ID: 7KK6). The root mean square deviation (RMSD) value between the two is 1.124 A, suggesting that the constructed structure is reasonable. Docking calculations were performed on the wildtype – wildtype and K97 acetylated - K97 acetylated PARP1 dimer structures using RosettaDock2, respectively. The calculation results show that the Total Score of the wildtype - wildtype PARP1 dimer is -2122.237, while that of the K97 acetylated - K97 acetylated PARP1 dimer is -2197.490 (Fig. 5F). Docking simulations yielded 10 distinct conformers per docking condition, where lower total scores correlated with enhanced binding stability. This inverse relationship aligns with RosettaDock’s physical energy minimization principle. To verify the outcomes of molecular docking, plasmids encoding acetylated-mimicking K97Q PARP1 and non-acetylatable K97R PARP1 were transfected into 293T cells. The Co- P assay results were consistent with the predictions of molecular docking. The acetylated - mimicking K97Q PARP1 exhibited a greater number of interactions, whereas the non - acetylated K97R PARP1 showed fewer interactions with K97Q PARP1 (Fig. 5G). This suggests that acetylation of K97 enhances the binding strength between PARP1 proteins, which aligns with the experimental results.

PARylation of PARP1 may influence its capacity to recruit downstream proteins, prompting us to assess PARP1-interacting proteins using protein-protein interaction databases (Fig. 5H). Among which, X-ray repair cross-complementing protein 1 (XRCC1) and DNA Ligase 1 (LIG1) were identified as key DNA damage repair proteins potentially recruited by PARP1 [26, 27]. To determine if PARP1 PARylation affects XRCC1 and LIG1 recruitment, IP assays showed that NAT10 significantly enhanced the interaction between PARP1 and XRCC1 or LIG1, while NAT10 KD reduced this interaction (Figs. 5I, J). Consistent with previous results, immunofluorescence staining also showed that co-localization of XRCC1 or LIG1 with PARP1 in the presence of NAT10 (Figs. 5K, L). These findings suggest that NAT10 enhances DNA damage repair by mediating PARP1 acetylation, which may be the molecular mechanism behind NAT10-mediated chemoresistance. To confirm this conclusion, we knocked down PARP1 in Eca109 cell and reintroduced the WT-PARP1-His or K97R-PARP1-His plasmids into PARP1 KD ECa109 cells. IP results revealed that the K97R mutation abolished NAT10’s effect on the interaction between PARP1 and XRCC1 or LIG1 (Figs. 5M, N).

Furthermore, PARP1 KD or K97R mutation abolished the impact of NAT10 OE on ESCA cell invasion (Supplementary Figs. 2C, D). Similarly, cell apoptosis and scratch assays also indicated that PARP1 KD or K97R mutation eliminated the differential responses to oxaliplatin (Supplementary Figs. 2E–G). In summary, NAT10 induced chemoresistance by regulating PARP1 acetylation at K97, thereby influencing its recruitment of DNA damage repair-related proteins.

NAT10 overexpression promoted tumor formation of Eca109 cells in response to oxaliplatin in vivo

To investigate the in vivo effects of NAT10 on esophageal cancer’s response to oxaliplatin, blank or NAT10 OE Eca109 cells were subcutaneously injected into immunodeficient BALB/c nude mice (Fig. 6A). Although no significant differences in body weight changes were observed between the two groups (Fig. 6B), the tumors with NAT10 OE grew faster and showed reduced sensitivity to oxaliplatin (Fig. 6C). Living fluorescent imaging revealed that tumors in NAT10 OE group were significantly larger than those in vector group (Fig. 6D). Moreover, tumor size and weight in NAT10 OE group were significantly larger than in vector group (Figs. 6E–F). Consistent with the in vitro results, immunofluorescent indicated there is increased co-localization of PARP1 with XRCC1 or LIG1 in NAT10 OE tumors compared to vector group (Figs. 6G–I; Supplementary Fig. 2B). We also examined the DNA damage repair marker γH2AX. The results revealed lower levels of γH2AX in NAT10 OE tumors, suggesting enhanced DNA repair following NAT10 overexpression (Figs. 6I–J). Interestingly, in tumor samples from patients with poorer response to oxaliplatin neoadjuvant therapy, we observed not only elevated NAT10 level but also a noticeable increased level of PARP1, along with a higher co-localization between NAT10 and PARP1 (Fig. 6K). The above findings further highlight that NAT10 regulates acetylated PARP1, promoting DNA damage repair in vivo and contributing to chemoresistance.

Fig. 6.

NAT10 overexpression resulted in increased resistance to oxaliplatin treatment in ESCA xenograft model. A Schematic diagram of tumor inoculation and treatment in vivo. 14 days after cancer cells injection, mice were treated with oxaliplatin at a dosage of 5 mg/kg/3 day for consecutive 15 days to the endpoint. B The body weight changes of mice injected with control and NAT10 OE cells (n = 5). C The tumor volumes during mice treated with oxaliplatin (n = 5). D In vivo living fluorescent imaging analysis revealed larger size of tumors in the NAT10 OE group as compared to the control group (n = 5). E The tumor images and F tumor weights from mice explanted with control and NAT10 OE cells (n = 5). G, H Immunofluorescence staining showed NAT10 OE increased the protein levels of PARP1 and more co-localization of PARP1 and XRCC1 or LIG1. I, J Western blotting assay and quantification of γH2AX levels in control and NAT10 OE tumors (n = 4). K Tumor samples from patients with poor responses to platinum-based neoadjuvant therapy showed higher expression and co-localization of NAT10 and PARP1. Data were presented as the mean ± SD and statistical significance were performed using Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Discussion

Platinum-based drugs, such as cisplatin and oxaliplatin, play a fundamental role in the treatment of solid tumors, especially in neoadjuvant and postoperative therapies [28, 29]. However, the suboptimal response to platinum-based drugs in a subset of patients can lead to poor prognoses. Besides acquired resistance, primary resistance substantially attenuates the efficacy of platinum-based drugs, particularly during initial treatment [30, 31]. Although several studies have suggested that numerous factors, including genetic mutations, signaling pathway regulation, and alterations in the tumor microenvironment, contribute to primary chemoresistance [32, 33], many mechanisms underlying it remain unknown [34, 35].

Esophageal cancer samples initially treated with platinum - based drugs in neoadjuvant therapy represent the optimal subjects for investigating primary chemoresistance. This study discovered an obvious inverse correlation between NAT10 expression levels and the degree of tumor shrinkage in ESCA treated with platinum-based neoadjuvant therapy. Additionally, NAT10 overexpression enhanced clonogenic and invasive capabilities in the presence of oxaliplatin. This finding indicates a strong correlation between NAT10 and primary resistance of ESCA to platinum-based drugs. Mechanistically, PARP1 was identified as a downstream target of NAT10 and oxaliplatin significantly enhanced the interaction between NAT10 and PARP1. Moreover, NAT10 elevated PARP1 K97 acetylation and diminished its ubiquitination, thereby stabilizing PARP1 in ESCA, highlighting the crucial role of PARP1 acetylation in chemoresistance.

PARP1, a key protein in DNA damage repair, recognizes the sites of DNA single- or double-strand breaks and recruits a variety of DNA repair proteins [36–38]. In cancer biology, PARP1 has attracted considerable attention for its roles in promoting DNA repair and influencing cancer cell survival. PARP1 inhibitors, for example, Olaparib, have been clinically used in BRCA-mutated cancers [39–42]. Considering its therapeutic potential, the regulation of PARP1 activity via post-translational modifications remains an active research area. The NAT10-mediated acetylation of PARP1 K97 significantly promotes the formation of PARP1 dimers, and then increases the PARylation level of PARP1. Elevated PARylation in PARP1 enhanced its recruitment of DNA repair proteins XRCC1 or LIG1, which results in resistance to platinum-based drugs.

Although both PARylation and acetylation are common types of protein modifications, the relationship between these two modifications remains unclear. While some studies have indicated that inhibiting PARP activity or overexpressing poly (ADP - ribose) glycohydrolase results in a reduction of the overall acetylation levels of histones H3 [43, 44], no research has reported on the interplay between acetylation and PARylation occurring on the same protein. Our study for the first time demonstrates that the acetylation of PARP1 can promote its own PARylation, and these two modifications augment the function of PARP1 in DNA damage repair through a cascading amplification mechanism. In vivo experiments further demonstrated that NAT10 promoted chemoresistance in the presence of oxaliplatin. Furthermore, higher levels of PARP1 and greater colocalization of PARP1 with XRCC1 or LIG1 were observed in tumors formed by NAT10 OE cells. In summary, both in vivo and in vitro experiments have corroborated that the crosstalk between acetylation and PARylation of PARP1 plays a crucial role in NAT10-induced primary resistance (Fig. 7). While our data indicate a strong correlation between NAT10 expression and platinum-based drug resistance, further clinical studies are warranted to establish definitive causal relationships.

Fig. 7.

The mechanism by which NAT10 promoted chemoresistance to platinum-based drugs in esophageal cancer

This study reveals a novel mechanism driving primary resistance to Platinum-based drugs in ESCA patients. Our findings have the potential to optimize the treatment protocols for ESCA. NAT10 levels show potential as a molecular marker for predicting the response of ESCA patients to platinum-based drugs. Considering the widespread use of platinum-based drugs in solid tumor therapy, our findings offer novel insights into the mechanisms of primary resistance to platinum - based drugs in various cancers. Based on our findings, we propose that further development of inhibitors targeting the NAT10-PARP1 interaction may potentially reduce platinum-based drug resistance in esophageal cancer patients. However, the discovery of such high-efficacy and low-toxicity inhibitors necessitates extensive future research. Although commercially available NAT10 inhibitors exist, their use is currently restricted to research settings due to significant toxicity concerns. For instance, Remodeline exhibits strong cytotoxic effects. Moreover, small-molecule inhibitors targeting the catalytic site of NAT10 may induce broad cytotoxicity by suppressing NAT10’s enzymatic activity. Therefore, developing protein-protein interaction (PPI) inhibitors that specifically disrupt the binding between NAT10 and PARP1 could represent a promising therapeutic strategy. However, there are limitations to our study. First, the limited number of ECSC cases treated with neoadjuvant platinum drugs calls for more extensive clinical studies. Second, the reasons why NAT10 overexpression does not increase resistance to other anti-cancer drugs, such as paclitaxel, remain unknown.

Supplementary information

Below is the link to the electronic supplementary material.

Abbreviations

ESCA

Esophageal cancer

GNAT

Gcn5-related N-acetyltransferase

KD

Knockdown

LIG1

DNA Ligase 1

L-OHP

Oxaliplatin

NAM

Nicotinamide

NAT10

N-acetyltransferase 10

OE

Overexpression

PTMs

Protein post-translational modifications

PARP1

poly (ADP-ribose) polymerase 1

TSA

Trichostatin A

XRCC1

X-ray repair cross-complementing protein 1

Author contributions

Yu Song and Heng Li conceived and designed the study. Sizhen Hou, Wenjing An, Yake Chen, YangYang, Boyuan Jiang, Jie Yin and Qiaocen Geng performed the experiments. Xinyue Huang, Zhijian Deng, Yisi Fan, and Di Han, and Heng Li reviewed the manuscript. Yu Song wrote this manuscript, and all authors approved the final version.

Data availability

All data supporting the findings of this study are available in the article and Supporting Information.

Declarations

Ethics approval

All animal experiments were performed according to the Ethic Requirements of Institutional Animal Care and Use Committee (XXMU2020070) at Xinxiang Medical University.

Competing interests

The authors declare no competing financial interests.