Acetyl-CoA carboxylase 1 knockdown promotes esophageal squamous cell carcinoma metastasis via the CXCL8–NET axis

Abstract

Esophageal squamous cell carcinoma (ESCC) is associated with variable clinical outcomes, even among tumours with similar characteristics, complicating treatment. Here, we identify acetyl-CoA carboxylase 1 (ACC1), a key lipogenic enzyme, as a factor influencing this variability. Specifically, ACC1 knockdown in ESCC cells induces histone acetylation, activating c-Fos and C-X-C motif chemokine ligand 8 (CXCL8) transcription, which in turn promotes epithelial–mesenchymal transition (EMT) and enhances migration and invasion via CXCL8–C-X-C motif chemokine receptor 1/2 (CXCR1/2)-mediated PI3K/AKT and MEK/ERK signalling. Additionally, ACC1 knockdown stimulates neutrophil recruitment and neutrophil extracellular trap (NET) formation through CXCL8-dependent paracrine signalling, further enhancing tumour cell migration and invasion. In ESCC xenografts, ACC1 knockdown increases neutrophil infiltration and NET formation, accelerating metastasis. Clinically, low ACC1 expression and high CXCL8 levels are linked to poor prognosis in patients with ESCC, while NET formation further correlates with reduced survival. These findings highlight the ACC1–CXCL8–NET axis as a potential therapeutic target and prognostic marker in ESCC.

ACC1 loss in ESCC promotes metastasis by enhancing histone acetylation and activating c-Fos/CXCL8, driving EMT, neutrophil recruitment, and NET formation, highlighting the ACC1–CXCL8–NET axis as a prognostic and therapeutic target.

Introduction

Oesophageal cancer is one of the most aggressive malignant tumours of the digestive system, associated with high morbidity and mortality rates¹. Its major histological subtypes are oesophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC)². In China, ESCC is the predominant subtype, with the highest incidence and mortality rates reported globally^3,4. Despite advances in diagnosis and treatment, the 5-year survival rate following surgery for patients with ESCC remains below 20%, primarily because of tumour metastasis. However, the mechanisms underlying ESCC metastasis are not fully understood, and effective biomarkers or targeted therapies are lacking. Therefore, investigating the molecular basis of ESCC metastasis may offer potential therapeutic strategies aimed at improving patient outcomes.

Acetyl-CoA carboxylase 1 (ACC1) is a rate-limiting enzyme in fatty acid synthesis that catalyses the carboxylation of acetyl-CoA to malonyl-CoA, which is subsequently utilised by fatty acid synthase to produce fatty acids–crucial for cell membrane formation and energy production^5,6. Acetyl-CoA also acts as the sole donor of acetyl groups in cells, participating in the acetylation of lysine residues in proteins, thereby regulating gene transcription through epigenetic mechanisms⁷. As ACC1 utilises acetyl-CoA as a catalytic substrate during fatty acid synthesis, it can influence both fatty acid synthesis and protein acetylation. ACC1 plays diverse roles in tumour progression across multiple cancer types. It has been shown to promote tumour growth by enhancing fatty acid metabolism, as evidenced by its crucial role in CDK13-induced lipid accumulation in prostate cancer and in supporting non-small cell lung cancer cell viability^8,9. Conversely, reduced ACC1 activity may also facilitate tumour progression by increasing intracellular acetyl-CoA levels and enhancing protein acetylation. For instance, ACC1 phosphorylation is associated with increased metastatic potential in breast cancer, and ACC1 inhibition enhances breast cancer cell invasion through Smad acetylation driven by elevated acetyl-CoA levels¹⁰. These findings suggest that the role of ACC1 in tumour progression is tissue-specific and regulated by metabolic signalling pathways. However, its function in oesophageal cancer remains largely unexamined.

Histone acetylation induces a relaxed chromatin structure, enabling transcription factors to access and bind to target gene sequences, thereby promoting gene transcription and influencing tumour metastasis⁵. Although a previous study reported that reduced histone acetylation in various tumour tissues is associated with an increased risk of tumour progression¹¹, other reports have shown that elevated histone acetylation correlates positively with disease progression in patients with prostate cancer and ESCC^12,13. These contrasting findings underscore the context-dependent and diverse biological roles of histone acetylation in the initiation and progression of different tumour types.

CXCL8 is a pro-inflammatory chemokine that plays a key role in promoting neutrophil chemotaxis and degranulation. In cancer, tumour cell-derived CXCL8 can induce autocrine signalling by binding to G protein-coupled receptors CXCR1 and CXCR2. This interaction activates several downstream pathways–such as PI3K/AKT, PLC/PKC, ERK/MAPK, and JAK–that contribute to tumour progression^14–17. Elevated levels of CXCL8 and its receptor CXCR2 have been associated with shorter survival and poorer prognosis in patients with ESCC^18,19. Moreover, tumour-associated macrophages release CXCL8, which enhances the migration and invasion of ESCC cell lines via CXCR1/2 receptors, further underscoring the role of the CXCL8–CXCR2 axis in the tumour microenvironment (TME)^20,21. These findings indicate that targeting this signalling pathway may represent a promising therapeutic strategy to inhibit tumour invasion and metastasis in ESCC.

Neutrophils, the body’s first line of defence against infections, also respond to inflammatory signals associated with tumour development. Chemokines from the TME recruit neutrophils to tumour sites, where they differentiate into tumour-associated neutrophils (TANs)²², known for their functional diversity and plasticity. In certain contexts, such as acute inflammation or immune-based therapies, neutrophils can exert antitumour effects by initiating immune responses²³. Conversely, neutrophil-generated enzymes, including myeloperoxidase (MPO), neutrophil elastase (NE), neutrophil collagenase, and gelatinase B, promote tumour progression, as observed in hepatocellular carcinoma^24,25. In ESCC, a high intratumoural neutrophil presence has been identified as an independent poor prognostic factor, and a high peritumoural neutrophil-to-CD8⁺ lymphocyte ratio is significantly associated with disease progression²⁶. These findings suggest the potential of TANs in driving ESCC progression.

Activated by various stimuli—such as chemokines and cytokines secreted by tumour cells within the TME–TANs undergo a range of morphological and functional changes. One key response is the release of neutrophil extracellular traps (NETs), which are unique network structures composed of depolymerised chromatin (DNA and citrullinated histones) combined with proteases such as NE and MPO^22,26–29. Numerous studies have shown that NETs promote tumour progression and metastasis. For instance, NETs activate the EGFR/ERK signalling pathway, enhancing pancreatic cancer cell migration and invasion³⁰. In gastric cancer, NET formation is induced by the hypoxic microenvironment via the HMGB1/TLR4/p38 MAPK pathway, leading to increased cancer cell migration and invasion³¹. Additionally, NE released from NETs activates Toll-like receptor 4 (TLR4) on cancer cells, upregulating PGC1α, promoting mitochondrial biogenesis, and supporting hepatocellular and colorectal tumour growth³². These findings suggest that NETs contribute to tumour progression through multiple mechanisms. However, the specific mechanisms by which NETs influence ESCC progression remain poorly understood.

In this study, we aimed to investigate the mechanisms by which ACC1 knockdown promotes migration and invasion in ESCC cells. Specifically, we hypothesised that ACC1 knockdown would upregulate CXCL8 expression, which in turn would facilitate ESCC cell migration and invasion through autocrine signalling. Additionally, we explored the paracrine role of CXCL8 in recruiting neutrophils and promoting NET formation. Our findings elucidate the critical role of the ACC1/CXCL8/NETs axis in ESCC metastasis and may guide the identification of novel therapeutic strategies and molecular markers targeting ESCC progression.

Results

Low ACC1 expression is significantly associated with poor prognosis in ESCC

In our initial investigation of molecular markers linked to ESCC metastasis and prognosis, ACC1 expression was found to be significantly lower in tumour tissues compared to adjacent normal tissues. To validate this observation, we performed a reverse transcription (RT)-qPCR analysis on 98 ESCC clinical specimens, which revealed that ACC1 mRNA levels were significantly reduced in tumour tissues of patients with poor prognosis compared to those with good prognosis, across both individual stages and overall (Fig. 1A). A clinicopathological correlation analysis showed that patients classified as N₁ exhibited lower ACC1 mRNA levels than those classified as N₀ (Fig. 1B and Supplementary Table 1). Kaplan−Meier (KM) analysis further showed that lower ACC1 mRNA levels were associated with significantly shorter overall survival in patients with ESCC (Fig. 1C). To assess ACC1 protein expression, we performed immunohistochemical (IHC) staining using a tissue microarray (TMA) of ESCC patient samples (Fig. 1D and Supplementary Table 2). The IHC results showed that ACC1 protein levels were significantly lower in tumour tissues than in adjacent normal tissues (Fig. 1E) and that reduced ACC1 protein expression was significantly correlated with poorer overall survival (Fig. 1F). Together, these findings suggest that decreased ACC1 expression is associated with aggressive clinicopathological features and unfavourable survival outcomes in ESCC, supporting its potential as a prognostic biomarker.

Fig. 1. Low expression of ACC1 is significantly associated with poor prognosis in ESCC.

A RT-qPCR analysis of ACC1 mRNA levels in tumour tissues of patients with the same stage but different prognosis (n = 98). B mRNA levels of ACC1 in tumour tissues of ESCC patients with different lymph node metastasis statuses. C Kaplan–Meier analysis of the overall survival of ESCC patients with high or low ACC1 expression. D Immunohistochemistry analysis of ACC1 levels in ESCC tissues. (n = 91). Scale bar, 100 µm. E H-score of ACC1 in tumour tissues and adjacent tissues. F Kaplan–Meier analysis of the overall survival of ESCC patients with high or low ACC1 expression in tumour tissues. Data are presented as mean ± SEM. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

ACC1 knockdown promotes ESCC cell migration and invasion by upregulating CXCL8

To investigate the mechanism by which ACC1 contributes to ESCC progression, we first assessed its protein levels across various ESCC cell lines (Fig. 2A and Supplementary Fig. 1A). KYSE-450 and KYSE-150 cells, which showed relatively high ACC1 expression, were selected to generate ACC1 knockdown cell lines. Notably, ACC1 knockdown significantly enhanced the migratory and invasive capacities of these cells, without affecting their proliferation (Fig. 2B, C, Supplementary Fig. 1B, and Supplementary Fig. 2D, E).

Fig. 2. ACC1 knockdown contributes to ESCC cells' migration and invasion by upregulating CXCL8.

A Western blot showing ACC1 protein levels across various ESCC cells. B, C Representative images and statistical analysis of migration and invasion in KYSE-450 shNC/shACC1 cells and KYSE-150 shNC/shACC1 cells. D RT-qPCR analysis of CXCL8 expression in KYSE-450 shNC/shACC1 and KYSE-150 shNC/shACC1 cells. E Representative images and statistical analysis of migration and invasion of KYSE-450 and KYSE-150 cells influenced by CM from KYSE-450 shNC/shACC1 and KYSE-150 shNC/shACC1 cells. F, G Representative images and statistical analysis of migration and invasion of KYSE-450 (F) and KYSE-150 (G) cells influenced by CXCL8 antibody-neutralised CM from KYSE-450 and KYSE-150 shACC1, with IgG-neutralised CM from KYSE-450 shNC and KYSE-150 shNC cells serving as controls. CM: conditional medium. Data are presented as mean ± SD. Each experiment was repeated in triplicate. Scale bar, 100 µm. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

To investigate the mechanism underlying ACC1 knockdown-induced migration and invasion, transcriptome sequencing was performed on KYSE-450 shNC and shACC1 cells. The analysis revealed a marked upregulation of CXCL8 mRNA levels in shACC1 cells compared to shNC controls (Supplementary Fig. 1C), which was subsequently validated by RT-qPCR (Fig. 2D and Supplementary Fig. 2F). Although CXCL1, CXCL2, and CXCL3 were also upregulated in ACC1 knockdown cells (Supplementary Fig. 1C, D), and TCGA data showed significant overexpression of all four chemokines in tumour tissues relative to adjacent normal tissues, Kaplan–Meier analysis revealed that only elevated CXCL8 mRNA levels were significantly associated with shorter overall survival (Supplementary Fig. 1F, G). Therefore, we focused on investigating the specific role of CXCL8 in our subsequent experiments.

Given that CXCL8 is a cytokine, an ELISA was performed to measure CXCL8 levels in the CM from shNC and shACC1 cells. Consistent with RT-qPCR results, CXCL8 levels were significantly higher in shACC1 CM than in shNC CM (Supplementary Fig. 1E). Moreover, shACC1 CM markedly facilitated the migration and invasion of KYSE-450 and KYSE-150 cells compared with shNC CM (Fig. 2E). These effects were attenuated when a CXCL8-neutralising antibody was applied to the CM (Fig. 2F, G), providing further evidence that ACC1 knockdown-induced migration and invasion are CXCL8-dependent.

ACC1 knockdown promotes ESCC cells migration and invasion by activating PI3K/AKT and MEK/ERK1/2 signalling pathways via the CXCL8–CXCR1/2 axis

To further investigate whether ACC1 knockdown-induced migration and invasion of ESCC cells are mediated via CXCL8-triggered activation of CXCR1/2 signalling, cells were transfected with CXCL8 siRNA or treated with the CXCR1/2 inhibitor reparixin. The efficacy of CXCL8 knockdown was confirmed by RT-qPCR, and CXCL8 levels in the CM were measured using ELISA (Fig. 3A). Both CXCL8 knockdown and reparixin treatment partially reversed the ACC1 knockdown-induced migration and invasion in ESCC cells (Fig. 3B–E). In addition, ACC1 knockdown attenuated the expression of the epithelial marker E-cadherin while increasing the expression of the mesenchymal markers vimentin and PAI-1 and the transcription factor Slug, indicating that ACC1 knockdown induces a shift towards an EMT phenotype (Supplementary Fig. 2A, C). Furthermore, both CXCL8 knockdown and reparixin treatment reversed the elevated levels of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) induced by ACC1 knockdown (Supplementary Fig. 2B), indicating that CXCL8 activates the PI3K/AKT and MEK/ERK1/2 signalling pathways via CXCR1/2. To further elucidate the mechanistic pathways involved, we conducted additional experiments using the selective PI3K inhibitor LY294002 and the selective MEK inhibitor U0126. Treatment with either inhibitor partially reversed the enhanced migration and invasion observed in ACC1-knockdown ESCC cells (Supplementary Fig. 3A–D). As expected, LY294002 effectively inhibited AKT phosphorylation, while U0126 reduced ERK1/2 phosphorylation. Moreover, both inhibitors mitigated the EMT-associated protein expression profile induced by ACC1 knockdown (Supplementary Fig. 3E, F).

Fig. 3. ACC1 knockdown promotes ESCC cells' migration and invasion by activating PI3K/AKT and MEK/ERK1/2 signalling pathways via the CXCL8–CXCR1/2 axis.

A RT-qPCR and ELISA analysis demonstrating CXCL8 knockdown efficiency. B, C Representative images and statistical analysis of migration and invasion of KYSE-450 shNC/shACC1 cells and KYSE-150 shNC/shACC1 cells after CXCL8 knockdown. D, E Representative images and statistical analysis of migration and invasion of KYSE-450 shNC/shACC1 cells and KYSE-150 shNC/shACC1 cells after reparixin treatment. Each experiment was conducted three times. Data are presented as mean ± SD. Scale bar, 100 µm. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

Collectively, these results indicate that ACC1 knockdown leads to elevated CXCL8 expression, which in turn activates the PI3K/AKT and MEK/ERK1/2 signalling pathways via CXCR1/2. This activation promotes EMT and ultimately enhances ESCC cell migration and invasion.

ACC1 knockdown enhances cell migration and invasion by upregulating c-Fos-mediated CXCL8 expression

CXCL8 expression is regulated by various mechanisms, including the transcription factor c-Fos, which forms the AP-1 complex with Jun family proteins. This complex regulates CXCL8 transcription by directly binding to its promoter and facilitating its activation alongside other regulatory proteins. In this study, transcriptome sequencing revealed a marked increase in c-Fos mRNA levels in shACC1 cells compared to shNC cells (Fig. 4A), a finding further validated by RT-qPCR (Fig. 4B). To determine whether the elevated CXCL8 expression following ACC1 knockdown is mediated by c-Fos, we performed a dual-luciferase reporter assay, which showed that c-Fos enhances the transcriptional activity of the CXCL8 promoter (Fig. 4C). This finding was further supported by ChIP experiments, which confirmed that c-Fos directly binds to the CXCL8 promoter (Fig. 4D). To evaluate the role of c-Fos in augmenting CXCL8 levels induced by ACC1 knockdown, cells were transfected with c-Fos siRNA. The efficacy of c-Fos knockdown was confirmed by RT-qPCR (Fig. 4E). Notably, c-Fos silencing attenuated the increase in CXCL8 mRNA and CXCL8 protein levels observed following ACC1 knockdown (Fig. 4F). Moreover, c-Fos knockdown partially reversed the enhanced migration and invasion of KYSE-450 and KYSE-150 cells induced by ACC1 knockdown (Fig. 4G, H). It also mitigated changes in EMT-related protein expression and reduced the levels of p-AKT and p-ERK1/2 induced by ACC1 knockdown (Fig. 4I). Collectively, these findings highlight the pivotal role of c-Fos in mediating CXCL8 upregulation following ACC1 knockdown, thereby contributing to the migratory and invasive phenotypes of ESCC cells.

Fig. 4. ACC1 knockdown enhances cell migration and invasion by upregulating c-Fos-mediated CXCL8 expression.

A RNA-seq results demonstrating up-regulation of c-Fos expression by ACC1 knockdown. B RT-qPCR assays assessing the levels of c-Fos in KYSE-450 shNC/shACC1 cells and KYSE-150 shNC/shACC1 cells. C Dual-luciferase reporter assays in 293 T cells transfected with CXCL8 promoter reporter and c-Fos transcription factor. D ChIP assay showing c-Fos binding to the CXCL8 promoter. E RT-qPCR analysis of c-Fos knockdown efficiency. F RT-qPCR assays assessing the level of CXCL8 in KYSE-450 shNC/shACC1 cells and KYSE-150 shNC/shACC1 cells after c-Fos knockdown, and ELISA assays measuring the level of CXCL8 in CM from these cells. G, H Statistical analysis (G) and representative images (H) of migration and invasion of KYSE-450 shNC/shACC1 and KYSE-150 shNC/shACC1 cells after c-Fos knockdown. I Immunoblotting analysis depicting the effect of sic-Fos on p-AKT, p-ERK1/2, and EMT-related protein levels in KYSE-450 shNC/shACC1 and KYSE-150 shNC/shACC1 cells. Data are presented as mean ± SD. Each experiment was repeated three times. Scale bar, 100 µm. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

ACC1 knockdown increases c-Fos and CXCL8 levels via a mechanism involving histone acetylation

We investigated the impact of ACC1 knockdown on fatty acid synthesis in ESCC cells, given its central role in this pathway. Surprisingly, ACC1 knockdown did not significantly alter cellular fatty acid levels, likely because of compensatory upregulation of sterol regulatory element-binding protein 1 (SREBF1) and fatty acid synthase (FASN) (Fig. 5A–C). However, ACC1 knockdown resulted in elevated levels of cellular acetyl-CoA and increased histone H3 acetylation (Fig. 5D and Supplementary Fig. 2A, C). To elucidate whether the sequential increase in c-Fos and CXCL8 levels was driven by enhanced histone acetylation, we employed the histone acetyltransferase inhibitor C646. Notably, C646 partially reversed the ACC1 knockdown-induced increase in c-Fos and CXCL8 mRNA levels (Fig. 5E, F), as well as the associated enhancement in cell migration and invasion (Fig. 5H, I). Consistent with the RT-qPCR results, C646 also reduced the elevated CXCL8 levels in the CM from shACC1 cells (Fig. 5G). Additionally, C646 treatment attenuated histone acetylation, c-Fos expression, and changes in EMT-related proteins, along with p-AKT and p-ERK1/2 levels induced by ACC1 knockdown (Supplementary Fig. 4G). To identify the specific histone acetyltransferase responsible for ACC1 knockdown-induced increase in histone acetylation, endogenous p300 was silenced in ESCC cells via siP300 transfection, and knockdown efficacy was confirmed by RT-qPCR (Supplementary Fig. 4C). Notably, p300 knockdown partially reversed the ACC1 knockdown-induced migration and invasion (Supplementary Fig. 4A, B), as well as the increased c-Fos and CXCL8 mRNA levels (Supplementary Fig. 4D, F) in ESCC cells. Consistent with the RT-qPCR data, CXCL8 levels in the CM from shACC1 cells were also reduced following p300 knockdown (Supplementary Fig. 4E). Additionally, p300 knockdown attenuated histone acetylation, c-Fos expression, and changes in EMT-related proteins and levels of p-AKT and p-ERK1/2 induced by ACC1 knockdown (Supplementary Fig. 4H). Taken together, these findings indicate that ACC1 knockdown enhances histone acetylation by increasing acetyl-CoA levels through a p300-dependent mechanism. This, in turn, upregulates the transcription factor c-Fos, which stimulates CXCL8 expression, ultimately promoting EMT, migration, and invasion in ESCC cells.

Fig. 5. ACC1 knockdown increases c-Fos and CXCL8 levels via a mechanism involving histone acetylation.

A Free fatty acid (FFA) levels in shNC and shACC1 of KYSE-450 and KYSE-150 cells. B, C RT-qPCR assays assessing the levels of FASN (B) and SREBF1 (C) in shNC and shACC1 of KYSE-450 and KYSE-150 cells. D Acetyl-CoA levels in shNC and shACC1 of KYSE-450 and KYSE-150 cells. E qPCR assays of c-Fos mRNA expression levels in shNC and shACC1 of KYSE-450 and KYSE-150 cells after C646 treatment. F qPCR assays of CXCL8 mRNA expression levels in shNC and shACC1 of KYSE-450 and KYSE-150 cells after C646 treatment. G ELISA assays of CXCL8 protein levels in shNC and shACC1 of KYSE-450 and KYSE-150 cells after C646 treatment. H, I Representative images of migration and invasion of KYSE-450 and KYSE-150 shNC/shACC1 cells after C646 treatment, along with statistical analysis. Each experiment was conducted three times. Data are presented as mean ± SD. Scale bar, 100 µm. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

ACC1 overexpression inhibits cell migration and invasion in ESCC cells

To further support our findings, we utilised KYSE-140 cells, which naturally express lower levels of ACC1 compared to the human oesophageal epithelial cell line HET-1A (Fig. 2A), to generate ACC1-overexpressing KYSE-140 cells. The efficacy of ACC1 overexpression was confirmed by RT-qPCR and western blotting (Supplementary Fig. 5A, F). Our results showed that ACC1 overexpression significantly suppressed CXCL8 expression, as well as migration and invasion in KYSE-140 cells (Supplementary Fig. 5B–D). Moreover, the CM from ACC1-overexpressing cells inhibited the migration and invasion of KYSE-140 cells (Supplementary Fig. 5E). Western blot analysis further revealed that ACC1 overexpression induced changes in EMT-related molecular markers, including increased levels of the epithelial marker E-cadherin and decreased levels of the mesenchymal markers vimentin and PAI-1, as well as the transcription factor Slug. Additionally, ACC1 overexpression reduced histone acetylation, suppressed c-Fos expression, and decreased p-AKT and p-ERK1/2 levels (Supplementary Fig. 5F). Collectively, these findings suggest a regulatory role for ACC1 in modulating key pathways involved in cell migration, invasion, and EMT in ESCC.

ACC1 knockdown in ESCC cells facilitates neutrophil recruitment and NET formation via a CXCL8-dependent paracrine pathway

The findings outlined above indicate that ACC1 knockdown promotes ESCC migration and invasion via CXCL8-mediated autocrine signalling. However, growing evidence suggests that CXCL8 secreted by tumour cells can also act via paracrine signalling, particularly by modulating immune cell behaviour in the TME. As a key chemotactic factor for neutrophils, CXCL8 plays a major role in their recruitment and activation. To assess the paracrine effects of CXCL8 released from shACC1 cells on neutrophils, human peripheral blood neutrophils were isolated, and their purity was confirmed by flow cytometry (Supplementary Fig. 6A). Notably, CM from shACC1 cells significantly enhanced neutrophil migration (Supplementary Fig. 6B). This effect was attenuated upon the addition of a CXCL8-neutralising antibody to the CM (Fig. 6A), confirming the critical role of CXCL8 in neutrophil recruitment.

Fig. 6. ACC1 knockdown in ESCC cells facilitates neutrophil recruitment and NET formation via a CXCL8-dependent paracrine pathway.

A Representative images and statistical analysis of neutrophil migration influenced by CXCL8 antibody-neutralised CM from KYSE-450 shACC1, with IgG-neutralised CM from KYSE-450 shNC serving as control. Scale bar, 100 µm. B Representative fluorescence microscopy images of NETs induced by NEi, PAD4i, and Dnase I-treated CM from KYSE-450 shNC/shACC1 cells. Scale bar, 50 µm. C Representative fluorescence microscopy images of NETs induced by CXCL8 antibody-neutralised CM from KYSE-450 shNC/shACC1 cells, with IgG-neutralised CM from KYSE-450 shNC serving as control. Scale bar, 50 µm. D Representative fluorescence microscopy images of NETs induced by reparixin in combination with CM from KYSE-450 shNC/shACC1 cells. Scale bar, 50 µm. E Representative images and statistical analysis of KYSE-450 cells penetrated in the Transwell upper chamber membrane when neutrophils were combined with CM from KYSE-450 shNC/shACC1 cells, with or without NEi, PAD4i, and Dnase I, in the lower chamber. Scale bar, 100 µm. F Representative images and statistical analysis of KYSE-450 cells penetrated in the Transwell upper chamber membrane when neutrophils were combined with CM from KYSE-450 shNC/shACC1 cells, with or without reparixin, in the lower chamber. Scale bar, 100 µm. NETs: stained with cit-H3 (green). Data are presented as mean ± SD. Each experiment was repeated three times. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ***P  <  0.001.

Neutrophils can release NETs upon activation by stimuli such as chemokines and cytokines. NETs are known to contribute to tumour metastasis, promote EMT, and reactivate dormant cancer cells. To investigate whether CM from shACC1 cells enhances NET formation, neutrophils were co-cultured with the indicated CM. As anticipated, CM from shACC1 cells promoted NET formation (Supplementary Fig. 6C). This effect was significantly reduced by treatment with a PAD4 inhibitor (PAD4i), NE inhibitor (NEi), or DNase I, all of which specifically interfere with NET formation (Fig. 6B). Similarly, this effect was markedly diminished upon addition of a CXCL8-neutralizing antibody or reparixin, which blocks CXCL8 binding to its receptor CXCR1/2 (Fig. 6C, D). We next investigated whether CM-induced NETs contribute to ESCC cell migration. Co-culturing ESCC cells with the indicated CM showed that CM from shACC1 cells significantly promoted ESCC cell migration compared to CM from shNC cells, both in the presence and absence of neutrophils. However, the enhanced migratory effect was significantly reduced by treatment with PAD4i, NEi, or DNase I (Fig. 6E). Similarly, it was markedly suppressed by reparixin, which inhibits CXCL8 binding to its receptor CXCR1/2 (Fig. 6F). Overall, these findings suggest that CXCL8 derived from ACC1 knockdown ESCC cells recruits neutrophils and induces NET formation, thereby facilitating ESCC cell migration.

ACC1 knockdown promotes neutrophil infiltration, NET formation, and invasive behaviour in ESCC xenograft tumours

To investigate the impact of ACC1 knockdown on ESCC tumour progression in vivo, subcutaneous xenograft tumours were established in BALB/c nude mice by injecting KYSE-150 shNC and shACC1 cells. Although ACC1 knockdown had no significant effect on tumour growth (Supplementary Fig. 7A–C), consistent with the in vitro proliferation assay results (Supplementary Fig. 1B), a notable increase in neutrophil infiltration and NET formation within the tumour tissue was observed (Supplementary Fig. 7D, E). This was further confirmed by immunofluorescence co-staining for cit-H3 and MPO (Supplementary Fig. 7G, H). Notably, a significant negative correlation was identified between neutrophil count and ACC1 expression (Supplementary Fig. 7F). Interestingly, tumours from the shACC1 group exhibited distinct features, including blurred boundaries between cancerous and adjacent tissues, compared to those from the shNC group (Supplementary Fig. 7D), suggesting that ACC1 knockdown promotes cancer cell invasion into surrounding tissue. At the molecular level, shACC1 tumours showed elevated CXCL8 mRNA levels (Supplementary Fig. 7I) and increased cit-H3 and MPO protein levels (Supplementary Fig. 7J) relative to shNC tumours. Additionally, ACC1 knockdown induced significant changes in tumour tissues, including reduced E-cadherin expression and increased vimentin, PAI-1, and Slug levels (Supplementary Fig. 7J), indicative of enhanced EMT. These changes may be driven by elevated c-Fos expression, potentially resulting from increased histone acetylation induced by ACC1 knockdown.

ACC1 knockdown drives ESCC metastasis via NETs in vivo

To further investigate the impact of ACC1 knockdown on ESCC metastasis in vivo and to assess the role of NETs in this process, we established a lung metastasis model by injecting shNC and shACC1 cells via the tail vein, followed by PAD4i treatment (Fig. 7A). In vivo imaging revealed a significant increase in both the number and size of lung metastatic nodules in the shACC1 + saline group compared to the shNC + saline group. Notably, this metastatic enhancement was effectively inhibited by PAD4i treatment (Fig. 7C). These observations were further validated by histological examination of lung tissue sections using haematoxylin and eosin (H&E) staining (Fig. 7D, E). Consistent with the in vitro findings and observations from the subcutaneous tumour model, lung nodules in the shACC1 group exhibited elevated levels of NETs compared to those in the shNC + saline group (Fig. 7F). Importantly, PAD4i treatment effectively suppressed this increase (Fig. 7F), with no significant changes observed in mouse behaviour or body weight and no abnormalities detected in the liver, kidney, or spleen tissues (Fig. 7B, Supplementary Fig. 8A). Collectively, these findings underscore the role of ACC1 downregulation in promoting neutrophil recruitment, NET formation, and tumour metastasis in ESCC. Moreover, they suggested NET inhibition may represent a promising therapeutic strategy for patients with low ACC1-expressing ESCC.

Fig. 7. ACC1 knockdown drives ESCC metastasis via NETs in vivo.

A Schematic diagram illustrating PAD4i treatment in the shNC/shACC1 lung metastasis tumour model. B Body weight curve of BALB/c nude mouse during the experiment. C In vivo imaging reveals lung metastases in the lung tissues. D HE staining showing ESCC metastases in lung tissues. Scale bar, 500 µm. E Statistical analysis of metastatic area as a percentage of lung area and the number of metastatic nodules in lung tissues. F Representative images of immunofluorescence staining for NETs, co-stained with cit-H3 (green) and MPO (red). Scale bar, 50 µm. n  =  3. Data are presented as mean ± SEM. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

Elevated CXCL8 and NET levels in ESCC tissues are associated with poor outcomes

To comprehensively investigate the role of CXCL8 in ESCC progression within a clinical context, CXCL8 mRNA levels were examined by RT-qPCR in samples obtained from 98 patients with ESCC. In contrast to ACC1 (Fig. 1A–C), CXCL8 expression showed an opposite trend, with significantly higher levels detected in tumour tissues from patients with poor prognosis compared to those with good prognosis, across various stages or overall (Fig. 8A). A clinicopathological correlation analysis revealed that patients classified as N₁ had higher CXCL8 mRNA levels than those classified as N₀ (Fig. 8B and Supplementary Table 3). Kaplan–Meier analysis further showed that elevated CXCL8 mRNA levels were predictive of shorter overall survival (Fig. 8C). In addition, a significant negative correlation was observed between ACC1 and CXCL8 expression levels (Fig. 8D).

Fig. 8. Elevated CXCL8 and NET levels in ESCC tissues are associated with poor outcomes.

A RT-qPCR analysis of CXCL8 mRNA levels in tumour tissues of patients with the same stage but different prognosis (n = 98). B mRNA levels of CXCL8 in tumour tissues of ESCC patients with different lymph node metastasis statuses. C Kaplan–Meier analysis of the overall survival of ESCC patients with high or low CXCL8 expression. D Correlation analysis of CXCL8 and ACC1 expression. E Immunofluorescence analysis of NET levels in ESCC tissues and adjacent invasive tissues. NETs are co-stained with cit-H3 (green) and MPO (red) (n = 91). Scale bar, 100 µm. F, G Kaplan–Meier analysis of the overall survival of ESCC patients with high or low NETs expression in tumour tissues (F) and adjacent invasive tissues (G). H Correlation analysis of ACC1 and NETs expression. Data are presented as mean ± SEM. Significance levels are indicated as follows: ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001.

To observe NETs in ESCC tumour tissues, immunofluorescence co-staining for cit-H3 and MPO was performed on an ESCC TMA containing tumour and matched adjacent tissues from 91 patients. Both tumour and adjacent tissues from patients with clinical stage T₃N₁M₀ and less than 1-year survival exhibited higher levels of NETs compared to those from patients with stage T₃N₀M₀ and more than 7-year survival (Fig. 8E). Although NET levels were not significantly correlated with other clinicopathological features (Supplementary Tables 4 and 5), Kaplan–Meier analysis revealed a significant correlation between elevated NET levels and shorter overall survival, in both tumour tissues (Fig. 8F) and adjacent tissues (Fig. 8G). Moreover, a significant negative correlation was observed between ACC1 and NET levels (Fig. 8H).

These findings suggest that elevated CXCL8 levels in individuals with low ACC1 expression may drive ESCC metastasis by inducing NET formation, ultimately contributing to a poorer prognosis. Our study further highlights the presence of increased CXCL8 levels and elevated NETs in ACC1-low ESCC tissues. The downregulation of ACC1 appears to stimulate neutrophils to generate and release extensive NETs by upregulating CXCL8 expression, thereby facilitating metastatic progression in ESCC.

Discussion

Despite consistent tumour characteristics and treatment modalities, ESCC remains a clinical challenge because of significant variations in patient prognosis. Notably, some patients diagnosed at an early stage experience rapid metastasis, which contradicts traditional TNM staging expectations. We hypothesised that individual differences in metastatic susceptibility among patients may be driven by specific signalling molecules and pathways. Identifying such markers could refine TNM staging and enhance both diagnostic accuracy and therapeutic strategies.

In our study, we identified ACC1 as a critical determinant of ESCC metastasis by modulating the epigenetic regulation of CXCL8 and promoting NET formation. Our findings revealed a significant association between reduced ACC1 expression and earlier lymph node metastasis, as well as shorter overall survival in patients with ESCC. Moreover, we demonstrated that ACC1 knockdown enhanced ESCC cell migration and invasion through p300-mediated epigenetic pathways, leading to the up-regulation of CXCL8, which, in turn, induced EMT via autocrine and paracrine mechanisms involving NETs.

ACC1, a critical enzyme in the fatty acid synthesis pathway, has been implicated in hepatocellular carcinoma development because of its role in facilitating fatty acid synthesis in tumour cells^32–34. In breast cancer, ACC1 inhibition or knockdown has been shown to significantly promote tumour progression by enhancing histone acetylation^35–37. Previous studies have established a positive correlation between intracellular acetyl-CoA levels and histone acetylation³⁸. In our study, ACC1 knockdown enhanced histone acetylation by elevating intracellular acetyl-CoA levels (Fig. 5), which serve as acetyl donors for histone modification. Interestingly, ACC1 knockdown did not reduce intracellular fatty acid levels in ESCC cells. This may be explained by the activity of SREBF1, a key regulator of lipid metabolism that activates the transcription of genes involved in fatty acid synthesis, including FASN, thereby maintaining cellular fatty acid homoeostasis^9,10. Our results revealed that ACC1 knockdown upregulated the expression of SREBF1 and FASN, suggesting a compensatory increase in fatty acid synthesis to maintain the production of essential fatty acids required for cellular function (Fig. 5A–C). The lack of a significant change in cell proliferation following ACC1 knockdown may be attributed to this compensatory effect on fatty acid synthesis (Supplementary Fig. 1B). Furthermore, our study showed that ACC1 downregulation of ACC1 enhanced the migration and invasion of ESCC cells through increased histone acetylation. This effect was validated by using the p300 inhibitor C646 or by directly targeting p300, a key member of the histone acetyltransferase family responsible for acetylating histone H3. Although ACC1 knockdown did not alter p300 expression levels, it promoted histone acetylation in a p300-dependent manner in the presence of elevated acetyl-CoA levels. This finding is consistent with previous studies indicating that the regulation of histone acetylation by acetyl-CoA metabolism primarily depends on intracellular acetyl-CoA levels³⁹.

Transcriptome sequencing identified 208 genes that showed significantly altered expression upon ACC1 knockdown, including four CXC chemokine ligands (CXCLs): CXCL1, CXCL2, CXCL3, and CXCL8. RT-qPCR analysis confirmed the differential expression of these four CXCLs (Supplementary Fig. 1). Analysis of the TCGA database revealed that among these, only CXCL8 showed a significant correlation with oesophageal cancer progression (Supplementary Fig. 1). Therefore, CXCL8 was selected for further investigation as a downstream effector potentially involved in promoting cell migration and invasion following ACC1 knockdown.

CXCL8, secreted by cancer cells, acts in an autocrine manner by binding to G protein-coupled receptors CXCR1 and CXCR2 on the surface of cancer cells¹⁵. This interaction triggers a cascade of downstream signalling pathways, including PI3K/AKT, PLC/PKC, ERK/MAPK, and JAK, ultimately promoting cancer progression^40,41. Our study showed that ACC1 knockdown led to increased CXCL8 expression and secretion in ESCC cells (Fig. 2). The CXCL8–CXCR1/2 pathway was shown to mediate ACC1 knockdown-induced migration and invasion of ESCC cells, as confirmed by CXCL8 knockdown, CXCL8 antibody neutralisation, and CXCR1/2 inhibitor treatment (Figs. 2 and 3). Furthermore, the PI3K/AKT and MEK/ERK1/2 signalling pathways were identified as downstream targets of the CXCL8–CXCR1/2 axis, promoting ESCC cell migration and invasion following ACC1 knockdown (Supplementary Fig. 2, 3).

The CXCL8 promoter region harbours binding sites for several transcription factors, such as AP-1 and NF-κB, which are pivotal in modulating CXCL8 gene expression⁴². Based on our transcriptome sequencing data, ACC1 knockdown led to the upregulation of c-Fos, a member of the AP-1 transcription factor family known to regulate CXCL8 transcription. Elevated c-Fos levels were detected following ACC1 knockdown. Using a dual-luciferase assay, we confirmed that c-Fos directly binds to the CXCL8 promoter region. Importantly, targeting c-Fos abolished the upregulation of CXCL8 induced by ACC1 knockdown, highlighting its essential role in this regulatory pathway. RNA-seq analysis revealed significant upregulation of additional CXCL8 regulators, JunB and C/EBPβ, which are known to act through AP-1 or C/EBP motifs, respectively^15,43–46. This upregulation was confirmed by qPCR (Supplementary Fig. 5G). Although their functional roles were not directly examined in this study, their consistent induction suggests that ACC1 knockdown may engage a broader transcriptional regulatory network contributing to CXCL8 expression. Previous studies have shown that increased histone acetylation at the c-Fos promoter enhances transcription factor accessibility to DNA, thereby promoting c-Fos transcription^47,48. Histone acetyltransferases, such as p300, can acetylate histones, resulting in a more open chromatin structure⁴⁹. Consistent with these findings, our results showed that inhibiting or targeting p300 reduced the ACC1 knockdown-induced increase in c-Fos levels, suggesting that ACC1 knockdown upregulates c-Fos expression by enhancing histone acetylation.

Additionally, previous studies have shown that recruitment of p300/CBP to the CXCL8 promoter induces histone acetylation and chromatin remodelling^50,51, thereby facilitating the binding of transcription factors such as AP-1 and NF-κB. This process enhances CXCL8 transcription, highlighting the essential role of histone acetylation in regulating CXCL8 promoter activity and enabling full gene activation. Taken together, our data indicate that ACC1 knockdown promotes histone acetylation, which in turn enhances the transcriptional activation of c-Fos and increases its expression. Moreover, this histone modification may loosen the chromatin structure at the CXCL8 promoter, enabling greater c-Fos binding, and sustaining high levels of CXCL8 expression over time.

CXCL8, secreted by cancer cells, exerts its effects through both autocrine and paracrine pathways. Its receptors, CXCR1 and CXCR2, are highly expressed on neutrophils, monocytes, and macrophages within the TME. The CXC domain of CXCL8 contains an ELR motif (Glu–Leu–Arg), which plays a crucial role in attracting neutrophils to the TME⁵¹. In our study, the culture medium from ACC1 knockdown ESCC cells significantly promoted neutrophil recruitment via CXCL8, which also acts as a key chemokine in inducing NET formation (Fig. 6). Although the primary function of NETs was originally identified as trapping and killing pathogens, they have since emerged as significant contributors to cancer initiation and progression⁵². Studies have shown that NETs are abundant in liver metastases of breast and colon cancers, and serum NET levels can predict liver metastases in early-stage breast cancer⁵³. NET-derived DNA acts as a chemotactic factor, attracting cancer cells to distant sites rather than merely trapping them⁵⁴. In oesophageal cancer, elevated levels of circulating NETs are observed in patients with advanced disease^55,56. NETs formed at the primary tumour site promote ESCC metastasis⁵⁵. Moreover, elevated NET levels are associated with poor prognosis in patients with oesophageal cancer⁵⁶. However, the molecular mechanisms driving these associations in oesophageal cancer remain largely unexplored. Our findings suggest that ACC1 knockdown promotes NET formation via the CXCL8-mediated paracrine pathway, thereby facilitating ESCC metastasis.

The tumour margin, a dynamic region within the TME, plays a key role in driving aggressive tumour behaviour^57,58. In hepatocellular carcinoma, it is enriched with invasive tumour cells and macrophages that contribute to cancer progression⁵⁹. In our subcutaneous tumour-bearing model with ACC1 knockdown cells, we observed an accumulation of highly invasive tumour cells at the tumour margins, accompanied by increased neutrophil infiltration and elevated NET levels (Supplementary Fig. 7). These findings indicate strong tumour–neutrophil interactions that may promote ESCC progression. This was further supported by tail vein metastasis experiments, where ACC1 knockdown enhanced lung metastasis. Notably, the inhibition of PAD4 reversed this effect, confirming that ACC1 downregulation promotes ESCC lung metastasis through neutrophil infiltration and NET formation (Fig. 7). Despite the promising effects of PAD4i inhibition in our in vivo model, a key limitation of this study is the lack of in vivo validation using CXCL8- or CXCR2-specific inhibitors. While our in vitro findings with a CXCL8-neutralising antibody support the relevance of this signalling axis, further studies using pharmacological inhibition of CXCL8/CXCR2 in ESCC metastasis models are needed to confirm its therapeutic potential.

Clinically, ACC1 expression was negatively correlated with CXCL8 and NET in tumour tissues from patients with ESCC, suggesting a potential link between low ACC1 expression, elevated CXCL8 levels, and NET formation (Fig. 8). Future studies should focus on analysing additional tissue samples and employing multi-fluorescent staining technology to better characterise the ACC1–CXCL8–NET axis in patients with ESCC. Although preliminary clinical data from our cohort support the prognostic significance of this axis, validation in larger, independent ESCC cohorts is required. Integrating prospective clinical data with multi-omics profiling will further clarify the robustness and generalisability of this axis as a therapeutic target.

Targeting the ACC1–CXCL8–NET axis offers a promising therapeutic strategy for ESCC, offering a potential alternative or complement to existing therapies such as surgery, chemotherapy, radiotherapy, and immunotherapy. Traditional therapies are often limited by chemotherapy resistance and variable responses to immune checkpoint inhibitors^60–62. Disrupting this axis may interfere with key signals driving cancer cell migration and metastasis. Integrating this targeted approach with existing treatment strategies may enhance therapeutic efficiency and improve patient outcomes.

Our study highlights the pivotal role of reduced ACC1 expression in driving ESCC progression. ACC1 knockdown induces histone acetylation, leading to CXCL8 upregulation through c-Fos activation. This signalling cascade promotes ESCC metastasis via both autocrine and paracrine pathways, enhancing migration, invasion, and NET formation. Monitoring ACC1 expression, along with CXCL8 and NETs, may serve as a potential biomarker strategy for predicting ESCC prognosis. Furthermore, targeting CXCL8 or inhibiting NET formation represents a promising therapeutic approach to counteract ACC1 downregulation-driven ESCC progression.

Methods

ACC1 knockdown cell line construction

The lentiviral vector LV3-(H1/RFP & Puro), expressing either a negative control shRNA (shNC) or ACC1-targeting shRNA (shACC1), as well as virus packaging and preparation, were provided by GenePharma (Shanghai, China). The viral titre was 1 × 10⁸ TU mL⁻¹. For cell line establishment, 3 × 10⁵ of ESCC cells were infected with 20 µL of shNC or shACC1 virus in the presence of 5 µg mL⁻¹ polybrenne (40804ES76, Yeasen, Shanghai, China). The transduced cells were selected with 4 µg mL⁻¹ puromycin (P8230, Solarbio, Beijing, China). Additional details are provided in the Supplemental Methods.

siRNA transfection

siRNAs targeting ACC1 (siACC1), CXCL8 (siCXCL8), c-Fos (sic-Fos), p300 (siP300), as well as a negative control siRNA (siNC) were obtained from GenePharma. A total of 3 × 10⁵ cells were seeded per well in a 6-well plate, and transfection was performed using 5 µL of 20 µM siRNA and 5 µL Lipo2000 (11668019, Thermo Fisher, Waltham, MA, USA). Additional details are provided in Supplementary Methods.

Cell-conditioned medium preparation

shNC and shACC1 cells were seeded in 35-mm culture dishes at a density of 3 × 10⁵ cells and cultured overnight. The medium was then replaced with RPMI-1640 medium containing 1% foetal bovine serum (FBS), and the cells were incubated for an additional 24 h. Subsequently, the culture supernatant was collected and centrifuged to remove cell debris. The resulting supernatant, hereafter referred to as conditioned medium (CM), was either used directly in the subsequent Transwell assay or stored at −20 °C.

Transwell assays

Transwell chambers with 8-µm pore-size membranes (353097, Corning, New York, NY, USA) were used for the ESCC cell Transwell assay. A total of 1 × 10⁴ ESCC cells were seeded in the upper wells with or without Matrigel-coated membranes (356231, Corning), depending on the experimental purpose. Additionally, 650 µL of complete medium or CM collected from shNC/shACC1 cells was added to the lower chamber. Additional details and the neutrophil Transwell assay are provided in the Supplementary Methods.

Western blot

Cells were collected and washed with cold PBS. For total protein extraction, cells were lysed in RIPA buffer (AR0105, Beyotime, Shanghai, China) supplemented with 1% protease inhibitor (AR1182, Boster, Wuhan, China), 1% phenylmethanesulfonyl fluoride (AR1192, Boster, Wuhan, China), and 5% phosphatase stop (4906845001, Roche, Shanghai, China). The lysates were centrifuged at 10,000 rpm for 15 min at 4 °C. Protein concentration was quantified using the BCA Protein Assay Kit (BCA02, Ding Guo, Beijing, China). Equal amounts of total protein (20 μg per sample) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 2 h and transferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for 2 h. Subsequently, the PVDF membranes were blocked with 5% fat-free milk in TBST for 1 h at room temperature to prevent nonspecific binding. After overnight incubation with specific primary antibodies, the membranes were washed with 1% Tris-buffered saline Tween-20 (TBST) for 30 min. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies–anti-Mouse HRP (bs-0296G-HRP, Bioss, Beijing, China) or anti-Rabbit HRP (bs-0295G-HRP, Bioss, Beijing, China)–for 1 h at room temperature. Detection was performed using a chemiluminescence imaging system (Amersham Imager 600, New York, NY, USA). β-actin was used as an internal control. Antibody details are provided in the Supplementary Methods.

For Western blot analyses, representative images from independent experiments are shown. Slight variations in band intensities across different figures may have occurred because of differences in exposure times or imaging conditions during blot development. All comparisons within a single experiment were made using blots that were processed and imaged under identical conditions. The original uncropped blots are provided in the Supplementary Information.

cDNA preparation and quantitative real-time PCR

Total RNA was extracted from each sample using TRIzol reagent (343911, Solarbio). For RNA extraction from patient samples, tissues from 98 patients with ESCC were used, and patient characteristics are summarised in Supplementary Tables 1 and 3. cDNA synthesis was performed using All-In-One MasterMix (G492, abm, Vancouver, Canada) on an Applied Biosystems^TM Veriti^TM 96 thermal cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: 25 °C for 10 min, 42 °C for 15 min, and 85 °C for 5 min. Specific products of ACC1, CXCL8, c-Fos, and GAPDH were amplified using Blastaq^TM 2× qPCR MasterMix (G891, abm). GAPDH served as a normalisation control. Quantitative real-time PCR (qPCR) was performed in a QuantStudio™ Dx Real-Time PCR Instrument (Applied Biosystems) using the following thermal cycling conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 35 s. Ct values were recorded during the exponential amplification phase. Relative gene expression level (fold change) was calculated using the 2^−ΔΔCt method and normalised to the internal control. Additional details are available in Supplementary Methods.

Chromatin immunoprecipitation (ChIP)

ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (9003, Cell Signalling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. KYSE-450 shACC1 cells were seeded in 8 cm culture dishes at a density of 1 × 10⁶ cells to reach approximately 80% − 90% confluency (4 × 10⁶ cells) at the time of collection. Before starting, the protease inhibitor cocktail (PIC) and glycine were thawed, and PBS containing PIC and formaldehyde was prepared for cross-linking. Formaldehyde (final concentration, 1%) was used to cross-link proteins to DNA, followed by quenching with glycine and washing with PBS. For nuclei preparation and chromatin digestion, cells were resuspended in buffer A containing DTT and PIC, centrifuged, and digested with 0.25 µM Micrococcal Nuclease. After stopping the reaction with EDTA, the nuclei were lysed by sonication using an ultrasonic cell disruptor (JY92-IIN, Scientz, Ningbo, China) with three 20 s pulses at 20% power, interspersed with 30 s pauses. Chromatin was clarified via centrifugation and stored at −80 °C. For immunoprecipitation, 10 µL of c-Fos antibody (2250, Cell Signalling Technology) was incubated with Protein G magnetic beads, with anti-rabbit IgG used as a negative control. After washing, DNA was quantified by qPCR (G891, abm), and results were expressed as fold changes. Primer details are provided in the Supplementary Methods.

Animal models

Female BALB/c nude mice weighing 14–18 g were purchased from Vital River (Beijing, China). The mice were acclimatised under standard conditions for 7 days before the experiment and were then randomly assigned to different groups. Animals were randomly selected from different cages for testing. All procedures involving animal care were approved by the Research Ethics Committee of the First Affiliated Hospital of Henan Medical University (permit number: EC-023-458). In accordance with the Committee’s guidelines, the maximal tumour diameter was not permitted to exceed 1.5 cm at any point during the study, and this limit was strictly adhered to in all experiments. We have complied with all relevant ethical regulations for animal use. Additional information is provided in Supplementary Methods.

TMA and immunofluorescence

A total of 97 ESCC cases were selected for the construction of a TMA, which was prepared by Outdo Biotech Co., Ltd. (Shanghai, China). After excluding samples with tissue loss during TMA preparation or staining, 91 samples were analysed. All tissue samples were obtained from The First Affiliated Hospital of Henan Medical University, and none of the recruited patients received preoperative treatments. Patient characteristics are summarised in Supplementary Tables 2 and 4. Authorisation for the use of ESCC samples was obtained from the Ethics Committee of the First Affiliated Hospital of Henan Medical University (permit number: EC-023-457), and informed consent was obtained from all participants. All ethical regulations relevant to human research participants were followed. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed according to standard protocols. The antibodies used in this study included Anti-Cit-H3 (1:1000, ab5103, Abcam, Cambridge, UK) and anti-MPO (1:1000, AF3667, R&D Systems, Minneapolis, MN, USA). After TMA staining, samples were scanned using the Digital Pathology Slide Scanner (Pannoramic MIDI, Budapest, Hungary) by Servicebio (Wuhan, China) for high-resolution imaging. Aipathwell® software was used to analyse the immunofluorescence colocalisation positive area ratio to derive IF scores. The colocalisation positive area ratio was defined as the area of overlapping pixels (yellow, where red and green fluorescence signals coincide) divided by the sum of red- and green-positive areas, minus the overlapping area.

Statistics and reproducibility

Statistical analyses were conducted using GraphPad Prism 8.0, with error bars representing either the standard error of the mean (SEM) or the standard deviation (SD). Two-tailed Student’s t-tests (for normally distributed data) or Mann−Whitney tests (for non-normally distributed data or paired comparisons) were used to assess differences between two groups, while one-way analysis of variance (ANOVA) was applied for comparisons among multiple groups. The homogeneity of variances was evaluated before conducting parametric tests. When the assumption of equal variances was not met, appropriate non-parametric tests were applied. Survival curves were generated using the KM method and compared using the log-rank test. The association between ACC1 and CXCL8 expression levels, neutrophil infiltration, and clinical features were analysed using the chi-squared test. Correlation analyses were performed using Spearman’s rank correlation method. A significance level of P < 0.05 was applied, and the differences meeting this threshold were considered statistically significant. To ensure reproducibility, all experiments were designed with adequate sample sizes to achieve sufficient statistical power. The number of replicates for each experiment is specified in the relevant figure legends and corresponding sections of the manuscript. For in vitro experiments, replicates were defined as independent experiments conducted on different days using separate cell cultures. For in vivo studies, replicates were based on the number of animals used in each experimental group. The data presented are representative of at least three independent experiments, unless otherwise stated. Specific sample sizes and replicate details for each experiment are provided in the corresponding Methods subsections.

Ethics approval and consent to participate

All animal care and experimental procedures were approved by the Institutional Research Ethics Committee of The First Affiliated Hospital of Henan Medical University (Permit number: EC-023-458). Authorisation for the use of ESCC samples in this study was obtained from the Committees for Ethical Review of Research at The First Affiliated Hospital of Henan Medical University (Permit number: EC-023-457). Informed consent was obtained from all participants. All procedures involving human tissues were conducted in accordance with institutional and national ethical standards.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

Jiaping Tang: data curation, formal analysis, funding acquisition, investigation, methodology, resources, software, validation, visualisation, writing—original draft, and writing—review and editing. Qi Bo: conceptualisation, data curation, formal analysis, investigation, methodology, validation, visualisation, and writing—review and editing. Qingya Zhuo: data curation, formal analysis, investigation, methodology, resources, software, validation, and visualisation. Shuhua Huo: data curation, formal analysis, and investigation. Xinchen Dang: data curation, formal analysis, and investigation. Tianbao Pang: data curation, formal analysis, and investigation. Kexin Cao: data curation, formal analysis, and investigation. Yuzhen Liu: conceptualisation, project administration, supervision, visualisation, writing—original draft, and writing—review and editing. Baosheng Zhao: conceptualisation, funding acquisition, project administration, supervision, and writing—review and editing.

Peer review

Peer review information

Communications Biology thanks Wang-Kai Fang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Johannes Stortz. A peer review file is available.

Data availability

The authors affirm that all data underpinning the conclusions of this study are accessible within the article and its Supplementary Information files. The raw sequencing data have been deposited in the Genome Sequence Archive for Human (GSA-Human) at the National Genomics Data Center with the accession number HRA013632⁶³, and the numerical source data for graphs and charts are provided in the Supplementary Data file. Additionally, the experimental data can be obtained from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Consent for publication

Informed consent was obtained from each patient for the use of their oesophageal cancer tumour tissue in the experimental research described in this manuscript. Patients were fully informed about the nature of the study, the use of their tissue samples for scientific research, and the potential for publication of the research findings, including any accompanying images or data. The consent process included an explanation of how the tumour tissue would be used in the experiments and the importance of the research in advancing the understanding and treatment of oesophageal cancer.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-09020-9.