LEF1/NFE2L3/TACC1 axis activates the AKT-mTOR pathway to promote the progression of esophageal squamous cell carcinoma

Introduction

Esophageal cancer is the ninth most common cancer and the fifth leading cause of cancer-related death in China (1). Esophageal squamous cell carcinoma (ESCC) accounts for over 90% of all histologic subtypes of esophageal cancer in China (2). Due to its high recurrence rate, extensive local invasion, and frequent metastasis, the overall 5-year survival rate for ESCC remains dismal, at less than 20% (3). Therefore, novel therapeutic targets urgently need to be identified to improve ESCC treatment outcomes.

Epithelial-mesenchymal transition (EMT) is a biological process whereby epithelial cells acquire mesenchymal characteristics. It plays essential roles in various physiological and pathological processes, including tissue remodeling, wound healing, and tumorigenesis (4). EMT is characterized by the loss of epithelial traits, including apical-basal polarity and intercellular junctions, together with the acquisition of mesenchymal features such as increased migratory capacity and extracellular matrix degradation. These changes enhance the invasiveness and metastatic potential of carcinoma cells (5,6). The molecular mechanisms governing the initiation and progression of EMT in ESCC have not yet been elucidated.

Lymphoid enhancer-binding factor 1 (LEF1) is a member of the high-mobility group transcription factor family and plays a central role in the Wnt/β-catenin signaling pathway (7). Our previous study demonstrated that LEF1 upregulation is associated with abnormal clinicopathological characteristics and a poor prognosis in patients with ESCC (8). LEF1 is highly expressed in ESCC, where it promotes cellular invasion, migration, and EMT through cooperation with the OCT4 transcription factor. Further, our study revealed that LEF1 facilitates ESCC tumorigenesis, a cancer stem cell (CSC)-like phenotype, and chemoresistance both in vitro and in vivo by activating the TGF-β signaling pathway (9). However, the downstream regulatory mechanisms underlying the oncogenic role of LEF1 remain unclear and warrant further investigation.

Nuclear factor erythroid 2-like 3 (NFE2L3) belongs to the Cap ‘n’ Collar basic region leucine zipper family of transcription factors. A recent study has shown that NFE2L3 participates in diverse biological processes, including cell differentiation, inflammatory responses, oxidative stress, lipid homeostasis, transcriptional regulation, immune responses, and tumor growth (10). NFE2L3 has been identified as a critical regulator of the development and prognosis of several cancers. It is overexpressed in clear cell renal cell carcinoma, hepatocellular carcinoma, and bladder cancer, where its dysregulation is closely associated with tumor progression and poor prognosis (11-13). NFE2L3 also promotes inflammation and the development of colitis-associated colorectal cancer (14). Our recent findings revealed that the methyltransferase like 3 (METTL3)-mediated methylation of NFE2L3 activates the Wnt/β-catenin signaling pathway, thereby promoting tumorigenesis and chemoresistance in lung adenocarcinoma (15). However, research on the role of NFE2L3 in ESCC is limited.

Transforming acidic coiled-coil-containing protein 1 (TACC1) is a member of the TACC protein family, characterized by a conserved C-terminal coiled-coil domain (the TACC domain). TACC1 is functionally linked to transcription, translation, and centrosome dynamics through its interactions with multiple protein complexes (16,17). The dysregulation of TACC1 has been implicated in the development of several malignancies, including rhabdomyosarcoma, breast cancer, gastric cancer, head and neck squamous cell carcinoma, and ovarian cancer (18-22).

Previous studies have primarily explored the individual roles of LEF1 or NFE2L3 (8,9,14,15); however, their mutual regulatory mechanisms and the integrated function of the downstream TACC1-PARP1-AKT/mTOR signaling axis in ESCC have not yet been elucidated. This study was the first to demonstrate that LEF1 directly regulates the transcription of NFE2L3. Our findings highlight the pivotal role of the LEF1/NFE2L3/TACC1/PARP1 axis in ESCC progression and suggest that it may serve as a promising therapeutic target for this malignancy. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0789/rc).

Methods

Clinical tissue and specimens

Ten fresh ESCC tissue samples were collected during surgical resection at The First Affiliated Hospital of Naval Medical University (Shanghai, China). Seventy-three paraffin-embedded specimens were obtained from patients diagnosed with primary ESCC who underwent esophagectomy between 2024 and 2025 at Changhai Hospital. None of the patients had received preoperative chemotherapy or radiotherapy. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Naval Medical University (No. CHEC2025-306), and all patients provided written informed consent.

Single-cell RNA-sequencing (scRNA-seq) analysis

scRNA-seq data from the Gene Expression Omnibus (GEO) database (GSE273127) were analyzed using the ‘Seurat’ R package, which provides a comprehensive suite for single-cell data quality control, normalization, and clustering. We retained genes expressed in at least three cells, and excluded cells with fewer than 200 or more than 5,000 genes, cells with an RNA count (nCount_SNA) below 300, cells with a mitochondrial percentage (pMT) exceeding 15%, and cells with a hemoglobin percentage (pHB) greater than 1%. After filtering, a total of 54,894 cells remained. The ‘DoubletFinder’ R package was used to identify and remove doublets. Data normalization was performed using the NormalizeData function, and the FindVariableFeatures function was applied to identify 2,000 highly variable genes. The ScaleData function was used to scale the data.

Principal component analysis (PCA) was employed for dimensionality reduction. To integrate multiple samples and remove batch effects, we applied the ‘Harmony’ R package, which uses an iterative clustering and correction algorithm to align datasets in a shared low-dimensional space, enabling robust downstream analysis. Following batch-effect correction, t-distributed stochastic neighbor embedding (t-SNE) was used for visualization, resulting in the identification of seven clusters. Cell clusters were annotated based on known marker genes using the ‘SingleR’ R package, which automatically assigns cell identities by comparing transcriptomic profiles with reference datasets. The malignancy level of each cluster was inferred using the ‘CopyKAT’ R package, which predicts copy number variations (CNVs) from single-cell transcriptomic data to distinguish malignant from non-malignant cells; non-diploid cells were removed. Pseudo-temporal analysis was performed using the ‘Slingshot’ R package, which infers developmental trajectories by constructing lineage graphs and ordering cells along continuous paths. Stemness markers [sex determining region Y-box 2 (SOX2) and cluster of differentiation 44 (CD44)] were compared with NFE2L3 expression using CytoTRACE analysis, a computational method that predicts cellular differentiation potential based on gene count features.

RNA-seq analysis

RNA-seq data from the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn) were used to compare the differential expression of the NFE2L3 gene in tumor and normal tissues. A differential expression analysis was performed using a cutoff of P<0.05 and log₂ fold change >1. NFE2L3 expression was also evaluated in the GEO dataset (GSE161533) to corroborate the findings. Differentially expressed genes (DEGs) were identified in the GEO dataset using the “limma” R package. TACC1 was selected for further analysis after intersecting the downregulated genes.

The expression data from 33 tumor types from The Cancer Genome Atlas (TCGA) database were analyzed, and the differential expression of TACC1 between tumors and normal tissues was assessed using the Wilcoxon rank-sum test. A gene set enrichment analysis was conducted using the “ClusterProfile” R package to investigate the biological processes associated with low versus high TACC1 expression in esophageal cancer. Spearman correlation analyses were performed to examine the relationships between TACC1 expression and immune cell infiltration, tumor immune checkpoints, and tumor mutational burden.

Cell culture

Human ESCC cell lines (Eca109, TE-1) and normal human esophageal epithelial cells (Het-1A) were obtained from the Shanghai Cell Bank (Shanghai, China). All the cell lines were cultured in Dulbecco’s Modified Eagle Medium (Gibco, 11995040, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco, 10099141, Grand Island, USA) and maintained at 37 ℃ in a humidified incubator with 5% CO₂.

siRNA, transfection, and lentivirus infection

Lentiviral vectors expressing LEF1 or NFE2L3 (ov-LEF1, ov-NFE2L3) and shRNA targeting NFE2L3 (sh-NFE2L3) were designed by HeYuan Biotechnology Company (Shanghai, China). TACC1 knockdown siRNA and negative control plasmids were transfected using Lipofectamine 2000 (GLPBIO, GK20005, Monterey Park, USA). Cells were harvested for RNA analysis two days after transfection and for protein analysis three days post-transfection.

Chromatin immunoprecipitation (ChIP) seq

ChIP assays were conducted using the SimpleChIP^® Enzymatic ChIP Kit (CST, 9003, Danvers, USA). Briefly, 4×10⁶ ESCC cells were cross-linked with 1% formaldehyde. Chromatin was digested with 0.5 µL of micrococcal nuclease per 4×10⁶ cells. Immunoprecipitation was performed using 10 µL of LEF1 antibody, 1 µL of normal rabbit immunoglobulin G, and 10 µL of histone H3 rabbit mAb (CST, 4620, Danvers, USA) at 4 ℃ overnight. Chromatin was eluted from the antibody-protein G magnetic beads, and cross-links were reversed. DNA was then amplified by polymerase chain reaction using specific primers for NFE2L3 [5'-TGCTGGGGAGGTGCTATT-3' (F), and 5'-TCCTTATCCGTTGTTTCCTTGAG-3' (R)].

Cell proliferation and colony formation assays

Cell proliferation was measured using the Cell Counting Kit-8 (Dojindo, Japan, TL616). ESCC cells (5,000 cells/well) were seeded in 96-well plates and cultured for four days. Absorbance was measured at 450 nm. For colony formation assays, cells were seeded in 6-well plates and incubated for 14 days. Colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Colonies containing ≥50 cells were counted.

Wound healing assays

ESCC cells were seeded at a density of 5×10⁵ cells/well in 6-well plates. When the cells reached near 100% confluence, a 200-µL sterile pipette tip was used to create wounds. The cells were cultured in medium containing 2% serum. The wound area was observed and photographed at 0 and 24 hours using an inverted phase contrast microscope. Wound surface areas were quantified using ImageJ software.

Migration and invasion assays

A total of 1×10⁵ ESCC cells in 200 µL of serum-free medium were seeded into the upper Transwell chamber (8.0 µm pore, Corning, Corning, USA) pre-coated with Matrigel. The lower chamber contained 500 µL of culture medium with 20% serum. After 24 hours, migrated/invaded cells in the lower chamber were fixed with 4% paraformaldehyde for 30 minutes and stained with 1% crystal violet for 30 minutes. The cells were counted using ImageJ software.

Flow cytometric apoptosis analysis

Apoptosis was assessed using an Annexin V-FITC/PI apoptosis kit (KenGEN, KGA1101-20, Beijing, China). ESCC cells (5×10⁵) were seeded in 6-well plates overnight, digested with EDTA-free trypsin (Beyotime, C0205, Shanghai, China), and resuspended in 500 µL of binding buffer. The cells were incubated with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) for 30 minutes at room temperature. Flow cytometry was performed to analyze apoptosis.

Immunofluorescence

The cells were cultured in 12-well glass chambers, washed with phosphate buffered saline (PBS), and fixed in 4% paraformaldehyde for 30 minutes at room temperature. The cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, T8532, St. Louis, USA) for 20 minutes at room temperature, washed with PBS, and blocked with 2% bovine serum albumin (Sigma-Aldrich, A4503, St. Louis, USA) for 1 hour at 37 ℃. The cells were incubated with primary antibodies overnight at 4 ℃ and washed in PBS before incubation with fluorophore-conjugated secondary antibodies for 2 hours at room temperature. Nuclei were stained with DAPI (Beyotime, C1006, Shanghai, China), and images were analyzed using ImageJ software.

Immunohistochemistry (IHC)

The tumor tissues were preserved in 4% paraformaldehyde, and then dehydrated and embedded in paraffin. Endogenous peroxidases were deactivated with 3% hydrogen peroxide, and non-specific binding was blocked using 5% bovine serum albumin for 30 minutes. The tumor samples were incubated overnight with primary antibodies against LEF1 (Abcam, ab137872, Cambridge, UK), NFE2L3 (Merck, HPA055889, Darmstadt, Germany), and TACC1 (Proteintech, 13862-1-AP, Wuhan, China). After washing, secondary antibodies were applied for 1 hour at room temperature, followed by staining with DAB (Beyotime, P0203, Shanghai, China) substrate. IHC staining intensity and positivity were quantified using a 5-point scale.

Immunopurification, MS, and silver staining

Immunoprecipitation was conducted using the Pierce Classic Magnetic IP/co-immunoprecipitation (co-IP) Kit (ThermoFisher, 88804, Waltham, USA). Protein was extracted using immunoprecipitation lysis buffer, incubated with specific primary antibodies overnight at 4 ℃ with rotation, and incubated with protein A/G beads for 1 hour at room temperature. Immunoprecipitates were washed, denatured in boiling loading buffer (Beyotime, P0015L, Shanghai, China), and visualized by silver staining. Mass spectrometry (MS) was performed by SpecAlly Life Technology C (Wuhan, China).

Western blot and co-IP

Cell or tissue lysates were prepared using SDS lysis buffer containing protease and phosphatase inhibitors. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4 ℃, followed by incubation with secondary antibodies. Protein bands were visualized by chemiluminescence and quantified using ImageJ software. Co-IP assays were performed using similar methods, with immunoprecipitation confirmed by Western blotting.

Animal models and in vivo tumor formation assay

Ten 6-week-old male BALB/c nude mice (purchased from Shanghai Meibo Biotechnology Co., Ltd.) were randomly assigned to the control group (ov-NC Eca109) and the overexpressing NFE2L3 (ov-NFE2L3) Eca109 group, with 5 mice in each group. All mice were housed in specific pathogen-free (SPF) conditions with controlled temperature and humidity, adequate ventilation, and free access to food and water. Eca109 cells stably overexpressing NFE2L3 (ov-NFE2L3) and control cells (ov-NC) were harvested during the logarithmic growth phase at 80–90% confluence. Single-cell suspensions were prepared, and the cell concentration was adjusted to 8×10⁷ cells/mL. The cell suspension was mixed with Matrigel at a 1:1 volume ratio at 4 ℃ to obtain a final concentration of 4×10⁷ cells/mL. The mixture was subcutaneously injected into the left dorsal flank of each mouse, and tumor growth was monitored continuously for 25–35 days. Tumor volume was measured weekly and calculated using the formula: volume = (length × width²)/2. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology Co., Ltd (No. YS-JL-001), in compliance with the institutional guidelines for the care and use of animals.

Statistical analysis

The statistical analysis was performed using GraphPad 9. The data are presented as the fold changes or percentages relative to controls, with standard deviations from three independent experiments. The Student’s t-test was used for normally distributed data, and the Chi-squared test was used to assess correlations in IHC staining. A P value <0.05 was considered statistically significant, with significance levels indicated as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Results

LEF1 binds to the promoter of NFE2L3, upregulating its expression

The RNA-seq data analysis revealed a significant increase in NFE2L3 expression following the overexpression of LEF1. The correlation between LEF1 and NFE2L3 expression was confirmed in tissue samples from 73 ESCC patients. Immunohistochemical consecutive sections revealed a close correlation between LEF1 and NFE2L3 expression (Figure 1A). Among the patients with high LEF1 expression, 73% exhibited concurrent high expression of NFE2L3 (Table 1).

Figure 1 LEF1 directly binds to the NFE2L3 promoter. (A) IHC shows a positive correlation between LEF1 and NFE2L3 expression in tumor tissues from ESCC patients. (B) IF demonstrates the co-localization of LEF1 and NFE2L3 in the nucleus of Eca109 cells. (C) Analysis of NFE2L3 mRNA levels in esophageal carcinoma using the GEPIA and GEO databases. (D) NFE2L3 protein expression is elevated in tumor tissues. (E) LEF1 expression is positively correlated with NFE2L3 levels at the cellular level. (F) Representative image of histological staining with IHC of high expression of LEF1 and NFE2L3 in tumor tissues of patients, compared to low expression in adjacent non-tumor tissues. (G) ChIP assay assessing the binding of LEF1 to the NFE2L3 sequence in Eca109 cells. *, P<0.05; **, P<0.01; ***, P<0.001. ChIP, chromatin immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; ESCA, esophageal carcinoma; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; IF, immunofluorescence; IgG, immunoglobulin G; IHC, immunohistochemistry; LEF1, lymphoid enhancer-binding factor 1; NFE2L3, nuclear factor erythroid 2-like 3.

The relationship between NFE2L3 expression and clinicopathological features in ESCC was then examined. The proportion of NFE2L3-positive cells was significantly higher in the ESCC tumor tissues than in the adjacent normal tissues (P<0.01) (Table 2). Additionally, patients with high NFE2L3 expression had poorer histological differentiation (P=0.04), a more advanced pathological T stage (P<0.001), and a higher tumor-node-metastasis (TNM) stage (P<0.001). NFE2L3 expression in the GEPIA and GEO datasets (GSE161533) confirmed that NFE2L3 was upregulated in the ESCC tissues compared to the normal tissues (Figure 1B).

To further verify these findings, we analyzed NFE2L3 expression in patient tumor samples, ESCC cell lines (Eca109 and TE-1), and normal esophageal epithelial cells (Het-1A) by Western blot. The results showed that NFE2L3 was highly expressed in tumor tissues, with significantly increased expression levels in tumor cell lines compared to normal cells (Figure 1C,1D), a finding that was also corroborated by IHC (Figure 1E). Further, immunofluorescence staining revealed that NFE2L3 and LEF1 colocalized in the nucleus (Figure 1F), suggesting a potential regulatory interaction between the two proteins.

Given that LEF1 can regulate gene expression as a transcription factor, we predicted that the promoter region of the NFE2L3 gene contains potential binding sites for LEF1. We verified this through ChIP-qPCR. The experimental results were consistent with our hypothesis (Figure 1G). Therefore, NFE2L3 was confirmed as a target gene of LEF1 and was selected for further investigation.

NFE2L3 as a potential stemness marker in ESCC

To analyze the role of NFE2L3 in ESCC, a scRNA-seq analysis was performed on nine ESCC samples from the GEO dataset (GSE273127). Through unsupervised clustering along with canonical marker genes for various cell types (23) (Figure 2A), 54,894 cells were divided into seven clusters (Figure 2B), including epithelial cells, fibroblasts, endothelial cells, T cells, B cells, myeloid cells, and mast cells. Epithelial cells were classified as malignant or normal based on chromosomal CNVs predicted by CopyKAT. Malignant cells exhibited a higher frequency of CNVs compared to normal cells (Figure 2C). Four malignant cell clusters were separated from the epithelial cell cluster using the subset function and unsupervised clustering. Cluster 0 was selected as the main tumor cell cluster, which was further analyzed to delineate its subcluster distribution (Figure 2D).

Figure 2 NFE2L3 serves as a potential stemness marker in esophageal squamous cell carcinoma. (A) t-SNE plot depicting the landscape of ESCC cells and the tumor microenvironment. (B) Bubble plot showing the specific marker genes for each cell cluster. (C) CopyKat analysis discriminated between diploid and aneuploid cells, with aneuploid cells defined as malignant tumor cells. (D) UMAP visualization of the major tumor cell cluster (Cluster 0) following re-clustering after extraction. (E) CytoTRACE analysis predicting the stemness score of NFE2L3(+) tumor cells. (F) UMAP plot showing the distribution of NFE2L3(+) and NFE2L3(−) tumor cells, alongside tumor stemness, across tumor subpopulations. (G) Expression distribution of common stem cell marker genes within the tumor subpopulations. (H) Slingshot analysis (red to blue indicates the start to the end of differentiation) positioned NFE2L3(+) tumor cells at the starting point of the differentiation trajectory. ESCC, esophageal squamous cell carcinoma; NFE2L3, nuclear factor erythroid 2-like 3; tSNE, t-distributed stochastic neighbor embedding; UMAP, Uniform Manifold Approximation and Projection.

To investigate the role of NFE2L3, the main tumor cell cluster was divided into NFE2L3⁺ and NFE2L3⁻ tumor cells based on NFE2L3 expression. A CytoTRACE analysis was performed to assess the differentiation potential of NFE2L3⁺ tumor cells, and a pseudotime trajectory analysis of the tumor cell subclusters was conducted using Slingshot. The results showed that NFE2L3⁺ tumor cells had a higher differentiation potential, and NFE2L3 expression decreased during pseudotime progression (Figure 2E-2G). We also analyzed the distribution of tumor stemness markers, such as sex determining region Y-box 2 (SOX2) and CD44 (24), and observed significant overlap with NFE2L3 expression, suggesting its potential as a tumor stemness marker (Figure 2H).

NFE2L3 promotes the proliferation, migration, invasion, and EMT progression of ESCC cells in vitro

CSCs are closely linked to the occurrence of EMT (25). To clarify the role of NFE2L3 in ESCC progression, we knocked down NFE2L3 (sh-NFE2L3) and overexpressed NFE2L3 (ov-NFE2L3) in the Eca109 and TE-1 cells. The knockdown of NFE2L3 significantly inhibited the proliferation and colony formation ability of both the Eca109 and TE-1 cells (Figure 3A,3B). The Transwell and wound healing assays showed that NFE2L3 knockdown significantly impaired cell migration and invasion ability (Figure 3C and Figure S1).

Figure 3 NFE2L3 promotes proliferation, migration, invasion, and EMT in human ESCC cells. (A) CCK-8 assay indicated that NFE2L3 overexpression increased the proliferation rate of Eca109 and TE1 cells, while its knockdown reduced it. (B) Colony formation assay with crystal violet staining showed that NFE2L3 overexpression enhanced, while its knockdown suppressed, the proliferation of Eca109 and TE1 cells in 6-well plates (whole-well view). (C) Crystal violet staining of Transwell assays demonstrated that NFE2L3 overexpression promoted, while its knockdown inhibited, the migration and invasion of ESCC cells in vitro (scale bar, 100 µm). (D) NFE2L3 overexpression upregulated N-cadherin and downregulated E-cadherin expression in ESCC cells, and NFE2L3 knockdown produced the opposite effect. (E,F) Crystal violet staining of rescue experiments illustrated the regulation of NFE2L3 by LEF1 (scale bar, 100 µm). *, P<0.05; **, P<0.01. ESCC, esophageal squamous cell carcinoma; LEF1, lymphoid enhancer-binding factor 1; NC, negative control; NFE2L3, nuclear factor erythroid 2-like 3; OD, optical density; ov, overexpression.

Further, the Western blot analysis revealed that silencing NFE2L3 reduced the expression of the mesenchymal marker N-cadherin and increased the expression of the epithelial marker E-cadherin (Figure 3D). Conversely, the overexpression of NFE2L3 promoted the proliferation and colony formation of both the Eca109 and TE-1 cells (Figure 3A,3B). NFE2L3 overexpression significantly increased cell migration and invasion (Figure 3C and Figure S1). The Western blot analysis showed that NFE2L3 overexpression increased N-cadherin expression while decreasing E-cadherin expression (Figure 3D). These data indicate that NFE2L3 activation promotes the proliferation, migration, invasion, and EMT progression of ESCC cells.

Subsequently, we conducted a rescue experiment to determine whether NFE2L3 could reverse the effects of LEF1 on the proliferation, migration, and invasion of ESCC. We found that the knockdown of NFE2L3 in ESCC and TE-1 cells overexpressing LEF1 reversed the pro-proliferative, pro-migratory, and pro-invasive effects of LEF1 on the ESCC cells (Figure 3E,3F). These results indicate that NFE2L3 mediates the pro-proliferative, pro-migratory, and pro-invasive effects of LEF1 on ESCC cells, as well as the occurrence of EMT.

NFE2L3 promotes tumor growth in vivo

To examine the role of NFE2L3 in ESCC growth in vivo, 10 BALB/c nude mice were randomly assigned to the ov-NC Eca109 group (control group) and the ov-NFE2L3 Eca109 group, with 5 mice in each group. Tumor volume was measured weekly. The results showed that tumor growth, volume, and weight were significantly increased in the ov-NFE2L3 group, compared with the control group (Figure 4A,4B).

Figure 4 NFE2L3 promotes the tumorigenicity of ESCC cells in vivo. (A,B) Tumor growth was promoted in vivo by cells overexpressing NFE2L3 (ov-NFE2L3). (C,D) IHC staining showed that NFE2L3 expression remained consistently high within the mouse tumors formed by Eca109 cells overexpressing ov-NFE2L3. *, P<0.05; **, P<0.01. ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; NC, negative control; NFE2L3, nuclear factor erythroid 2-like 3; ov, overexpression.

On day 28, tumor tissues were excised, and the NFE2L3 expression in the tumor tissues of each group was analyzed via Western blot and IHC. The expression level of NFE2L3 and the number of NFE2L3-positive cells were significantly higher in the tumor tissues of the ov-NFE2L3 group, compared with the control group (Figure 4C,4D). These findings suggest that NFE2L3 promotes the growth of ESCC cells in vivo.

TACC1 expression is associated with the immunosuppressive tumor microenvironment in esophageal cancer

To identify the target genes of NFE2L3, we analyzed gene expression changes using RNA-seq data from NFE2L3 knockdown cells (GEO accession: GSE226941) (Figure 5A). By intersecting the downregulated DEGs from the aforementioned dataset, we identified nine genes co-downregulated with NFE2L3, including PER1, ETV1, GSKIP, and TACC1 (Figure 5B).

Figure 5 TACC1 acts as a downstream effector of NFE2L3 and is associated with the formation of an immunosuppressive tumor microenvironment. (A) Volcano plots of differentially expressed genes from the GEO database upon NFE2L3 knockdown. (B) Genes such as TACC1, PER1, and ETV1 were co-downregulated with NFE2L3. (C) TACC1 mRNA expression in tumor versus normal tissues from the TCGA database. (D) Pan-cancer analysis of the association between TACC1 and 22 types of immune cells. (E) TACC1 inhibits the epithelial phenotype pathway in ESCA. (F) Pan-cancer analysis of the association between TACC1 and immune checkpoints. (G) Pan-cancer analysis of the association between TACC1 and TMB. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ESCA, esophageal carcinoma; FC, fold change; GEO, Gene Expression Omnibus; NK, natural killer; TACC1, transforming acidic coiled-coil-containing protein 1; TCGA, The Cancer Genome Atlas; TMB, tumor mutational burden.

Given that NFE2L3 promotes EMT and TACC1 is negatively correlated with epithelial cell phenotypes such as epidermal cell differentiation and keratinocyte differentiation (Figure 5C), we selected TACC1 as a target gene of NFE2L3. We analyzed the role of TACC1 using TCGA database. TACC1 was upregulated in digestive system tumors, including esophageal carcinoma (ESCA), cholangiocarcinoma (CHOL), and liver hepatocellular carcinoma (LIHC) (Figure 5D). Compared with paracancerous tissue, TACC1 expression was increased in LIHC and ESCA (Figure S2).

Immunotherapy is currently used to treat solid tumors. Patients who respond to immunotherapy have higher long-term survival rates and fewer adverse effects than those receiving traditional drugs (26). However, only a minority of patients benefit from this approach. Therefore, we analyzed the impact of TACC1 on immune cells.

The Spearman correlation analysis revealed that in ESCA with high TACC1 expression, CD8⁺ T cell infiltration was decreased, while pro-tumorigenic M2 macrophage infiltration was increased (Figure 5E), and the expression of immune checkpoint molecules programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immunoglobulin and ITIM domain (TIGIT), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) was elevated (Figure 5F). These results suggest that TACC1 expression in esophageal cancer regulates the infiltration of immunosuppressive cells, contributing to an immunosuppressive tumor microenvironment. Additionally, TACC1 showed a negative correlation with tumor mutational burden in nine cancer types, including ESCA, lung adenocarcinoma, and stomach adenocarcinoma (STAD) (Figure 5G), indicating a poor response to immunotherapy in esophageal cancer and potential effects on tumor prognosis and immunotherapy efficacy. Thus, TACC1 plays a pivotal role in the progression of esophageal cancer.

NFE2L3 promotes TACC1 expression

To clarify the relationship between NFE2L3 and TACC1, we performed a Western blot analysis on Eca109 cells with NFE2L3 knockdown, and observed that TACC1 expression was significantly reduced following NFE2L3 knockdown (Figure 6A). The relationship between NFE2L3 and TACC1 expression was further validated in tumor specimens from 20 patients. Immunohistochemical consecutive sections revealed a strong correlation between NFE2L3 and TACC1 expression (Figure 6B), with 11 tumor tissues co-expressing high levels of NFE2L3 and TACC1, and three tumor tissues co-expressing low levels of both proteins (Table 3).

Figure 6 NFE2L3 promotes TACC1 expression, and TACC1 binds PARP1 to activate the AKT-mTOR pathway. (A) TACC1 expression decreased after knockdown of NFE2L3. (B) Representative image of histological staining with IHC showing high expression of NFE2L3 and TACC1 in tumor tissues of ESCC patients, compared to low expression in adjacent non-tumor tissues (scale bars, 500 µm/200 µm). (C) TACC1 expression is positively correlated with NFE2L3 levels at the cellular level. (D) Western blot analysis of TACC1 protein expression in Eca109 cells transfected with TACC1 siRNA or control siRNA. (E) Transwell assay with crystal violet staining showed that knockdown of TACC1 inhibits the migration and invasion of Eca109 cells (scale bar, 100 µm). (F) Cell lysates from Eca109 cells were immunoprecipitated with an anti-TACC1 antibody, followed by silver staining analysis. (G) IF shows the co-localization of TACC1 and PARP1 in the cytoplasm of Eca109 cells. (H) Cell lysates from Eca109 cells were immunoprecipitated with an anti-TACC1 or an anti-PARP1 antibody, followed by Western blotting. (I) Knockdown of TACC1 promotes apoptosis in Eca109 cells. (J) Flow cytometry shows that knockdown of TACC1 promotes cell apoptosis. (K) Knockdown of TACC1 inhibits the activation of the AKT-mTOR pathway. (L) Schematic diagram. LEF1 directly regulates NFE2L3, which upregulates TACC1. TACC1 then binds to PARP1 and activates the AKT-mTOR signaling pathway to promote the progression of ESCC. **, P<0.01. DAPI, 4',6-diamidino-2-phenylindole; EMT, epithelial-mesenchymal transition; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblotting; IF, immunofluorescence; IgG, immunoglobulin G; IP, immunoprecipitation; LEF1, lymphoid enhancer-binding factor 1; mTOR, mammalian target of rapamycin pathway; NC, negative control; NFE2L3, nuclear factor erythroid 2-like 3; PARP1, proline-rich acidic protein 1; TACC1, transforming acidic coiled-coil-containing protein 1; V-FITC, V-fluorescein Isothiocyanate.

The Spearman correlation analysis revealed a positive correlation between NFE2L3 and TACC1 expression (R=0.606, P<0.01). Additionally, the Western blot analysis confirmed the high expression of NFE2L3 and TACC1 in the Eca109 and TE-1 cell lines (Figure 6C). To examine the role of TACC1 in ESCC progression, we knocked down TACC1 in Eca109 cells (si-TACC1) and verified knockdown efficiency via Western blot (Figure 6D). Transwell assays indicated that TACC1 knockdown inhibited the migration and invasion of Eca109 cells (Figure 6E).

TACC1 promotes the progression of ESCC by activating the AKT-mTOR pathway through PARP1

To further investigate the mechanisms by which TACC1 promotes ESCC progression, we performed immunoprecipitation of interacting protein complexes using an anti-TACC1 antibody in the Eca109 cells. Silver staining and immunoprecipitation-mass spectrometry (IP-MS) analysis identified proline-rich acidic protein 1 (PARP1) as a potential interacting protein of TACC1 (Figure 6F). Moreover, immunofluorescence staining revealed cytoplasmic co-localization of TACC1 and PARP1 in the Eca109 cells, suggesting an interaction between them in vivo (Figure 6G). We confirmed the direct binding of TACC1 to PARP1 through co-IP assays (Figure 6H).

Given that PARP1 is a key regulator of the apoptosis pathway (27), we investigated whether TACC1 inhibits apoptosis in ESCC cells. Flow cytometric apoptosis assays revealed that the proportion of early and late apoptotic cells increased following TACC1 knockdown (Figure 6I). The Western blot analysis demonstrated that TACC1 knockdown decreased the ratio of the anti-apoptotic protein Bcl-2 to the pro-apoptotic protein Bax, and increased the levels of the key executioner enzyme Caspase3, further confirming that TACC1 inhibits apoptosis in ESCC cells via PARP1 (Figure 6J).

Guan et al. demonstrated that increased PARP1 activity activates the PI3K/AKT/mTOR signaling pathway, thereby promoting head and neck squamous cell carcinoma progression (28). Through Western blot analysis, we confirmed that TACC1 activates the AKT/mTOR signaling pathway (Figure 6K). In summary, LEF1 promotes NFE2L3 transcription, thereby upregulating TACC1, which in turn binds to PARP1 to activate the AKT-mTOR pathway and promote ESCC progression (Figure 6L).

Discussion

NFE2L3 expression is upregulated in several cancers, including lung adenocarcinoma, hepatocellular carcinoma, and bladder cancer. Our study showed that LEF1 binds directly to the NFE2L3 promoter to enhance its transcription and translation. ChIP assays identified the LEF1 binding site at positions 1321 to 1334 of the NFE2L3 promoter. However, research on NFE2L3 in ESCC remains limited.

Chen et al. found that NFE2L3 regulates the radiosensitivity of ESCC cells through the IL-6-STAT3 signaling pathway (29). In the present study, we observed that NFE2L3 was highly expressed in ESCC specimens, and that high NFE2L3 expression was correlated with poorer histological differentiation, a higher pathological T stage, and a more advanced TNM stage. Further, NFE2L3 was shown to enhance the proliferation, migration, invasion, and EMT of ESCC cells in vitro, and promote tumor growth in vivo. Additionally, the scRNA-seq analysis suggests that NFE2L3 may serve as a potential tumor stemness marker, highlighting its potential as a novel therapeutic target for ESCC.

Based on the RNA-seq analysis of NFE2L3 knockdown, we found that TACC1 expression decreased following NFE2L3 suppression. Recent studies have shown that the dysregulation of TACC1 is linked to the development of various malignancies, including breast cancer, head and neck squamous cell carcinoma, and ovarian cancer (18-22). An analysis of TCGA data revealed that TACC1 was upregulated in the ESCA tissues compared to the normal and paired adjacent tissues, and contributes to the formation of an immunosuppressive microenvironment in esophageal cancer, suggesting its oncogenic role. Given that NFE2L3 promotes EMT and TACC1 suppresses epithelial phenotypes, these findings led us to select TACC1 for further investigation.

Co-IP assays were used to study molecular interactions, through which we identified and validated the interaction between TACC1 and PARP1. The AKT/mTOR signaling pathway is widely recognized as one of the most frequently dysregulated pathways, playing a critical role in regulating cell growth and tumorigenesis in various cancers. Guan et al. showed that increased PARP1 activity activates the PI3K/AKT/mTOR signaling pathway, promoting tumor progression (28). Therefore, it is plausible that TACC1 activates the AKT/mTOR pathway by binding to PARP1.

To examine the effect of TACC1 on this pathway, a Western blot analysis was performed to examine the expression of key signaling factors. As expected, TACC1 knockdown in the ESCC cells reduced the phosphorylation of proteins in the AKT/mTOR pathway, confirming that TACC1 activates AKT/mTOR signaling to mediate ESCC progression.

In summary, our study revealed that the LEF1/NFE2L3/TACC1 axis promotes ESCC progression by binding to PARP1 and activating the AKT/mTOR signaling pathway. Blocking this axis may offer a potential therapeutic strategy to delay ESCC progression.

We conclude that LEF1 directly regulates NFE2L3, thereby upregulating TACC1. TACC1 subsequently binds to PARP1 and activates the AKT-mTOR signaling pathway, promoting ESCC progression. These findings suggest that NFE2L3 may be a promising therapeutic target for ESCC treatment.

Conclusions

This study established a novel LEF1/NFE2L3/TACC1/PARP1 signaling axis that promotes ESCC progression through EMT induction, stemness maintenance, and AKT/mTOR pathway activation. NFE2L3 serves as a critical mediator of LEF1 oncogenic function and represents a promising therapeutic target for ESCC treatment. The immunosuppressive role of TACC1 highlights its potential as a target for combined targeted and immunotherapy strategies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0789/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0789/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0789/prf

Funding: This study was supported by a Youth Research Grant from the Shanghai Municipal Health Commission (2023QN28 to Q.L.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0789/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by Ethics Committee of The First Affiliated Hospital of Naval Medical University (No. CHEC2025-306) and informed consent was taken from all the patients. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology Co., Ltd (No. YS-JL-001), in compliance with the institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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