FANCI promotes esophageal squamous cell carcinoma progression and cell cycle regulation and interacts with FANCD2

Introduction

Esophageal squamous cell carcinoma (ESCC) is a major subtype of esophageal cancer, particularly prevalent in Eastern Asia, and is characterized by high aggressiveness and poor survival (1-3). Multimodal therapy has advanced, but the prognosis of advanced ESCC is unsatisfactory. Therefore, it is urgently needed to identify key molecular drivers and effective treatment targets for ESCC (4).

The Fanconi anemia (FA) pathway is an important mechanism of DNA damage response and is essential for maintaining genomic integrity (5,6). FA group I protein (FANCI), a core component of the FA pathway, forms the ID2 complex with FA group D2 protein (FANCD2), which is central to the activation of the pathway upon DNA insult (7,8). Emerging evidence suggests that FANCI may possess oncogenic properties across malignancies beyond its typical role in DNA repair (9,10). For instance, FANCI upregulation is linked to unsatisfactory prognosis and chemoresistance in several cancers (11,12). However, a comprehensive understanding of FANCI’s role in ESCC, including its clinical significance, impact on the tumor immune microenvironment, functional mechanisms, and particularly its relationship with FANCD2, is still lacking.

This study employed integrated multi-omics and functional approaches to elucidate the oncogenic functions of FANCI in ESCC. Its significant overexpression and prognostic value in ESCC were confirmed. Furthermore, we uncovered its critical role in promoting genomic instability and fostering an immunosuppressive tumor microenvironment (TME). Meticulous in vitro and in vivo investigations demonstrated that FANCI drove malignant phenotypes and elucidated a key mechanism that FANCI interacted with FANCD2 to regulate cell cycle progression. Our findings support FANCI as a potential oncogenic biomarker and suggest that the FANCI-FANCD2 axis may represent a candidate therapeutic target in ESCC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0535/rc).

Methods

Data acquisition and preprocessing

To comprehensively investigate FANCI in ESCC, we obtained RNA-seq expression data, clinical annotations, and somatic mutation profiles from The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov/) via the TCGAbiolinks package. A total of 95 ESCC patients with complete survival metadata were retained for downstream analyses. Gene expression profiles were normalized as transcripts per million and log2-transformed for comparability. The limma package was utilized for the differential expression analysis (13).

For independent validation, we downloaded the bulk RNA-seq dataset GSE53624 from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). Pan-cancer expression and clinical data harmonized with GTEx normal tissues were further retrieved from the UCSC Xena platform (14). All analyses were completed in R4.3.1.

Prognostic analysis

Clinical and transcriptomic data of the TCGA-ESCC cohort were used for survival analyses. Patients were stratified into high- and low-expression groups based on the median expression of FANCI or FANCD2, respectively. Kaplan-Meier survival analysis and log-rank tests were performed using the survival and survminer packages. Disease-free interval (DFI) was analyzed using the same grouping strategy. Cox proportional hazards regression was conducted in TCGA pan-cancer cohorts to evaluate the association between FANCI expression and overall survival (OS) across cancer types, and the results were visualized as a forest plot. For external validation, Kaplan-Meier analysis was performed in the GSE53624 cohort to assess the relationship between FANCI expression and OS.

Single-cell RNA-seq

Single-cell RNA-seq data of ESCC were obtained from GSE188900. The Seurat package was utilized for quality control and downstream analyses (15). Cells with <200 or >5,000 detected genes, or mitochondrial gene content >10% were excluded. Data normalization, variable gene selection, and scaling were carried out by standard procedures. We conducted dimensionality reduction with principal component analysis, followed by clustering via the Louvain algorithm. The Harmony algorithm was used to correct the batch effects (16). Cell clusters were annotated using canonical cell-type markers, and FANCI activity was scored with AUCell (17).

Pseudotime trajectory analysis

To reconstruct transcriptional dynamics of epithelial cells stratified by FANCI expression, a pseudotime trajectory analysis was performed with Monocle2. Genes with high dispersion and expression (dispersion ≥1, mean expression ≥0.1) were used to order cells along developmental trajectories (18). Branch expression analysis modeling was further applied to identify differentially expressed genes between trajectory branches, providing mechanistic insights into FANCI-related lineage programs.

Protein-protein interaction (PPI) and functional annotation

A PPI network centered on FANCI was created in STRING. FANCD2, a core interactor, was further evaluated for expression patterns and prognostic value in ESCC. The clusterProfiler package was utilized for the functional enrichment analysis of FANCI-associated genes by Gene Ontology (GO) and KEGG reference gene sets.

Somatic mutation and genomic instability analysis

We acquired data on somatic mutation in MAF format from TCGA and visualized by the maftools package (19). Tumor mutation burden (TMB) was calculated as the number of nonsynonymous mutations per megabase. Mutation landscapes were compared between the FANCI-high and FANCI-low groups. In addition, FANCI copy-number status and continuous GISTIC CNV values were used to evaluate the association between CNA and FANCI expression. Gene-level SCNA burden metrics (Amp_burden, Deep_deletion_burden, Del_burden, High_level_amp_burden, and total SCNA_burden) were compared between FANCI-high and FANCI-low groups (median cutoff) and correlated with FANCI expression using Spearman correlation.

Immune infiltration and TME analysis

To assess the tumor immune microenvironment, the immune cell fractions were estimated by CIBERSORTx (20) in bulk RNA-seq data from the Stromal score, immune score, and tumor purity. Immune checkpoint and immunosuppressive gene expression were compared between FANCI-high and FANCI-low groups. We quantified pathway activities by gene set variation analysis (GSVA) (21), and identified enriched biological processes by gene set enrichment analysis (GSEA) (22).

Cell culture and transfection

Human ESCC cell lines (KYSE-150, Eca-109, KYSE-30, TE-1, COLO-680N) and the normal esophageal epithelial cell line HET-1A were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) in March, 2025, all authenticated by short tandem repeat profiling within 18 months prior to manuscript submission. Cells were cultured with RPMI-1640 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under 5% CO₂ at 37 °C. All cell lines were confirmed to be free of mycoplasma contamination prior to use. To establish stable FANCI-knockdown cell lines, Eca-109 and TE-1 cells were infected with GV493 lentiviruses (hU6-MCS-CBh-gcGFP-IRES-puromycin; GeneChem, Shanghai) encoding shRNAs targeting human FANCI (LV-FANCI-RNAi#1, 5'-ACGGGCATCTGGGAGATATAG-3'; LV-FANCI-RNAi#2, 5'-ATGTAAGCTCGGAGCTAATAT-3'; LV-FANCI-RNAi#3, 5'-CTAGTTCCTCATAGATCTTAT-3') or non-targeting control shRNA (sh-NC, 5'-TTCTCCGAACGTGTCACGT-3'). Following infection, stable transformants were selected by puromycin (2–4 µg/mL) for one week. Knockdown efficiency was validated by Western blotting.

Immunohistochemistry (IHC)

After being fixed with formalin and embedded in paraffin, the sections of ESCC and adjacent normal tissues were deparaffinized and rehydrated, followed by antigen retrieval. After endogenous peroxidase activity was blocked, the sections were incubated with an anti-FANCI primary antibody (1:4000, Abcam, ab245219) at 4 °C overnight. Then they were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, and developed with diaminobenzidine, followed by hematoxylin counterstaining. Two pathologists independently assessed the staining intensity and percentage of positive cells.

Western blotting

Total protein was extracted by RIPA lysis buffer with a protease inhibitor Cocktail, and its concentration was measured by a BCA assay kit. Following SDS-PAGE, an equal volume of protein was transferred onto PVDF membranes. After being blocked with 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against FANCI (1:2,000, Abcam, ab245219), FANCD2 (1:500, Proteintech, 28619-1-P), and GAPDH (1:10,000, Proteintech, 10494-1-AP). Following incubation with HRP-conjugated secondary antibodies, an enhanced chemiluminescence system was utilized to visualize protein bands.

Cell counting kit-8 (CCK-8) and colony formation assays

In the CCK-8 assay, the cells were seeded into a 96-well plate, added with 10 µL of CCK-8 solution (Dojindo Laboratories) in each well at 0, 24, 48, 72, and 96 h, and incubated for 2 h. The absorbance at 450 nm was measured by a microplate reader. In the colony formation assay, the cells were seeded into a 6-well plate (500 per well) and incubated for 14 days. The resulting colonies were mounted with methanol, stained with 0.1% crystal violet, and counted.

Transwell invasion and wound healing assays

We assessed cell invasion by 24-well Transwell chambers pre-coated with Matrigel (Corning). Briefly, 5×10⁴ cells were plated in the upper chamber, while medium containing 20% FBS was placed in the lower chamber as a chemoattractant. After incubation for 24 h, the non-invading cells were removed. The invading cells were fixed, stained, and counted under a microscope. In the wound healing assay, the cells were seeded into a 6-well plate. Upon reaching 90% confluence, a scratch wound was created by a sterile 200 µL pipette tip. The image of wound closure was acquired at 0 and 48 h, and the migration distance was measured.

Apoptosis and cell cycle analyses

In apoptosis analysis, the cells were stained using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s instructions. Then, flow cytometry (BD FACSVerse) was carried out. In cell cycle analysis, the cells were fixed in 70% ethanol at 4 °C overnight and treated with RNase A, followed by propidium iodide staining. Flow cytometry was carried out on DNA to determine the cell percentage across cell cycle phases (G0/G1, S, G2/M).

Co-immunoprecipitation (Co-IP) and immunofluorescence (IF)

To investigate the interaction and subcellular localization of FANCI and FANCD2, Co-IP and IF were performed. For Co-IP, cell lysates were immunoprecipitated with antibodies against endogenous FANCI or ectopically expressed Flag-FANCI, along with an IgG control, followed by capture with Protein A/G Plus Agarose beads. The precipitates were then immunoblotted with an anti-FANCD2 antibody. For IF, the cells fixed on coverslips were co-stained with primary antibodies against FANCI and FANCD2, and subsequently with fluorescent dye-conjugated secondary antibodies. DAPI staining was performed on nuclei, and images were acquired under a confocal laser scanning microscope.

Xenograft tumor model

All animal experiments were approved by the Laboratory Animal Ethics Committee of Xinjiang Medical University (approval No. IACUC-JT-20250527-34; Project No. SKT-HIDCA-2024-TT15) on May 27, 2025, and were performed in accordance with institutional guidelines for the care and use of animals. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Male BALB/c nude mice (5–6 weeks old) were randomly assigned to two groups (5 mice per group). Eca-109 cells stably expressing shRNA targeting FANCI (sh-FANCI) or sh-NC were subcutaneously injected into the flanks. We measured the tumor volume every 4 days: (Length × Width²) / 2. After 21 days, the mice were sacrificed, and the tumors were weighed.

Statistical analyses

R4.3.1 was used for statistical analyses. Continuous variables underwent the Wilcoxon rank-sum or Kruskal-Wallis test. Log-rank tests were conducted for Kaplan-Meier survival analysis. Univariate/multivariate Cox regression analyses were carried out. The associations of FANCI, immune infiltration, and pathway activity were evaluated by Spearman correlation coefficients. P<0.05 (two-sided) was deemed statistically significant.

Results

Expression profile of FANCI across cancers and in ESCC

To characterize the transcriptional landscape of FANCI, we first examined its expression across TCGA pan-cancer cohorts. FANCI was significantly upregulated in multiple malignancies, suggesting a potential oncogenic role (Figure 1A). Given the clinical aggressiveness of ESCC and the lack of reliable molecular markers, we next focused on ESCC. In both the TCGA-ESCC and GSE53624 cohorts, FANCI expression was significantly higher in ESCC tissues than in normal esophageal tissues (Figure 1B). ROC analysis further demonstrated strong diagnostic performance of FANCI expression in discriminating ESCC from normal tissues [TCGA-ESCC: area under the curve (AUC) = 0.940, 95% confidence interval (CI): 0.878–1.000; GSE53624: AUC = 0.975, 95% CI: 0.954–0.995; Figure 1C].

Figure 1 Expression landscape and high-expression validation of FANCI in ESCC. (A) Pan-cancer paired analysis across TCGA cohorts showed FANCI expression in tumor tissues versus matched normal tissues in 15 cancer types (BLCA, BRCA, COAD, ESCA, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, STAD, THCA, UCEC), with FANCI showing an overall upregulation trend in tumors; paired samples from the same patient are connected by gray lines. (B) Violin plots with embedded boxplots showing FANCI expression differences between ESCC and normal tissues in two independent cohorts (TCGA-ESCC and GEO GSE53624); FANCI was significantly higher in ESCC than in normal tissues in both datasets (Wilcoxon P<2.2e−16). (C) ROC curve analysis evaluating the diagnostic performance of FANCI expression for discriminating ESCC from normal tissues, performed separately in TCGA-ESCC and the independent validation cohort GSE53624. The AUC was 0.940 (95% CI: 0.878–1.000) in TCGA-ESCC and 0.975 (95% CI: 0.954–0.995) in GSE53624. (D) qPCR analysis of FANCI mRNA levels in the normal esophageal epithelial cell line HET-1A and five ESCC cell lines (Eca-109, TE-1, KYSE-30, KYSE-150, COLO-680N), showing higher FANCI mRNA expression in ESCC cell lines. (E) Western blot analysis of FANCI protein expression (149 kDa) with GAPDH (36 kDa) as a loading control in HET-1A and ESCC cell lines, together with densitometric (grayscale) quantification, confirming increased FANCI protein abundance in ESCC cell lines. (F) Representative IHC images showing weak/absent FANCI staining in normal squamous epithelium and stronger FANCI immunoreactivity in ESCC tissues; the magnified tumor region (×30) highlights positive staining in tumor cells. ns, not significant; **, P<0.01; ****, P<0.0001. AUC, area under the curve; CI, confidence interval; ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; IHC, immunohistochemistry; qPCR, quantitative polymerase chain reaction; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; WB, Western blot.

These transcriptome-level observations prompted experimental validation. Quantitative polymerase chain reaction (qPCR) confirmed that FANCI mRNA levels were markedly higher in multiple ESCC cell lines (Eca-109, TE-1, KYSE-30, KYSE-150, and COLO-680N) than in the normal esophageal epithelial cell line HET-1A (Figure 1D). Consistently, Western blotting with densitometric quantification demonstrated increased FANCI protein abundance in ESCC cell lines (Figure 1E). IHC further revealed stronger FANCI immunoreactivity in ESCC tissues compared with normal squamous epithelium (Figure 1F). Collectively, FANCI is aberrantly activated in ESCC at both mRNA and protein levels.

Prognostic significance and validation of FANCI

In addition to FANCI, FANCD2 expression was significantly higher in ESCC tumors than in normal tissues (Figure S1A). To assess the prognostic relevance of FANCI, we evaluated its survival association across cancers and in ESCC. Pan-cancer Cox analyses identified FANCI as a high-risk factor in several malignancies (Figure 2A). In the TCGA-ESCC cohort, Kaplan-Meier analysis showed that patients in the FANCI-high group had significantly worse OS than those in the FANCI-low group (Figure 2B). This unfavorable prognostic association was independently validated in the GSE53624 cohort (Figure 2C). In addition, high FANCI expression was associated with a shorter DFI in TCGA-ESCC, indicating a potential link between FANCI and tumor recurrence (Figure S1B). Together, these results support FANCI as a reproducible prognostic biomarker for ESCC.

Figure 2 Prognostic validation of FANCI in ESCC. (A) Forest plot of HR for FANCI across multiple TCGA cancer types, identifying FANCI as a high-risk factor in several malignancies. (B) Kaplan-Meier survival analysis for OS between FANCI-high and FANCI-low groups in the TCGA-ESCC cohort. (C) Kaplan-Meier survival analysis confirming the unfavorable prognosis associated with high FANCI expression in the validation cohort GSE53624. CI, confidence interval; ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; HR, hazard ratio; OS, overall survival; TCGA, The Cancer Genome Atlas.

FANCI bidirectionally regulates ESCC cell proliferation

To clarify the functional role of FANCI in ESCC progression, we performed bidirectional gain- and loss-of-function assays. qPCR and Western blotting confirmed efficient knockdown of FANCI using three independent shRNAs in Eca-109 and TE-1 cells (Figure 3A,3B). CCK-8 assays demonstrated that FANCI knockdown significantly inhibited cell proliferation at 48, 72, and 96 h (Figure 3C), and colony formation assays further showed markedly reduced clonogenic capacity (Figure 3D).

Figure 3 FANCI promotes ESCC cell proliferation via bidirectional validation. (A) qPCR and (B) Western blotting confirming the knockdown efficiency of three shRNAs (sh-FANCI#1/2/3) in Eca-109 and TE-1 cells, with GAPDH as a loading control. (C) CCK-8 assays showing that FANCI knockdown (SH2 and SH3) significantly inhibited cell proliferation at 48, 72, and 96 h. (D) Colony formation assays demonstrating reduced long-term clonogenic capacity following FANCI knockdown. (E) qPCR and (F) Western blotting (including Flag-tag) validating FANCI overexpression in KYSE-150 and COLO-680N cells. (G) CCK-8 assays indicating that FANCI overexpression significantly promoted cell growth. (H) Colony formation assays showing increased colony-forming ability upon FANCI overexpression. ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; OD, optical density; OE, overexpression; qPCR, quantitative polymerase chain reaction.

Conversely, qPCR and Western blotting validated successful FANCI overexpression in KYSE-150 and COLO-680N cells (Figure 3E,3F). FANCI overexpression significantly promoted cell growth in CCK-8 assays (Figure 3G) and increased colony-forming ability (Figure 3H). Together, these data indicate that FANCI promotes ESCC cell proliferation.

FANCI bidirectionally enhances ESCC cell migration and invasion

We next assessed whether FANCI contributes to ESCC cell motility and invasiveness. Wound-healing assays showed that FANCI knockdown significantly reduced wound closure in Eca-109 and TE-1 cells (Figure 4A). Matrigel-coated Transwell assays further demonstrated that FANCI silencing markedly decreased invasive capacity (Figure 4B).

Figure 4 FANCI enhances ESCC cell migration and invasion via bidirectional validation. (A) Wound healing assays (0, 24, and 48 h) showing that FANCI knockdown (SH2, SH3) significantly reduced wound closure in Eca-109 and TE-1 cells. Crystal violet staining; magnification: ×100. (B) Matrigel-coated Transwell invasion assays with crystal violet staining demonstrating that FANCI knockdown markedly decreased the number of invaded cells. Crystal violet staining; magnification: ×100. (C) Wound healing assays indicating that FANCI overexpression significantly accelerated wound closure in KYSE-150 and COLO-680N cells. Crystal violet staining; magnification: ×100. (D) Matrigel-coated Transwell invasion assays showing increased numbers of invaded cells following FANCI overexpression. Crystal violet staining; magnification: ×100. *, P<0.05; **, P<0.01; ****, P<0.0001. ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; NC, negative control; OE, overexpression.

In gain-of-function settings, FANCI overexpression accelerated wound closure in KYSE-150 and COLO-680N cells (Figure 4C) and increased the number of invaded cells in Transwell assays (Figure 4D). These findings suggest that FANCI enhances ESCC cell migration and invasion.

FANCI regulates apoptosis and promotes tumor growth in vivo

Given its oncogenic phenotypes in vitro, we further evaluated the effects of FANCI on apoptosis and tumor growth. Flow cytometry showed that FANCI knockdown increased the apoptotic fraction in TE-1 and Eca-109 cells compared with controls (Figure 5A). Conversely, FANCI overexpression reduced apoptosis in KYSE-150 and COLO-680N cells (Figure 5B).

Figure 5 FANCI regulates ESCC cell apoptosis and promotes tumor growth in vivo. (A) Flow cytometry analysis of apoptosis in TE-1 and Eca-109 cells following FANCI knockdown, showing an increased apoptotic fraction compared with the control group. (B) Flow cytometry analysis of apoptosis in KYSE-150 and COLO-680N cells following FANCI overexpression. (C) Representative images of mice and excised subcutaneous xenograft tumors comparing the control and FANCI knockdown groups. (D) Tumor growth curves showing that FANCI knockdown significantly suppressed ESCC tumor growth in vivo. ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; NC, negative control; OE, overexpression; SH, short hairpin RNA.

To validate the in vitro observations in vivo, xenograft assays demonstrated that FANCI knockdown suppressed tumor growth, as evidenced by representative tumor images and significantly reduced tumor growth curves (Figure 5C,5D). Collectively, FANCI promotes ESCC cell survival and tumor growth.

FANCI promotes ESCC progression through interaction with FANCD2 and cell cycle regulation

To explore the mechanisms underlying FANCI in ESCC, we constructed a PPI network and identified FANCD2 as a key interactor (Figure 6A). In TCGA-ESCC, FANCI expression was strongly and positively correlated with FANCD2 mRNA levels (Figure 6B). Clinically, higher FANCD2 expression predicted poorer OS and a shorter DFI (Figure S1C,S1D), supporting its clinical relevance in ESCC.

Figure 6 FANCI promotes ESCC progression through interaction with FANCD2 and cell cycle regulation. (A) PPI network of FANCI generated by STRING database analysis, with FANCD2 highlighted as a key nodal protein. (B) Correlation analysis demonstrated a strong positive association between FANCI and FANCD2 mRNA expression in TCGA-ESCC. (C) GSEA identified biological hallmark pathways including cell cycle and DNA repair enriched in the FANCI high group compared with the FANCI low group based on TCGA-ESCC data. (D) qPCR analysis showed synchronized mRNA expression patterns of FANCI and FANCD2 across ESCC cell lines. (E) Western blotting confirmed that FANCI knockdown or overexpression synchronously regulated FANCD2 protein levels in ESCC cell lines. (F) Co-IP assays confirmed the interaction between FANCI and FANCD2 proteins using exogenous and endogenous validation. (G) Subcellular co localization analysis of FANCI and FANCD2. IF double staining of FANCI in green and FANCD2 in red in Eca109 cells. Nuclei were counterstained with DAPI in blue. Images were acquired by confocal microscopy. The merged image demonstrates overlap of FANCI and FANCD2 signals in the nucleus. Scale bar 25 μm. (H) Flow cytometry analysis of cell cycle distribution in ESCC cells following FANCI knockdown or overexpression, demonstrating that FANCI regulates cell cycle progression. Co-IP, co-immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; ESCC, esophageal squamous cell carcinoma; FANCD2, Fanconi anemia group D2 protein; FANCI, Fanconi anemia group I protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSEA, gene set enrichment analysis; IF, immunofluorescence; NC, negative control; OE, overexpression; PPI, protein-protein interaction; qPCR, quantitative polymerase chain reaction; TCGA, The Cancer Genome Atlas.

At the pathway level, GSEA showed enrichment of cell cycle- and DNA repair-related hallmark pathways in the FANCI-high group, suggesting an association with altered DNA repair-related programs (Figure 6C). Consistently, single-sample gene set enrichment analysis (ssGSEA) demonstrated activation of cell-cycle/replication-stress/FA-DNA damage response (DDR) programs in FANCI-high tumors (Figure S1E), and FANCI expression correlated with the corresponding ssGSEA scores (Figure S1F). A correlation heatmap further summarized coordinated associations among FANCI, FANCD2, cell-cycle/DDR modules, and immune-related modules (Figure S1G), and FANCI showed positive correlations with representative cell-cycle/DDR genes (Figure S1H).

We next performed a series of experiments to confirm the interaction between FANCI and FANCD2 and its functional consequences. qPCR showed synchronized mRNA expression patterns of FANCI and FANCD2 across ESCC cell lines (Figure 6D). Western blotting demonstrated that FANCI knockdown or overexpression synchronously regulated FANCD2 protein levels (Figure 6E). Co-IP assays confirmed a direct interaction between FANCI and FANCD2 using both exogenous and endogenous validation (Figure 6F). IF double staining further revealed nuclear colocalization of FANCI and FANCD2 (Figure 6G). Furthermore, flow cytometric cell cycle analysis showed that FANCI knockdown or overexpression altered cell cycle distribution, demonstrating that FANCI regulates cell cycle progression in ESCC cells (Figure 6H). Together, these findings suggest that FANCI promotes ESCC progression through functional cooperation with FANCD2 and regulation of cell cycle progression.

Single-cell characterization of FANCI and its functional implications in ESCC

To gain further insight at the cellular level, we analyzed scRNA-seq data to construct an ESCC single-cell atlas. Uniform manifold approximation and projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) analyses identified major cell populations including epithelial cells, fibroblasts, and immune cells, and canonical marker genes validated cell type annotation (Figure 7A-7C). Pseudotime trajectory analysis revealed dynamic changes in cell states along pseudotime (Figure 7D). GO and pathway enrichment analyses of genes associated with cell state transitions highlighted biological processes including DNA damage checkpoint, cell cycle arrest, and immune-related processes (Figure 7E). Importantly, FANCI expression was predominantly enriched in epithelial cells (Figure 7F). Functional module analysis further demonstrated that FANCI-high cells exhibited higher activity in malignant functional programs, including PI3K/AKT signaling, invasion and metastasis, EMT, and apoptosis-related modules, compared with FANCI-low cells (Figure 7G). These findings support a role for FANCI in shaping malignant cell states and functional plasticity in ESCC.

Figure 7 Single cell characterization of FANCI expression and its regulatory roles in ESCC cell functionality. (A) UMAP plot showing the distribution of major cell populations in ESCC samples including epithelial cells fibroblasts and immune cells. (B) Heatmap of canonical marker genes validating cell type annotation with representative markers. (C) t-SNE plot further confirming the clustering consistency of major cell populations. (D) PCA-based pseudotime trajectory analysis showing cell state dynamics along pseudotime. (E) Gene Ontology and pathway enrichment analysis of differentially regulated genes associated with cell state transitions highlighting biological processes including DNA damage checkpoint cell cycle arrest and immune related processes. (F) UMAP plot showing the single cell expression landscape of FANCI across cell subpopulations and its enrichment in epithelial cells. (G) Bubble plot comparing the activity of malignant functional modules including PI3K AKT signaling invasion and metastasis EMT and apoptosis between FANCI high and FANCI low groups. EMT, epithelial-mesenchymal transition; ESCC, esophageal squamous cell carcinoma; FANCI, Fanconi anemia group I protein; PCA, principal component analysis; t-SNE, t-distributed stochastic neighbor embedding; UMAP, uniform manifold approximation and projection.

To further address the single-cell-level mechanism, we integrated epithelial trajectory with FANCI expression and functional scores. The trajectory proxy mapped onto the epithelial UMAP and recapitulated a continuous state progression (Figure S2A), and feature plots showed the spatial concordance of FANCI activity/expression with stemness, malignancy-like, cell-cycle, DDR, and immune-regulatory programs across epithelial cells (Figure S2B). Along the trajectory, stemness score increased (rho =0.31, P<0.01), whereas multiple programs displayed trajectory-dependent dynamics, including antigen presentation (rho =0.28, P<0.01) and hypoxia/stress (rho =0.06, P=0.03) (Figure S2C). Module-score comparisons further demonstrated differences in malignant, stemness-related, and immune-regulatory programs between FANCI-high and FANCI-low epithelial cells (Figure S2D). FANCI activity was correlated with FANCD2 expression (rho =0.32, P<0.01), EMT/invasion (rho =0.17, P<1×10⁻⁷), and malignancy program score (rho =0.11, P<0.01), while also showing associations with immune-regulatory features at the single-cell level (Figure S2E). FANCI-high epithelial cells were progressively enriched toward late trajectory bins (Figure S2F) and showed altered immune-regulatory module activities compared with FANCI-low cells (Figure S2G). Co-localization analysis further demonstrated correlations between FANCI activity and immune-regulatory module scores at the single-cell level (Figure S2H). Collectively, these analyses suggest that FANCI-associated epithelial states are linked to trajectory progression and coordinated changes in stemness/malignancy and immune-related programs in ESCC.

Genomic alterations, mutation landscape, and immune microenvironment associated with FANCI

To characterize FANCI-associated genomic alterations in ESCC, we analyzed somatic mutation profiles in TCGA-ESCC. A combined oncoplot summarized the top mutated genes and sample-level TMB distribution with FANCI-high/low annotations (Figure S3A). In addition, mutation type composition differed between FANCI-high and FANCI-low groups (Figure S3B), and group-wise comparison revealed distinct mutation frequencies across recurrently mutated genes (Figure S3C,S3D). Consistently, the FANCI-high group showed an overall trend toward higher TMB compared with the FANCI-low group (Figure S3E).

To examine immunological associations of FANCI, we performed CIBERSORT-based deconvolution to estimate immune cell infiltration fractions (Figure S3F). Correlation analyses further linked FANCI expression with immune-related features (Figure S3G). GSVA-based analyses showed that FANCI expression was negatively correlated with inflammatory response (tStudent( 89) =−2.31, P=0.02; Figure S3H) and interferon-gamma response signatures (tStudent(89) =−2.58, P=0.01; Figure S3I), suggesting potential immune modulation associated with FANCI expression. Taken together, FANCI is associated with an altered mutation profile and immune-related features in ESCC.

In addition, FANCI copy-number status was distributed across deletion, neutral, gain, and amplification categories in TCGA-ESCC (Figure S4A), and the proportions of these categories differed between the FANCI-high and FANCI-low groups (Figure S4B). FANCI expression differed significantly across copy-number status categories (Kruskal-Wallis P=0.01) and correlated positively with FANCI GISTIC CNV values (Spearman rho =0.33, P=0.001; Figure S4C,S4D). Gene-level SCNA burden metrics were also compared between FANCI-high and FANCI-low groups (Figure S4E). FANCI expression was positively correlated with Amp_burden (rho =0.21, P=0.041) and total SCNA_burden (rho =0.23, P=0.03; Figure S4F).

Discussion

Our integrated analysis indicates that FANCI is associated with oncogenic features and adverse prognosis in ESCC. Previous genomic studies have highlighted the occasional alteration of the FA pathway in cancer, but the multi-omics approach delineated a multifaceted role for FANCI in driving ESCC pathogenesis, not only by promoting cell-intrinsic proliferative, migratory, and invasive capacities but also by correlating with immunosuppressive TME remodeling and genomic instability. Importantly, our data suggest a non-canonical role for the FANCI-FANCD2 complex in supporting basal cell-cycle progression, even in the absence of exogenous DNA damage, thereby moving beyond its canonical context of genotoxic stress response.

The FANCI-FANCD2 heterodimer is a well-established core component of the FA pathway (23,24), classically activated by genotoxic stress to facilitate DNA interstrand crosslink repair (25,26). The prevailing model, largely derived from studies in FA patient cells, assumes that the complex’s primary function is in coordinating the DDR (27). Our work suggests a non-canonical function for this complex in ESCC biology. In ESCC, this interaction was critical for basal cell cycle homeostasis, which was independent of exogenous damage. This finding implies emerging roles for other DDR proteins, such as ATR and CHK1, in regulating unperturbed cell cycles (28,29), suggesting a broader theme where genome custodians acquire cell cycle functions in hyperproliferative contexts. A key finding is that FANCI knockdown robustly destabilized FANCD2 protein and triggered G2/M cycle arrest even in the absence of genotoxic insult. This interdependent stability contrasts with the model where FANCI and FANCD2 are co-regulated primarily by the FA core complex activation upon damage (30). These results suggest that FANCI may contribute to maintaining FANCD2 protein stability in ESCC. This indispensability for uninterrupted cell cycle progression signifies a fundamental functional expansion beyond the DNA damage-centric view of the FA core complex (31,32), strongly supporting a model with FANCI as a crucial stabilizing chaperone for FANCD2, which enhances the understanding of this core complex’s regulation.

Our findings that the FANCI-FANCD2 axis governed the G2/M checkpoint aligned with the emerging role of FA proteins in managing endogenous replication stress (33). For instance, FANCD2 has been implicated in stabilizing stalled replication forks under basal conditions (8,34). This study identified the FANCI-FANCD2 dimer as a central regulator of this process in ESCC. To bridge this mechanism with oncogenic phenotypes, bioinformatic and single-cell analyses were performed, and high FANCI expression was linked to the activation of PI3K-AKT, EMT, and apoptosis resistance. This connection is intriguing, as crosstalk between DDR pathways and PI3K-AKT signaling is increasingly recognized (35). For example, ATM can directly activate AKT (36). These data suggest that FANCI may be involved in this crosstalk. In summary, we proposed a unified model with FANCI as a central signaling node in ESCC, coordinating its canonical genome maintenance capacity with its newly identified cell cycle regulatory function. This dual capacity (sustaining proliferation while potentially fostering the genetic diversity that underlies genomic instability) may create a feed-forward loop that synergistically amplifies key oncogenic pathways (e.g., PI3K-AKT), accelerating tumor evolution and aggressiveness.

The association between FANCI expression, increased TMB, and an immunosuppressive TME suggests a potential link to immune evasion, although a causal relationship remains unproven. While high TMB is often associated with T-cell infiltration and response to immunotherapy in some cancers (37), this study highlighted that this relationship was complex and could be counteracted by specific immunosuppressive mechanisms, consistent with growing evidence in ESCC and other gastrointestinal malignancies. The CIBERSORT analysis revealed that FANCI-high tumors exhibited an immunosuppressive landscape, characterized by enriched M0/M2 macrophage infiltration and a reduction in activated NK and memory CD4⁺ T cells, a signature often linked to poor immunotherapy outcomes. We further identified a greatly positive correlation of FANCI with key immunomodulators, CCL2 and VEGFA. This is particularly relevant as the CCL2-CCR2 axis is a well-established pathway for recruitment of monocytes and macrophages (38), and VEGFA is known to suppress T-cell function and contribute to expansion of regulatory T-cells (39). Based on these established roles and our correlative data, we hypothesize that FANCI may promote the formation of an immunosuppressive niche, potentially via the upregulation of CCL2 and/or VEGFA, thereby facilitating TAM recruitment and angiogenic signaling. This proposed causal relationship, however, warrants future experimental validation to distinguish a direct role for FANCI in cytokine regulation from an indirect consequence of the pro-tumorigenic state it creates.

The pleiotropic nature of FANCI underscores its compelling clinical translational potential. First, its validated role as an prognostic biomarker provides an immediate tool for patient risk stratification (10,40), complementing existing clinical parameters. Based on this and its association with an immunosuppressive TME, FANCI expression could be integrated into a composite biomarker to predict responses to immune checkpoint inhibitors, similar to efforts combining TMB with PD-L1 or other immune signatures (41,42). Second, the central role of the FANCI-FANCD2 complex in DNA repair and cell cycle control positions FANCI-high tumors within a clear theranostic framework, consistent with the findings in ovarian and breast cancers (12,43). Based on prior studies, FANCI overexpression may be associated with resistance to DNA-damaging agents, including platinum-based chemotherapy and radiotherapy (12,44,45), which was a concept termed “oncogene-induced DNA dependency”. As a result, these tumors may be vulnerable to synthetic lethality with PARP inhibitors. Therefore, directly targeting the FANCI-FANCD2 axis or exploiting its associated vulnerabilities represents a promising strategy to overcome conventional therapy resistance in this aggressive ESCC subset. This offered a rationale for patient selection in future clinical trials of PARP inhibitors or other DDR-targeted agents.

Notwithstanding these insights, our study has limitations. The precise biochemical mechanism by which FANCI regulates FANCD2 protein stability, whether through direct physical protection, competition with degradation machinery, or influencing post-translational modifications, remains to be fully elucidated. In addition, rescue experiments were not performed, which limits causal inference regarding the FANCI-FANCD2 axis. Additionally, the use of immunodeficient mouse models, while ideal for assessing cell-intrinsic tumor growth, precluded a direct assessment of FANCI’s impact on the adaptive immune system in vivo. Future studies employing humanized patient-derived xenograft models or immunocompetent genetically engineered mouse models of ESCC are required for dissecting the role of FANCI in immunomodulation and validating the CCL2/VEGFA hypothesis. Furthermore, retrospective and prospective clinical validation correlating FANCI/FANCD2 protein with patient survival and treatment responses in large, multi-center ESCC cohorts is the next crucial step for clinical translation.

Conclusions

FANCI is upregulated in ESCC and is associated with unfavorable clinical outcomes. Functional analyses indicate that FANCI promotes ESCC cell proliferation, migration, invasion, apoptosis resistance, and xenograft tumor growth. FANCI is associated with FANCD2 expression and with cell-cycle- and DNA-repair-related molecular programs. At the transcriptomic and single-cell levels, FANCI-high states are also associated with malignant epithelial programs, mutation-related features, copy-number-related features, and altered immune-associated signatures. However, the genomic, immune, and single-cell findings are primarily associative, and the direct causal mechanisms require further experimental validation. Overall, FANCI may represent a candidate biomarker and potential therapeutic vulnerability in ESCC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0535/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0535/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0535/prf

Funding: This work was supported by the State Key Lab of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia Fund (No. SKL-HIDCA-2024-YY15) and Ili & Jiangsu Joint Institute of Health (No. yl2022py16).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0535/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. All animal experiments were approved by the Laboratory Animal Ethics Committee of Xinjiang Medical University (approval No. IACUC-JT-20250527-34; Project No. SKT-HIDCA-2024-TT15) on May 27, 2025, and were performed in accordance with institutional guidelines for the care and use of animals. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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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