CHMP2A as a potential prognostic biomarker for esophageal squamous cell carcinoma promotes tumor angiogenesis and cancer progression

Abstract

BACKGROUND

Esophageal squamous cell carcinoma (ESCC) is a highly prevalent and malignant tumor of the digestive system with poor prognosis. CHMP2A promotes formation and progression in multiple tumor types; however, its precise role and clinical relevance in ESCC remain unclear. High CHMP2A expression correlates with lymph node metastasis and immunosuppression in other tumors. We investigated whether CHMP2A is upregulated in ESCC tissues, its correlation with clinical prognosis and potential mechanisms in immune microenvironment modification, cell proliferation, metastasis, angiogenesis, migration, and invasion.

AIM

To investigate CHMP2A expression, prognostic value, and functional roles in ESCC progression.

METHODS

This immunological study integrated The Cancer Genome Atlas bioinformatics, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and immunohistochemistry on ESCC and paired normal tissues. Eca9706 ESCC cells were transiently transfected with CHMP2A-overexpression plasmids to establish the OE-CHMP2A group. Proliferation, migration, invasion, and angiogenesis were assessed, and data were analyzed by Student's t-test (SPSS 21.0/GraphPad Prism; P < 0.05).

RESULTS

CHMP2A mRNA/protein was upregulated in ESCC vs normal tissues. Elevated CHMP2A expression was associated with decreased infiltration of CD8⁺ T cells and NK cells into the tumor microenvironment and exhibited a negative connection with the expression of immunological checkpoint markers, including PD-1 and CTLA-4. Functional experiments demonstrated that OE-CHMP2A significantly enhanced the proliferation, migration, and invasion abilities of ESCC cells. Furthermore, the conditioned medium from OE-CHMP2A ESCC cells significantly promoted tube formation in human umbilical vein endothelial cells, suggesting a pro-angiogenic role.

CONCLUSION

CHMP2A is associated with ESCC tumor progression, including malignant proliferation, invasion, metastasis, angiogenesis, and immunosuppression, serving as a potential prognostic biomarker in ESCC.

Key Words: Esophageal squamous cell carcinoma; CHMP2A; Prognosis; Proliferation; Angiogenesis

Core Tip: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with a poor prognosis. This study revealed that CHMP2A is significantly upregulated in ESCC tissues and cell lines, and its high expression predicts poor survival in ESCC patients. Functional assays demonstrated that CHMP2A promotes ESCC cell proliferation, invasion, migration, and tumor angiogenesis. Our findings identify CHMP2A as a novel prognostic biomarker in ESCC, potentially informing new strategies for its diagnosis and treatment.

INTRODUCTION

Esophageal cancer is one of the most common malignant tumors of the digestive system worldwide, with approximately 511000 new cases and 445000 deaths reported in 2022[1-3]. China has the highest incidence and mortality, with esophageal squamous cell carcinoma (ESCC) accounting for over 90% of cases[2,4]. Despite recent advancements in multimodal therapy, the 5-year survival rate remains poor due to late diagnosis and variation in treatment response[5-9]. Therefore, identifying biomarkers of tumor progression and therapeutic response is an important direction for improving clinical management of ESCC.

CHMP2A is a member of the SNF7 protein family and a core component of the endosomal sorting complex required for transport (ESCRT-III). Its main functions are mediated by interactions with proteins such as CHMP4B and VPS4, which are essential for membrane remodeling and endosomal sorting[10]. CHMP2A can polymerize into spiral tubular structures, contributing to multivesicular body formation, and plays an important role in protein degradation, signal trafficking, and exosome biogenesis[10-14]. Moreover, CHMP2A is rapidly recruited to repair damaged plasma membranes for repair, maintaining cellular integrity[11]. Beyond membrane dynamics, CHMP2A also participates in nuclear envelope repair, cytokinetic abscission, and neuronal pruning[15]. In virology, CHMP2A is indispensable for the budding and release of enveloped viruses such as the hepatitis B virus and the human immunodeficiency virus[14]. Collectively, these findings indicate that CHMP2A, a key ESCRT-III factor, is a critical node in maintaining cellular homeostasis and signal regulation.

Recent studies have shown that CHMP2A is aberrantly expressed in several cancers, including endometrial carcinoma, colorectal cancer, and lung adenocarcinoma, and is closely associated with prognosis[16-18]. Functionally, CHMP2A plays a multifaceted role in tumor progression, including regulating exosome secretion, activating cancer-associated fibroblasts, facilitating invasion and metastasis, and fostering immune evasion[10,14,19]. Specifically, CHMP2A depletion in head and neck squamous cell carcinoma enhances NK cell-mediated cytotoxicity and upregulates inflammatory cytokines[20]. In endometrial cancer, its overexpression correlates with an advanced International Federation of Gynecology and Obstetrics stage and deep myometrial invasion, suggesting involvement in epithelial-mesenchymal transition (EMT)[16]. Proteomic analyses of urinary exosomes from colorectal cancer patients revealed decreased CHMP2A levels, supporting its potential as a non-invasive diagnostic biomarker[17]. In lung adenocarcinoma, CHMP2A expression shows significant association with pyroptosis-related gene signatures and patient survival outcomes[18]. Collectively, these findings establish CHMP2A as a key coordinator of fundamental oncogenic processes—including regulated cell death, exosome-mediated intercellular communication, and immune evasion—across diverse malignancies. This evidence positions CHMP2A as a prognostic biomarker and promising therapeutic target[21-23].

Based on these observations, the present study investigated CHMP2A’s expression in ESCC, association with clinical prognosis, and potential mechanisms. Its functions in angiogenesis, migration and invasion, cell proliferation, and immune microenvironment modification were given special focus to provide theoretical support for ESCC diagnosis, stratified treatment, and the creation of therapeutic targets.

MATERIALS AND METHODS

CHMP2A expression and clinical prognostic analysis

Cancer RNA-seq data were obtained from The Cancer Genome Atlas (TCGA) database. R (v4.2.1) software with the stats (v4.2.1) and car (v3.1-0) packages were used to analyze CHMP2A mRNA levels across cancers. Patients were grouped by the minimum P value method, and Kaplan-Meier curves were plotted using R (v3.6.3) software with the survival (v3.3.1) and ggplot2 (v3.4.4) packages. Nomograms predicting 1-, 3-, and 5-year survival were constructed using R (v4.2.1) program, with area under the curve (AUC) calculated.

Immune infiltration and gene function enrichment analysis

The correlation between CHMP2A expression and immune cell infiltration was analyzed using the single-sample gene set enrichment analysis (ssGSEA) algorithm from the R package GSVA (v1.46.0), the CIBERSORT algorithm (implemented via the CIBERSORT.R script), and the ESTIMATE package (v1.0.13). The association between CHMP2A expression and the expression levels of other immune checkpoints was also assessed. Using the Tumor Immune Single-cell Hub 2 database, we analyzed single-cell sequencing results to determine the distribution of different cell types and CHMP2A expression levels in the GSE160269 and GSE173950 datasets. The UALCAN database identified genes positively associated with CHMP2A expression in esophageal carcinoma (ESCA). Gene Ontology (GO) bioprocess analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R (v4.2.1).

Immunohistochemistry

Sections (4 μm) from formalin-fixed paraffin-embedded tissues were dewaxed, rehydrated, and subjected to antigen retrieval in EDTA buffer (Zsbio, Beijing, China). After blocking endogenous peroxidase activity and nonspecific binding, the slides were incubated overnight at 4 °C with a primary antibody against CHMP2A (Affbiotech, Changzhou, China), followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (Maxim, Fuzhou, China). Staining was visualized with a DAB kit (Maxim, Fuzhou, China) and counterstained with hematoxylin. Two senior pathologists independently evaluated the results. Final immunohistochemistry (IHC) scores were calculated by multiplying the staining intensity and percentage scores, categorized as low expression (0-3) or high expression (4-12) according to modified criteria from the immunoreactive score system proposed by Remmele and Stegner[24]. The staining intensity scoring criteria were as follows: 0 = no staining; 1 = weak positive, pale yellow; 2 = moderate positive, brownish-yellow; and 3 = strong positive, dark brown. Scoring criteria for the percentage of positive cells: 0 = no positive cells; 1 = ≤ 10% positive cells; 2 = 11%-50% positive cells; 3 = 51%-80% positive cells; and 4 = > 80% positive cells. The final IHC score was calculated as the product of the staining intensity score and the percentage of positive cells score. Based on this scoring system, patients were dichotomized into two groups: Low CHMP2A expression (total score 0-3) and high CHMP2A expression (total score 4-12). All procedures in this experiment were conducted in strict compliance with relevant guidelines and protocols. The present study was reviewed and approved by the Ethics Committee of Heping Hospital Affiliated to Changzhi Medical College [Approval No. (2026)057], and all patients provided informed consent. Clinicopathological characteristics and follow-up information for all enrolled patients are summarized in Supplementary Table 1.

Cell culture

The Clinical Research Center Laboratory of Peace Hospital, Changzhi Medical College, supplied the human umbilical vein endothelial cells (HUVECs) and the human ESCC cell line Eca9706. The Eca9706 cells were maintained in RPMI-1640 media (Gibco, Thermo Fisher Scientific, United States) supplemented with 10% fetal bovine serum (FBS). Conversely, HUVECs were cultivated in endothelial cell-specific medium (iCell Bioscience Inc., China) with 5% FBS. All cell lines were cultured in a controlled environment at 37 °C in 5% CO₂.

Transient transfection

Briefly, cells were seeded in 6-well plates 24 hours prior to transfection. At 60%-70% confluence, 2.5 μg of plasmid DNA (GenScript, Nanjing, China) and 7.5 μL of Lipofectamine^TM 3000 (Invitrogen, Thermo Fisher Scientific, United States) were separately diluted in Opti-MEM medium (Gibco, Thermo Fisher Scientific, United States), combined, and incubated at room temperature for 15 minutes to form transfection complexes. The complexes were then added to the cells. Following a 6-hour transfection period, the medium was substituted with new complete media. Cells were collected 48 hours after transfection for further functional validation and experimental investigation.

Western blotting

Cell samples were processed according to established protocols. Proteins were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Electrophoresis was performed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels with 15 μg protein samples, which were subsequently transferred to polyvinylidene fluoride membranes. Following an overnight incubation with the primary antibody at 4 °C, the membrane was incubated for 2 hours at room temperature with HRP-conjugated secondary antibodies (Zsbio, Beijing, China). Bands were detected by chemiluminescence.

Plate colony formation assay

Cells (800/well) were inoculated into 6-well plates and cultured for 14 days. Following 30-minute paraformaldehyde fixation, plates were stained with 0.1% crystal violet. Finally, the number of individual colonies was counted. Colonies containing more than 50 cells were manually counted.

Transwell assay

For invasion assays, Matrigel was added to Transwell chambers. Cells (2 × 10⁴) resuspended in serum-free media were added to the upper chamber; lower chambers contained 10% serum medium. After 24-hour incubation at 37 °C, the migratory or invaded cells were fixed in 10% trichloroacetic acid and stained with 0.1% crystal violet. Cells were then photographed and enumerated microscopically.

Tube formation assay

Matrigel (50 μL/well) was added to 96-well plates and solidified at 37 °C for 2 hours. HUVECs (2 × 10⁴/well) were inoculated onto gels and incubated at 37 °C for 6-8 hours. Photographs were taken under a microscope, and the number of tubes was counted.

Statistical analysis

Statistical analyses were performed using SPSS 21.0 and GraphPad Prism 5.0. The data were expressed as the mean ± SD. Data were analyzed using Student's t-test; P < 0.05 was considered statistically significant.

RESULTS

CHMP2A expression is significantly upregulated in multiple tumors and correlates with immune cell infiltration levels

To study the function of CHMP2A in tumor growth, we conducted a pan-cancer investigation. The expression level of CHMP2A was found to be significantly upregulated in a variety of tumors, including breast invasive carcinoma, cholangiocarcinoma, ESCA, and kidney chromophobe (Figure 1A). CHMP2A exhibited a substantial correlation with worse prognosis in individuals with ESCA and low-grade glioma (Figure 1B and C). Further immune infiltration analysis showed that CHMP2A was significantly negatively correlated with the infiltration levels of multiple immune cells (Figure 1D). Thus, CHMP2A was highly expressed in a variety of tumors and correlated with the level of immune cell infiltration.

Figure 1 CHMP2A expression is significantly upregulated in multiple tumor types and correlates with the level of immune cell infiltration. A: CHMP2A mRNA levels across different cancer types from The Cancer Genome Atlas data; B and C: Correlation analysis of CHMP2A mRNA with overall survival and progression-free interval in tumor patients; D: Correlation between CHMP2A expression levels and the degree of immune cell infiltration. The numbers in the heatmap represent the corresponding P values of the correlation analysis. ^aP < 0.05; ^bP <0.01. TPM: Transcripts per million; ESCA: Esophageal carcinoma.

CHMP2A expression is significantly upregulated in esophageal cancer and positively correlates with lymph node metastasis

We first explored the expression level of CHMP2A in esophageal cancer using the TCGA database. The results showed that the CHMP2A mRNA expression was significantly upregulated in esophageal cancer (Figure 2A). CHMP2A expression was significantly increased in eight cases of ESCC tissues vs paired normal tissues (Figure 2B). Additionally, TCGA data showed CHMP2A positively correlated with lymphatic metastasis (Figure 2C).

Figure 2 CHMP2A expression is elevated in esophageal cancer and predicts poor prognosis. A and B: CHMP2A mRNA expression in esophageal cancer patients from The Cancer Genome Atlas data; C: Correlation of CHMP2A expression with N stage; D-F: Kaplan-Meier curves elucidate the correlation between CHMP2A expression and overall survival, disease-specific survival, and progression-free interval in patients with esophageal cancer; G: Nomogram showing patients with esophageal cancer projected to have 1-, 3-, and 5-year survival probability; H: Correction curve demonstrating the degree of agreement between predictions and actual observations; I: Receiver operating characteristic curve showing the diagnostic value of CHMP2A in patients with esophageal cancer. ^aP < 0.05; ^bP <0.01. AUC: Area under the curve; TPM: Transcripts per million; TPR: True positive rate; FPR: False positive rate.

Elevated CHMP2A expression is associated with poor prognosis in esophageal cancer patients

The relationship between CHMP2A expression and patient prognosis was then examined by plotting Kaplan-Meier curves. Compared to individuals with low CHMP2A expression in esophageal cancer, those with high CHMP2A expression exhibited noticeably poorer overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI; Figure 2D-F). These findings suggest that CHMP2A expression serves as an important prognostic indicator in esophageal cancer. We constructed a nomogram based on CHMP2A expression, T stage, N stage and M stage to predict 1-year, 3-year, and 5-year survival probabilities in esophageal cancer patients. The results showed that higher levels of CHMP2A expression and later T stage, N stage and M stage were predictive of poorer survival outcomes in esophageal cancer patients (Figure 2G). The calibration curves demonstrate a satisfactory alignment between the predictions and the actual observations (Figure 2H), and the receiver operating characteristic (ROC) curve demonstrated that CHMP2A exhibited good specificity for the diagnosis of ESCC patients (Figure 2I). Therefore, CHMP2A expression levels may be useful indicators for esophageal cancer prognosis and diagnosis.

CHMP2A expression is associated with the infiltration of multiple immune cells in ESCC

The ESTIMATE method was used to estimate the amounts of stromal and immune cell infiltration in ESCC tumor tissues. CHMP2A expression was negatively associated with both stromal and immune cell infiltration (Figure 3A). The ssGSEA and CIBERSORT algorithms revealed negative connections between CHMP2A expression and many immune cells, including T cells, NK cells, and macrophages. (Figure 3B and C). Subsequently, ESCC patients were divided into two groups based on their CHMP2A expression levels, and the extent of immune cell infiltration was evaluated. Patients with high CHMP2A expression demonstrated significantly reduced immune cell infiltration in comparison to the low CHMP2A expression group (Figure 3D).

Figure 3 CHMP2A expression is associated with infiltration of multiple immune cells in esophageal squamous cell carcinoma. A: Correlation of CHMP2A with immune, estimate, and stromal as analyzed by the ESTIMATE algorithm; B and C: Relationship between CHMP2A expression and immune cells as elucidated by the single sample gene set enrichment analysis and CIBERSORT algorithms; D: Elevated CHMP2A expression inhibited the infiltration of multiple immune cells; E: Relationship between CHMP2A expression and immune checkpoint expression levels, including CD274 (PD-L1), CTLA4, HAVCR2, LAG3, PDCD1 LG2, PDCD1 (PD-1), SIGLEC15, and TIGIT. ^aP < 0.05; ^bP < 0.01. ssGSEA: Single sample gene set enrichment analysis; TPM: Transcripts per million.

Subsequently, correlation analysis showed a negative correlation between the expression levels of CHMP2A and those of eight important immune checkpoint inhibitors, including CD274 (PD-L1), CTLA4, HAVCR2, LAG3, PDCD1 LG2, and TIGIT (Figure 3E). Furthermore, single-cell sequencing analysis based on CHMP2A expression levels demonstrated that CHMP2A expression was predominantly distributed among immune cells in ESCC tissues (Figure 4A). Subsequent investigation revealed that CHMP2A expression was mostly detected in T cells and dendritic cells. (Figure 4B-E).

Figure 4 Single-cell transcriptomic analysis of the distribution of CHMP2A expression in different cell types in the tumor microenvironment. A: The correlation between CHMP2A expression and cell type in GSE160269 and GSE173950 datasets; B and C: CHMP2A expression in multiple cell types in the GSE160269 dataset; D and E: CHMP2A expression in multiple cell types in the GSE173950 dataset. ESCA: Esophageal carcinoma; TPM: Transcripts per million.

Functional enrichment analysis of genes co-expressed with CHMP2A

We performed CHMP2A correlation analysis of genes co-expressed with CHMP2A obtained from the UALCAN database (Figure 5A). GO enrichment analysis indicated that CHMP2A co-expressed genes were linked to primary active transmembrane transporter activity, NADH dehydrogenase, and the activation of multiple signaling pathways (Figure 5B). Furthermore, KEGG enrichment analysis revealed that upregulation of CHMP2A co-expressed genes was associated with a variety of signaling pathways, including thermogenesis, metabolic pathways, and the AMPK signaling pathway (Figure 5C).

Figure 5 Functional enrichment study of genes co-expressed with CHMP2A. A: The CHMP2A gene was subjected to correlation analysis with the co-expressed genes obtained; B: Genes co-expressed with CHMP2A were analyzed using Gene Ontology enrichment; C: Analysis of Kyoto Encyclopedia of Genes and Genomes enrichment for genes co-expressed with CHMP2A. NAFLD: Non-alcoholic fatty liver disease; TPM: Transcripts per million.

CHMP2A expression is upregulated in ESCC and correlates with poor prognosis

To investigate the expression of CHMP2A in ESCC, we collected tumor tissues from 50 clinical ESCC patients and performed IHC staining. CHMP2A was mostly expressed in the cytoplasm (Figure 6A). The expression levels of CHMP2A in ESCC tissues and corresponding paracancerous tissues were also investigated in the 50 cases. In tumor tissues, CHMP2A was highly expressed in 31 cases and lowly expressed in 19 cases. In para-carcinoma tissues, CHMP2A was highly expressed in 17 cases and lowly expressed in 33 cases (Figure 6B and Table 1). In conclusion, CHMP2A expression was significantly higher in ESCC tissues than in para-carcinoma tissues.

Figure 6 CHMP2A expression is upregulated in esophageal squamous cell carcinoma and correlates with a poor prognosis. A and B: Immunohistochemistry detection of CHMP2A expression in esophageal squamous cell carcinoma (ESCC) tissues and para-carcinoma tissues; C: Correlation between CHMP2A expression and prognosis in ESCC patients (n = 50). ESCC: Esophageal squamous cell carcinoma.

Table 1 Correlation between CHMP2A expression and clinical-pathological parameters in patients with esophageal squamous cell carcinoma.

Characteristics	n	CHMP2A		P value
		High	Low
Total	50	31	19
Gender				0.895
Male	31	19	12
Female	19	12	7
Age (year)				0.109
≤ 60	10	4	6
> 60	40	27	13
Size (cm)				0.777
< 4	33	20	13
≥ 4	17	11	6
Pathological grading				0.042
I-II	13	5	8
III	37	26	11
Lymph node metastasis				< 0.001
Yes	19	15	4
No	31	16	15
Venous invasion				0.011
Positive	15	11	4
Negative	35	12	23
Nerve infiltration				0.081
Positive	12	10	2
Negative	38	21	17
Differentiation				0.214
Well	23	15	8
Moderate	17	8	9
Poor	10	8	2
Smoking				0.085
Yes	18	14	4
No	32	17	15
Alcohol use				0.287
Yes	12	9	3
No	38	22	16
Family history				0.026
Yes	7	7	0
No	43	24	19

The relationship between CHMP2A expression and the prognosis of ESCC patients was investigated by plotting Kaplan-Meier curves. In ESCC tissues, the survival time of patients with positive CHMP2A expression was significantly shorter than that of patients with negative expression (Figure 6C). This indicated that CHMP2A overexpression is associated with an unfavorable prognosis in ESCC patients. Furthermore, CHMP2A expression was significantly correlated with pathological grading, lymph node metastasis, venous invasion, and family history of tumors (Table 1). CHMP2A expression levels and relevant clinicopathological parameters were further incorporated into univariate and multivariate Cox regression analysis (Table 2). The results showed that lymph node metastasis, lymphovascular invasion, and high CHMP2A expression were all significantly associated with patient prognosis in the univariate analysis. However, in the multivariate analysis, CHMP2A expression, lymph node metastasis, and lymphovascular invasion did not demonstrate independent prognostic value, suggesting that the impact of CHMP2A on patient prognosis may be confounded by other clinicopathological factors.

Table 2 Univariate and multivariate Cox regression analysis of overall survival in esophageal squamous cell carcinoma patients.

Characteristics	Univariate analysis		Multivariate analysis
	HR (95%CI)	P value	HR (95%CI)	P value
Gender	1.909 (0.826-4.410)	0.130
Age (year)	1.266 (0.428-3.743)	0.670
Size (cm)	1.139 (0.478-2.716)	0.769
Pathological grading	1.331 (0.491-3.611)	0.575
Lymph node metastasis	4.428 (1.785-10.982)	0.001	3.416 (0.866-13.483)	0.079
Venous invasion	3.254 (1.384-7.654)	0.007	2.060 (0.816-5.205)	0.126
Nerve infiltration	1.073 (0.396-2.911)	0.889
Differentiation	1.854 (0.777-4.424)	0.164
Smoking	0.640 (0.250-1.636)	0.351
Alcohol use	0.676 (0.229-1.999)	0.479
Family history	1.060 (0.313-3.582)	0.926
CHMP2A expression	2.965 (1.090-8.065)	0.033	0.974 (0.219-4.327)	0.972

Additionally, ROC curve analysis was performed to evaluate the discriminatory ability of CHMP2A expression (Supplementary Figure 1). The survival status and follow-up duration used for this analysis are detailed in Supplementary Table 1. The AUC was 0.633 (95%CI: 0.524-0.742), indicating only modest discriminatory capacity.

Elevated CHMP2A levels correlated with a reduction in the infiltration of immune cells within ESCC tissues

To further validate the relationship between CHMP2A expression and tumor immune cell infiltration, we performed IHC staining for the classic immune cell markers CD3 and CD20 on the same cohort of ESCC patient tissues. The results demonstrated that the CHMP2A-low expression group exhibited a significantly higher number of CD3⁺ T lymphocytes and CD20⁺ B lymphocytes, along with greater infiltration density. In contrast, these immune cells were sparsely distributed and markedly reduced in number in the CHMP2A-high expression group (Figure 7). These findings indicate that high CHMP2A expression is associated with reduced T-cell and B-cell infiltration in ESCC tissues.

Figure 7  Levels of CD3⁺ T cells and CD20⁺ B cells in the same esophageal squamous cell carcinoma patient’s tumor tissues.

Elevated CHMP2A expression promotes the proliferation, migration, and invasion of ESCC

To investigate the biological function of CHMP2A in ESCC, an ESCC cell model with CHMP2A overexpression was successfully constructed (Figure 8A and B). The colony formation assay demonstrated that ESCC cells exhibited a significant increase in colony formation ability following CHMP2A overexpression (Figure 8C). Furthermore, the Transwell assay demonstrated that CHMP2A overexpressed ESCC cells exhibited enhanced migration and invasion capabilities in comparison with the control group (Figure 8D and E). In conclusion, high CHMP2A expression significantly promoted the proliferation, migration, and invasion of ESCC cells.

Figure 8 Elevated CHMP2A expression promotes the proliferation, migration, and invasion of esophageal squamous cell carcinoma. A and B: Protein expression level of CHMP2A detected by western blotting (n = 3); C: Plate colony formation test assessing the impact of CHMP2A expression on the proliferation of esophageal squamous cell carcinoma (ESCC) cells (n = 3); D and E: Influence of CHMP2A expression on the migratory and invasive capabilities of ESCC cells (n = 3), examined using Transwell assay. ^aP < 0.05.

Elevated CHMP2A expression significantly promotes angiogenesis in ESCC

To investigate the role of CHMP2A overexpression on ESCC angiogenesis, we collected ESCC conditioned medium (CM) from the OE-CHMP2A group and induced HUVECs for 48 hours. The results showed a significant increase in the migratory and invasive abilities of HUVECs in the OE-CHMP2A CM group (Figure 9A and B). In addition, the tube formation assay showed that the number of tube formations by HUVECs was significantly increased in OE-CHMP2A CM compared with the control group (Figure 9C). Thus, high expression of CHMP2A significantly promoted angiogenesis in ESCC.

Figure 9 Elevated CHMP2A expression significantly promotes angiogenesis in esophageal squamous cell carcinoma. A and B: Transwell test evaluating the impact of OE-CHMP2A conditioned medium (CM) on the migratory and invasive capabilities of human umbilical vein endothelial cells (HUVECs; n = 3); C: Tube formation assay detecting the effect of OE-CHMP2A CM on the tube formation of HUVECs (n = 3). ^aP < 0.05; ^bP < 0.01. CM: Conditioned medium.

DISCUSSION

Esophageal cancer represents a major public health issue and is a critical focus of clinical research. Research on esophageal cancer contributes to elucidating the mechanisms of invasion and metastasis, while providing insights into immune microenvironment remodeling, molecular signaling pathways, and resistance mechanisms. Accumulating evidence has revealed the multifaceted roles of CHMP2A in tumorigenesis[25]. As a core ESCRT-III component, CHMP2A is primarily involved in membrane scission and intracellular trafficking[15,26]; its aberrant activation may induce membrane dynamics alterations that promote tumor growth and migration[25]. CHMP2A may promote tumor cell invasiveness by regulating exosome release and transporting pro-metastatic molecules (e.g., MMPs, integrins) into the tumor microenvironment[27-29]. Furthermore, by modifying vesicular trafficking, CHMP2A may change oncogenic signaling turnover. For example, the ESCRT complex is essential for the endosomal sorting and degradation of receptors like EGFR, which controls downstream PI3K/AKT signaling activity and fosters a more malignant phenotype[30].

Our clinical sample analysis showed that CHMP2A was significantly upregulated in ESCC and strongly correlated with poor prognosis. Kaplan-Meier curves showed markedly shorter OS, DSS, and PFI in the high-expression group; ROC curves and the nomogram further supported its potential diagnostic and prognostic value. IHC results also confirmed the upregulation of CHMP2A at the protein level, which was consistent with worse clinical outcomes. Notably, aberrant upregulation of CHMP2A has also been reported in other malignancies, such as endometrial carcinoma, colorectal cancer, and lung adenocarcinoma, suggesting that its overexpression may be a common feature associated with progression and unfavorable prognosis across multiple cancer types.

From a tumor biology perspective, sustained proliferation constitutes a fundamental driver of tumor expansion and clonal evolution, and is clinically correlated with poor survival outcomes[31]. On the other hand, tumor cells’ migratory and invasive capacities, which depend on dynamic processes including the EMT, cytoskeletal remodeling, and extracellular matrix breakdown, represent crucial stages of the metastatic cascade[32,33]. In the present study, CHMP2A overexpression was shown to significantly enhance colony formation, migration, and invasion in ESCC cells.

Meanwhile, angiogenesis is considered a pivotal “switch” for the transition of solid tumors from dormancy to progression, as it not only supplies oxygen and nutrients but also provides routes for tumor cells to enter circulation[34,35]. Our study provides functional evidence that CHMP2A contributes to this process: CM from CHMP2A-high cells significantly promoted migration and tube formation in HUVECs, demonstrating its pro-angiogenic capacity.

Collectively, emerging evidence positions CHMP2A as a negative regulator of antitumor immunity. Studies across various models indicate that CHMP2A expression facilitates immune evasion by impairing NK cell cytotoxicity[20], inhibiting CD8⁺ T cell priming via suppression of dendritic cell cross-presentation[36], and fostering an immunosuppressive microenvironment characterized by reduced T cell infiltration and increased MDSC accumulation[20,37]. This study’s immune infiltration analysis revealed that reduced infiltration of CD8⁺ T cells, dendritic cells, and NK cells, among other immune cell types, was significantly associated with elevated CHMP2A expression. Furthermore, there were negative correlations between CHMP2A expression and several immune checkpoint molecules (PD-L1, CTLA4, HAVCR2), indicating that CHMP2A may promote an immunosuppressive microenvironment by decreasing immune cell infiltration and lowering the expression of checkpoint molecules, which would allow tumor cells to elude immune surveillance. Singlecell sequencing further indicated that CHMP2A is predominantly expressed in certain immune subsets rather than in malignant cells themselves, suggesting that its protumor effects could be mediated, at least in part, through immune modulation.

Abnormal tumor angiogenesis is a key driver of the immunosuppressive microenvironment[38], characterized by vascular structural disorganization and functional dysfunction, accompanied by marked hypoxia and acidosis. This pathological environment collectively restricts immune cell infiltration and function[39]. Additionally, proangiogenic factors that are overexpressed in tumor vasculature not only stop T-cell activation and function and slow down the maturation of dendritic cells, but they also help bring in inhibitory immune cells like M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells[40]. Therefore, by controlling the angiogenesis process in ESCC, CHMP2A may affect the infiltration behavior of various immune cells within tumor tissue.

To investigate the mechanistic role of CHMP2A in ESCC, we examined its co-expressed genes using KEGG and GO enrichment analysis. The results indicated significant enrichment in key biological processes, including transmembrane transporter activity, metabolic pathways, and the AMPK signaling pathway. AMPK serves as a central regulator of cellular energy sensing and metabolic reprogramming, playing a critical role in the tumor microenvironment under stress conditions such as hypoxia and nutrient deprivation. Previous studies have demonstrated that aberrant activation of AMPK under energetic stress promotes tumor cell survival, proliferation[41,42], and angiogenesis[43]. Therefore, we speculate that CHMP2A may regulate AMPK signaling to alter cellular energy homeostasis, drive metabolic reprogramming, and enhance malignant phenotypes in ESCC, including proliferation, migration, invasion, and angiogenesis. Investigating this potential mechanism may reveal new combination therapies targeting cellular trafficking and metabolism.

CONCLUSION

In conclusion, this work is the first to methodically examine CHMP2A’s expression pattern and functional involvement in ESCC. The findings indicated that CHMP2A is significantly upregulated in ESCC tissues and is closely associated with a poor prognosis, and functional analyses confirmed that CHMP2A enhances the proliferation, migration, invasion, and angiogenesis capabilities of ESCC cells. While our clinical data and bioinformatic analyses strongly suggest a role for CHMP2A in promoting ESCC progression, future studies involving animal models and cell-based assays are required to definitively establish the causal relationship and the precise molecular mechanisms involved. It should be noted that the ROC analysis yielded an AUC of 0.633, which suggests that CHMP2A alone has limited accuracy as a standalone predictive marker. Future studies incorporating multi-gene panels or combined clinical parameters may be necessary to improve predictive performance.