Investigating DOCK9 as a Potential Prognostic Marker: Implications for Angiogenesis and Immunity in Esophageal Squamous Cell Carcinoma

Abstract Background Esophageal squamous cell carcinoma (ESCC) remains a significant public health concern worldwide due to its high incidence and mortality rates. Consequently, developing a robust predictive risk model centered on RNA expression and identifying novel target genes in ESCC is paramount. While previous studies have implicated DOCK9 in tumor prognosis, its specific role in ESCC remains to be elucidated. This study aims to investigate the prognostic significance of DOCK9 and its biological functions in ESCC. Methods We reanalyzed RNA microarray datasets (GSE67269, GSE20347, GSE53625) from the Gene Expression Omnibus (GEO) database to identify potential survival-associated genes and assess their expression in ESCC. We also comprehensively analyzed single-cell RNA sequencing (scRNA-seq) data from GSE160269, GSE188990, and The Cancer Genome Atlas (TCGA) ESCC cohorts to explore potential molecular mechanisms. Kaplan-Meier analysis determined the correlation between DOCK9/CD31 and prognosis. Protein expression of DOCK9 in ESCC tissues was examined through immunohistochemistry and Western blot analyses in a small cohort of six ESCC patients. The co-expression of DOCK9 and CD31 was verified using Immunofluorescence (IF) analysis. Additionally, we investigated the functional impact of DOCK9 on human umbilical vein endothelial cells (HUVECs) proliferation, migration, and tube formation using cell counting kit-8 (CCK-8) assay, 5-ethynyl-2’-deoxyuridine (EdU) staining assay, wound-healing assay, and tube formation assay. Results Our study identified 21 genes from GSE67269, GSE20347, and GSE53625 datasets based on differential and univariate COX analyses, enabling us to construct a prognostic risk model for ESCC where DOCK9 plays a central role. DOCK9 expression was markedly lower in cancerous tissues than in ESCC patients' paracancerous tissues. Furthermore, DOCK9 emerged as a survival-related risk factor in ESCC, exhibiting high expression in tumo endothelial cells (TECs) and playing a role in angiogenesis and tumor-associated fibroblasts development. Our immunity analysis suggested that DOCK9 might influence the immune landscape, and the DOCK9/CD31 ratio could serve as an indicator for assessing the response to immunotherapy in ESCC. Functionally, our assays indicated that inhibiting DOCK9 expression curtailed the proliferation, migration, and tube formation of ANG-2-stimulated HUVECs, a process potentially related to the ANG-2/Tie2 axis. Conclusions Our study provides evidence that DOCK9 could serve as a potential prognostic biomarker associated with angiogenesis and immune therapy in esophageal squamous cell carcinoma, thereby opening avenues for improved therapeutic strategies.