As a prevalent gastrointestinal malignancy, gastric cancer (GC) is distinguished by its high incidence, aggressive progression profile and high mortality rates. There were >968,000 novel cases of stomach cancer in 2022 and close to 660,000 deaths, ranking the disease as fifth in terms of both incidence and mortality worldwide (1). Despite a decline in the global prevalence and death rate of GC, rates remain high in Eastern Asian countries (2). The majority of patients with GC are typically diagnosed already at advanced stages during consultations, resulting in a poor prognosis post-surgery. This scenario poses considerable public health challenges (3). Consequently, the quest for efficacious molecular markers for GC assumes paramount importance in facilitating precise treatment design, extending patient survival and enhancing their overall quality of life.

The primary approach for treating GC continues to involve conventional surgical procedures, followed by postoperative radiotherapy and chemotherapy (4). However, the 5-year survival rate following surgery remains low (<10%) (5). With the rapid advancement of molecular biology research, GC treatment methods have undergone continuous improvements, including the introduction of immunotherapy, such as programmed cell death protein (PD)-1/PD-L1 inhibitors and cytotoxic T lymphocyte antigen 4 inhibitors (6). Additionally, the emergence of chemotherapeutic and molecular-targeted drugs, such as oxaliplatin and herceptin, has instilled optimism among patients with intermediate and advanced stages of GC. Although these interventions have led to improved postoperative survival rates, the prognosis for GC, particularly in cases in, remains poor due to the severe toxic side effects, limited sensitivity and specificity of drugs (7). This is especially the case in patients at intermediate and advanced stages (7). The metastatic propensity of GC represents a formidable obstacle in the treatment paradigm (8).

Therefore, the exploration of effective strategies to inhibit GC's metastatic spread and identification of molecular markers for prognostic assessment is required.

E74-like factor 1 (ELF1), which is highly homologous to the Drosophila E74 factor (9) is a transcription factor inducible by ecdysone in Drosophila and a member of the Ets transcription factor family (9). ELF1 is located on chromosome 13q13 and consists of 619 amino acid residues, possessing the ability of both transcriptional activation and repression of target genes depending on the physiological context. Posttranslational processing determines its subcellular localization, biological activity and metabolic degradation (10). The functional regulation of ELF-1 is complex. ELF1 protein exists in a 80-kDa form in the cytoplasm and enters the nucleus in a 98 kDa form after phosphorylation and glycosylation (11). It is predominantly expressed in lymphocytes, where its posttranslational modifications bestow ELF1 with regulatory functions, enabling it to bind to gene promoters or enhancers critical for the selection, survival and maturation of diverse immune cells (12). The importance of ELF1 extends to its association with the development and metastasis of various malignancies (13), such as glioma, oral squamous cell carcinoma, endometrial carcinoma, nasopharyngeal carcinoma and prostate cancer, and colon cancer. Long non-coding RNAs (lncRNAs) are associated with cancer progression in GC (14). E2F transcription factor 1 has been shown to activate the transcription of terminal differentiation-induced non-coding RNA by binding to its promoter region, thereby promoting the proliferation of GC cells and inhibiting apoptosis (15). lncRNAs NONHSAT057282 and NONHSAG023333 can regulate genes associated with chemoresistance, such as GSTP1, BTG3, SOCS3, and BRAC2, by interacting with the transcription factors ELF1 and E2F1. ELF1 may become a new player in chemoresistance through its interaction with different lncRNA interactions involved in tumor therapy (16). So, it was hypothesized that ELF1 may be associated with GC.

Tumor cells, together with extracellular matrix (ECM), cancer-associated fibroblasts (CAF), vascular-associated smooth muscle cells, pericytes, endothelial cells, mesenchymal stem and immune cells collectively constitute the complex tumor microenvironment (TME) (17). The development, progression and ultimately the prognosis of malignancies, are profoundly influenced by the characteristics of tumor cell invasion and metastasis (18). A pivotal process in this cascade involves ECM degradation, which is tightly regulated by MMPs and tissue inhibitors of metalloproteinases (TIMPs) (19). Among MMPs, MMP-9 is of particular importance as a key protease responsible for ECM degradation, where it serves a crucial role in various types of cancers, such as breast cancer (20,21). During cancer progression, ECM homeostasis is dynamically disrupted by MMP9, enabling cancer invasion and metastasis through the ECM barrier. MMP2 and MMP9 have been previously found to be upregulated in GC tissues (22). MMP9 rs3918242 polymorphism has been associated with the risk of various cancers, including lung, prostate, breast, and colorectal cancers (23). The chromosomal location (20q12-1q13) where MMP9 is located, has been identified as one of the most common regions of genomic gain in GC (24). Therefore, the progression of GC is highly likely to be influenced by altered MMP9 expression.

However, the precise expression pattern, interaction with MMP9 and predictive value of ELF1 in GC remain elusive. Therefore, the present study aimed to explore the expression profile of ELF1 in GC, its relationship with MMP9 and its (combined with MMP9) associations with various clinicopathological parameters, survival and prognosis of patients with GC. Bioinformatics and clinical sample analyses would be performed. In addition, relevant mechanisms, such as the association of ELF1 with epithelial-mesenchymal transition (EMT), angiogenesis and immune infiltration, were explored.

GC is a common malignant tumor worldwide and is characterized by high incidence and mortality rates, with multifactorial associations encompassing dietary habits (including high-fat intake, elevated salt consumption, and smoking), infections (such as Helicobacter pylori and Epstein-Barr virus infections) and genetic predispositions (35,36). Familial clustering is discernible in GC development, with ~25% of autosomal dominant diffuse GC-prone families harboring E-cadherin mutations (37). The aggressive and metastatic attributes of GC are closely associated with the poor patient prognosis, with a median survival of <12 months (38). Although significant improvements have been made in identifying biomarkers and molecular targeted agents (trastuzumab, Bevacizumab, Panitumumab, Everolimus, etc.) for improving patient prognosis, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) (39), mTOR and PI3K (40), predictive biomarkers and corresponding targeted agents specifically addressing GC invasion and metastasis remain scarce.

Ets genes, known for their erythroblast transformation specificity, constitute a class of highly conserved oncogenes that serve important roles in regulating tumor infiltration and metastasis (41). Among the genes in the Ets transcription factor family, ELF1 is one of the most prominent members (42). ELF1 is a T cell-specific transcription factor that was originally cloned from a human T-cell library through hybridization to a probe encoding the DNA-binding structural domain (Ets structural domain) of human Ets-1 cDNA. This transcription factor mainly exerts its influence by binding to gene promoters or enhancers (42) and can function to either activate and repress the expression of target genes. In addition, it is unique in being subject to glycosylation and phosphorylation (10). Elevated levels of ELF1 expression have been previously associated with the initiation and progression of various malignancies, such as pancreatic and colon cancer (13,43). The high expression of ELF1 has been associated with poor prognosis in patients with endometrial (44) and ovarian cancer (45). ELF1 can also promote the malignant progression of glioma (46). In addition, ELF1 has been found to promote the proliferation of oral squamous cell carcinoma cells by increasing the expression of β-catenin mRNA (47). By contrast, ELF1 can activate the doublecortin-like kinase 1/Janus kinase/STAT signaling pathway, thereby promoting the malignant progression of pancreatic cancer (13). Another recent study has elucidated ELF1 effect on the biological behavior of colon cancer, which was by modulating the transcriptional activation of serine peptidase inhibitor kazal type 4, thereby promoting colon cancer progression (43). However, conflicting reports exist that also suggest ELF1 to serve an inhibitory role in certain cancers, such as prostate cancer (48) and Hodgkin's lymphoma (49).

The extracellular matrix (ECM) is divided into basement membrane and interstitial matrix, and is composed of various proteins such as type IV collagen, laminin, elastin, and hyaluronic acid, which provide structural support for cells and are also involved in the development of epithelial cells. The composition and stability of the ECM is closely related to the protein-protein and polysaccharide-protein binding in the extracellular matrix, which is one of the main barriers to prevent tumor metastasis. Degradation of ECM facilitates tumor cell invasion and metastasis and is a key trigger of EMT, where MMPs serve a key role because they can degrade almost all ECM components (50). The MMP family includes 26 different members, which can then be divided into various subtypes, including collagenases, gelatinases, matrix proteases and membrane-type MMPs (22). Previous studies have shown that MMP9 (a member of the gelatinase family), which can degrade the ECM and basement membranes, in addition to being associated with cancer cell adhesion and migration, is an important marker for predicting poor tumor prognosis in colorectal cancer (51) and endometrial carcinoma (44). In addition, MMP9 has been reported to positively correlate with the infiltration of a diverse range of immune cell types, including Th1 cells, neutrophils and macrophages, and regulates their transport in uveal melanoma and clear Cell Renal Cell Carcinoma, which is essential for establishing and coordinating the tumor immune environment (52,53). The influence of MMP9 has been documented to extend to angiogenic and lymphangiogenic factors, such as VEGF, TGF-β, tumor necrosis factor-α, ΙL-8 and EGFR (54). Elevated levels of MMP9 mRNA and protein expression have been frequently found in various cancer types, including ovarian (55), colorectal (22), breast (56) and lung cancers (57). High MMP9 expression is also consistently associated with advanced tumor stages and adverse clinical outcomes, underscoring its potential as a prognostic indicator for patients with cancer of uveal melanoma and breast cancer (54,56).

Ets family proteins enhance the expression of the urokinase-type plasminogen activator (uPA) gene by binding to its inducible enhancer region. uPA in turn activates a variety of MMPs (MMP1, MMP2 and MMP9) whilst promoting protein hydrolysis in the ECM (44). ELF1 serves a pivotal role in tumor angiogenesis, ECM remodeling and metastasis, by regulating various stages of neovascularization (which encompasses the generation of proangiogenic factors, MMPs and protease inhibitors) (58). In addition, ELF1 overexpression has been documented to contribute to a critical facet of tumor infiltration (pancreatic and colon cancer), namely ECM penetration. This was mediated through the transcriptional control of genes encoding enzymes involved in ECM degradation, such as MMP9(59). The aforementioned findings suggest that ELF1 may regulate tumorigenesis and progression through the TME, which is associated with EMT, angiogenesis and immune infiltration. The present study revealed the expression profiles of ELF1 in GC, the possible mechanistic interplay between ELF1 and MMP9, in addition to their collective effect on patient survival and prognosis.

Initial analysis using the GEPIA database and R 4.2.1 software indicated elevated ELF1 expression in GC. Subsequently, RT-qPCR analysis corroborated these findings, suggesting the diagnostic value of ELF1 expression in distinguishing GC from adjacent normal tissues. Therefore, it became possible to initially distinguish GC tissues from normal tissues based on ELF1 expression. Recent in vitro experiments showed that ELF1 overexpression directly activated MMP9 promoter activity, which identified an ELF1-triggered transcriptional mechanism by which neurotrauma upregulated MMP9 expression in the dorsal root ganglion (59). ELF1 has also been shown to activate forkhead box D3-antisense 1 to promote the migration, invasion and EMT of osteosarcoma cells by sponging microRNA-296-5p, preventing the inhibition of zinc finger CCHC-type containing 3(60). Transcription factor ELF1 activates MEIS1 transcription and promotes MEIS1 expression. Overexpression of MEIS1 increases growth factor independent protein 1) expression by activating the GFI1 enhancer, but decreases FBW7 expression and thus promoting glioma cell proliferation (decreased PCNA), migration and invasive ability (decreased MMP-9), and reducing apoptosis (increased capase-3) promotes glioma development (46). ELF1 is involved in prostate cancer tumor cell migration and EMT by interfering with the oncogenic ETS function of ETS/Activator protein-1 cis-regulatory motifs (48). To elucidate the association between ELF1 and EMT in GC, TMA-IHC was performed. The results revealed increased levels of ELF1 and MMP9 proteins in GC tissues compared with adjacent normal tissues, where both exhibited positive associations with key clinical parameters, including T, N and M stages, TNM staging, MVI, lymphatic invasion and blood CEA levels. Survival and prognostic analyses also revealed that patients with high expression levels of ELF1 and MMP9 proteins and advanced TNM staging had shorter survival and poorer prognoses. The findings of IHC revealed a positive correlation between the expression levels of ELF1 and MMP9. These findings suggest that high ELF1 expression may serve an important role in the pathogenesis and malignant progression of GC, which can be exploited to predict the survival and prognosis of patients with GC. However, the specific mechanistic interactions between ELF1 and MMP9 remain unclear, which require further study.

Based on heterogeneity of gastric cancer and the rapid development of molecular biology, TCGA proposed a molecular classification system for gastric cancer by analyzing data from multiple platforms, consisting of four distinct subtypes, including Epstein-Barr virus+ (accompanied by PIK3CA and ERBB2 amplification), microsatellite instability (usually accompanied by MLH1 silence, high-frequency mutation of ERBB2 and PIK3CA), genomically stable (accompanied by CDH1 gene mutation) and chromosome instability (accompanied by high mutation rates of TP53 and ERBB2 amplification (30). TIMP1, an endogenous inhibitor of MMP-9, executes a role not only in impeding the matrix degradation activity of MMP-9 whilst also activating pivotal cytokines, including VEGF, TGF-α,TGF-β (transforming growth factor-β) and CAM (cell adhesion molecule), binding cell surface protein CD63, activating FAK-PI3K/AKT (focal adhesion kinase-phosphatidylinositol 3 kinase/protein kinase B) signaling pathway and MAPK) signaling pathway, leading to tumor cell proliferation (61). VEGFA and KDR are involved in tumor angiogenesis in addition to molecular subtype identification of GC (62,63). The CDH1 gene encodes the vital cell adhesion molecule E-cadherin, which is crucial for maintaining epithelial tissue integrity. E-CAD reduction is associated with EMT), generation of stem-cell, and metastasis (64,65). Analysis of the GEPIA database in the present study revealed associations of ELF1 with MLH1, PIK3CA and CDH1. ELF1 has been previously reported to serve an indispensable role in the survival, differentiation and maturation of T and B lymphocyte cells, especially NK/T cells, where the absence of which results in T cell apoptosis (66). Findings from the TIMER database, CIBERSORT and IHC in the present study unveiled associations between ELF1 expression and B cells and CD4+ T cells. CD4⁺ and CD8+ T cells contribute to tumor growth and became effective targets for cancer survival, prognosis and treatment in lung cancer (67). B cells can produce cytokines (e.g., IL-10) that inhibit the antitumor response of T cells (inhibit CTL-mediated tumor clearance) and promote the production of immune complexes (generated by antitumor antibodies), inducing tumorigenesis. Breg (regulatory B) cells exhibit pro-tumorigenic activity due to a lack of response to CTLA4, resulting in a shortened overall survival in cutaneous melanoma, and produce adenosine, which is involved in the inhibition of T cell activation and/or inactivation influencing prognosis (such as urothelial bladder and gastric cancer) (68,69). CD4+ T cells can produce tumor cytokines (such as IL-4), associate with other cell types (myeloid-derived suppressor cells and tumor-associated macrophages) and transform into regulatory T cells, which ultimately exert pro-tumorigenic functions (69,70). Based on the aforementioned results, we hypothesized that ELF1 appeared to be associated with GC development, which are intricately intertwined with the TME including EMT, angiogenesis and immune infiltration.

However, the present study has several limitations. All the specimens used for this study were selected randomly, and the size and quality of specimens could not be controlled. The detection of protein expression using IHC may be affected by tumor heterogeneity and subjective scoring system analysis. The selected tumor location may not accurately represent the entire tumor due to intratumor heterogeneity. In addition, the r-value of the correlation analysis based on data from the GEPIA database was low, which require confirmation through further detailed exploration in subsequent studies. The majority of the studies on the involvement of ELF1 in EMT and angiogenesis in GC were based on bioinformatics, where the exact mechanism and related signaling pathways requires further experimental verification. However, the results may provide guidance for future prospective clinical trials. Any future studies should conduct comprehensive and in-depth studies at the cytological and molecular levels to provide a solid theoretical foundation for the study of GC molecular targets.