Cancer is a global health challenge that significantly impacts human well being and quality of life. According to World Health Organization (WHO) statistics, cancer is currently the leading cause of mortality worldwide (1). Further research has indicated that among all types of cancer, lung, breast, and stomach cancers exhibit higher incidence and mortality rates (2). Despite substantial advancements in cancer treatments, such as d targeted therapies, immunotherapy, and radiotherapy, the overall survival (OS) rate within a 5-year period remains sub-optimal for many patients (3). Additionally, the economic burden imposed by cancer on society is substantial and cannot be overlooked (4). Consequently, the identification of effective cancer biomarkers holds critical significance in improving prognostic outcomes for individuals diagnosed with cancer and alleviating the societal burden.

The human chorionic gonadotropin (hCG) beta-subunit, a hormone-specific entity, is produced by Placental syncytiotrophoblasts. Its encoding is facilitated by four distinct chorionic gonadotropin β genes (CGB, CGB5, CGB7, and CGB8), which are located within the LHB/CGB gene cluster on chromosome 19q13.3 (5). Among these four duplicated hCG beta genes, CGB5 and CGB8 exhibit the highest transcriptional activity, accounting for 62%−82% of the total beta subunit mRNA transcript pool (6). The role of CGB5 in tumors has garnered significant attention. Previous studies have provided evidence to supporting the notion that CGB5 plays a critical role in gastric cancer (7, 8), ovarian cancer (9), and squamous lung cancer (10), and is closely associated with patient prognosis. However, to date, there have been no comprehensive reports investigating the role of CGB5 across pan-cancer.

In the present study, a comprehensive analysis of the genomics, transcriptomics, epigenetics, immune microenvironment, and clinical and prognostic significance of CGB5 was conducted across various cancer types. Additionally, experimental studies were performed to examine the expression level of CGB5 in gastric cancer and to elucidate the potential mechanisms underlying its impacts on prognosis.

Our pan-cancer analysis demonstrates that aberrant CGB5 expression significantly correlates with poor prognosis across multiple cancer types, specifically in malignancies like GC and PAAD, potentially driven by genetic mutations, DNA methylation, and CNAs. Notably, CGB5 exhibits strong associations with immune cell infiltration, with distinct patterns and magnitudes observed in specific cancers. In GC, we validated CGB5 upregulation and further established its independent adverse impact on patient survival. Mechanistically, CGB5 over-expression likely influences tumor progression not only through modulation of immune infiltration but also via interactions with multiple signaling pathways. Collectively, this multi-dimensional study underscores the potential oncogenic role of CGB5 and provides a rationale for targeting CGB5 in future therapeutic strategies.

This investigation concurrently evaluated CGA (hCGα-encoded protein) to establish CG5′s functional specificity. Tumor-specific dysregulation of CGA significantly influenced prognosis across malignancies, with minimal overlap in affected cancer types between CGA and CGB5. Sex-stratified analyses further confirmed sex-independent associations between CGB5 overexpression and adverse outcomes, highlighting its tumor-specific functional profile and clinical translational potential.

Gene expression levels are influenced by numerous factors, among which genetic variations and epigenetic modifications are included (20, 21). It is notable that environmental exposures have the potential to induce epigenetic alterations, which can impact gene expression and modify the risks of diseases related to genetic variations (22). Studies suggest that CIAPIN1 overexpression promotes breast cancer by regulating cell cycle and DNA replication, while DBNDD1 hypomethylation may contribute to tumor progression by affecting DNA synthesis or damage repair (23, 24). In this study, we investigated the gene mutations and CNVs of the CGB5 gene across pan-cancer types. Gene mutations result in alterations in DNA sequences, thereby influencing the structure and function of encoded proteins. Such changes may give rise to abnormal protein functions, thereby disturbing cellular processes and gene expression regulation (25, 26). CNVs refer to the deletion or duplication of DNA segments, leading to an imbalance in gene dosage (27, 28). When genes are duplicated, the corresponding mRNA and protein levels may increase, resulting in overexpression. Conversely, gene deletions may cause a reduction in mRNA and protein production, leading to underexpression (29). Conclusively, both gene mutations and CNVs can significantly affect gene expression by influencing transcriptional regulation, altering the binding of transcription factors, and affecting mRNA stability, ultimately leading to changes in the abundance of specific gene transcripts (30–32). In addition, the methylation status of the CGB5 promoter exhibits variations among different tumor types. DNA methylation is a crucial epigenetic modification that primarily occurs at CpG dinucleotides, especially within CpG islands located in the promoter regions of genes (33, 34). High methylation levels in CpG islands can inhibit the interaction between transcription factors and DNA, thereby repressing gene transcription (35). In our study, this is supported by the negative correlation between CGB5 methylation and mRNA expression (Figure 4B), which may promote oncogenic signaling pathways, such as Wnt signaling, or immune suppression via PD-1 pathway activation (Figure 9C). These findings suggest that CGB5 methylation status could serve as a prognostic biomarker, with hypomethylation indicating aggressive tumor behavior. This mechanism provides important clues for understanding the changes in gene expression regulation during tumorigenesis. Through comprehensive analysis of gene mutations, CNVs, and DNA methylation status, we can more comprehensively reveal the role of the CGB5 gene in cancer and its potential biological significance (36).

An expanding body of research has underscored the critical role of the immune microenvironment in tumor initiation and progression. Studies have shown that the pan-immune inflammation value and albumin-to-globulin ratio are closely associated with the tumor immune microenvironment and serve as important prognostic indicators in colorectal cancer (37, 38). In our study, we observed a robust correlation between CGB5 expression across various cancer types and immune cell infiltration. The results derived from the estimation algorithm demonstrated that CGB5 expression was positively correlated with the tumor estimate score and stromal score but not significantly associated with the immune score. Typically, higher immune scores indicate a greater abundance of immune cells, which may contribute to an effective antitumor immune response (39). However, in this study, high CGB5 expression exhibited an inverse correlation with immune cell infiltration in different tumor types. Moreover, elevated EstimateScores and StromalScores in tumors generally reflect a higher proportion of stromal and non-immune cells within the TME, which may promote tumor progression without improving patient outcomes (40).

Compared with T cells, CGB5 expression exhibited a stronger positive correlation with the infiltration of B cells and cancer-associated fibroblasts (CAFs). Although direct mechanistic studies delineating the interaction between CGB5 and B cells remain scarce, accumulating evidence indicates that hCG family members can exert immunomodulatory effects on B cells. In a previous study, mucosal administration of Lactobacillus engineered to express hCGβ elicited robust anti-hCGβ antibody responses, indicating that trophoblastic antigens can effectively prime and activate B cells (41). This observation implies that aberrant CGB5 expression within the tumor microenvironment may similarly trigger B cell mediated immune responses. CAFs constitute a dominant stromal component of the tumor microenvironment, exerting pivotal roles through ECM remodeling, cytokine signaling modulation, and regulation of immune cell functionality. Accumulating evidence indicates that CGB5 and other hCG β-subunits can promote the conversion of resident fibroblasts into CAFs. Specifically, CGB5 disrupts canonical TGF-β signaling, thereby driving fibroblasts to acquire a CAF phenotype characterized by robust secretion of ECM proteins and pro-inflammatory cytokines (42), ultimately remodeling the ECM and facilitating tumor invasion (41, 43). Concurrently, these secreted factors recruit and activate stromal cells- including CAFs -to establish an inflammatory and fibrotic niche that further promotes immune evasion (42, 44).

For tumors to survive and progress, they must circumvent immune surveillance. CGB5-expressing tumor cells convert conventional CD4⁺ T cells into Tregs by up-regulating the immunosuppressive cytokines TGF-β and IL-10, together with the transcription factor FOXP3. These Tregs, via cell-to-cell contact and the release of IL-10 and TGF-β, suppress the functions of effector T cells and other immune subsets, thereby attenuating antitumor immunity. Mechanistic studies further demonstrate that exposing tumor cells to hCG augments FOXP3 expression and increases Treg differentiation, indicating that CGB5-mediated signaling is a key driver of the immune-evasion phenotype (41).

In recent years, immune-checkpoint inhibitors (ICIs) targeting molecules such as PD-L1 and CTLA-4 have substantially reshaped the therapeutic landscape for a broad spectrum of malignancies. However, the efficacy of these agents is critically contingent upon a pre-existing and robust antitumor immune response. For example, in the phase III IMvigor210 trial evaluating atezolizumab for urothelial carcinoma, patients with high CGB-family gene expression exhibited markedly reduced CD8⁺ T-cell infiltration and significantly shorter overall survival, indicating that these tumors are intrinsically less responsive to immune-checkpoint inhibition (42, 45). Collectively, these molecular events orchestrate an immune-evasive phenotype that enables tumor cells to sustain proliferation despite ongoing host immune surveillance.

Encouragingly, pre-clinical evidence now substantiates the clinical potential of CGB5-directed interventions. Vaccination protocols or antibody-based neutralization of hCGβ markedly attenuated tumor growth in experimental models, indicating that disruption of CGB5-dependent pathways can re-invigorate antitumor immunity and enhance responsiveness to immune-checkpoint blockade (41, 46). From a clinical standpoint, CGB5 exerts dual functions—driving intrinsic tumor aggressiveness while profoundly reshaping the immune microenvironment–thereby serving as both a prognostic biomarker and a compelling therapeutic target. Patients with elevated CGB5 expression can be stratified into a high-risk subset anticipated to derive limited benefit from monotherapy with immune checkpoint inhibitors, thus warranting investigation of rational combination regimens: first, combinatorial targeting of CGB5 or its downstream effectors—exemplified by TGF-β inhibitors and anti-IL-10 antibodies—together with immune-checkpoint inhibitors is anticipated to dismantle the immunosuppressive fortress. Second, circulating HCG (including CGB5-derived species) is readily quantifiable in plasma or urine, offering a non-invasive liquid-biopsy companion for real-time monitoring of therapeutic response and disease progression.

Previous studies have explored the role of CGB5 in gastric cancer, primarily focusing on gene set analyses while offering limited insight into its underlying mechanisms (7, 47, 48). In this study, we confirmed the robust expression of CGB5 in gastric cancer cells and further investigated its mechanism of action. Our results demonstrated that CGB5 facilitates gastric cancer progression via multiple pathways, with significant enrichment observed in the Wnt signaling pathway through both GO and KEGG analyses. The Wnt signaling pathway plays a critical role in various physiological processes, and its dysregulation is commonly implicated in gastric cancer. Aberrant activation of this pathway contributes to chemotherapy resistance, thereby promoting gastric carcinogenesis and resistance development, which highlights its significance in gastric cancer progression (49). Recent studies show that the Wnt signaling pathway reduces lipid peroxidation by up-regulating GPX4, an essential antioxidant enzyme that prevents cellular iron oxidation, ultimately inhibiting ferroptosis in gastric cancer cells (50). Furthermore, GSEA analysis suggests that CGB5 may promote gastric cancer progression by modulating the immune microenvironment, underscoring the pivotal role of immune infiltration in gastric cancer, findings that are consistent with prior research (8, 47).

This study possesses certain limitations that merit acknowledgment. First, the findings are primarily based on publicly accessible data from online databases rather than direct clinical observations, which may raise questions about the reliability of the data. To address this concern, we prioritized the use of large-scale and well-established databases, such as TCGA and GEO. Second, although CGB5 expression was confirmed in GC cell lines in this study, functional validation and further investigations in additional tumor types(such as PAAD) demand comprehensive in vitro and in vivo experiments utilizing relevant cancer cell lines and/or animal models. Moreover, discrepancies in algorithms or data sources across different databases could potentially lead to inconsistencies in the results. The inherent complexity of the TME necessitates acknowledgment of methodological constraints in immune cell quantification. Differences in computational frameworks—such as CIBERSORT's deconvolution-based model vs. XCELL's reliance on cell type-specific gene signatures—may inherently yield inter-algorithm variability for identical immune metrics. Concurrently, the bulk RNA-seq approach utilized here lacks spatial resolution to resolve CGB5 expression dynamics within specific cellular subpopulations or characterize cell-cell interactions. Consequently, we advocate future validation through scRNA-seq technology in tractable malignancies demonstrating significant CGB5 associations (e.g., GC or PAAD). This would specifically interrogate CG5′s functional modulation within cancer-associated fibroblasts and T-cell subpopulations, thereby advancing mechanistic insights for developing precision immunotherapies targeting CGB5. In summary, future studies should aim to overcome these limitations by conducting large-scale clinical trials or employing multi-source data integration for thorough validation.

To summarize, CGB5 expression was significantly up-regulated across a range of malignancies and exhibited a robust association with the prognoses of cancer patients, specifically in malignancies like GC and PAAD. This observation not only highlights its essential role in cancer development but also demonstrates a close association with the prognosis of cancer patients, specifically in malignancies like GC and PAAD. Extensive studies indicate that CGB5 expression in tumor tissues may facilitate tumorigenesis and progression by modulating the tumor immune microenvironment. Moreover, our experimental findings have further confirmed the elevated expression of CGB5 in GC and clarified its correlation with immune cell infiltration. Specifically, the expression level of CGB5 appears to directly affect the distribution and functional state of immune cells within the tumor microenvironment, offering valuable clues for elucidating the mechanisms underlying tumor immune evasion. Clinically, CGB5 expression could be integrated into diagnostic panels to stratify patients at higher risk of poor outcomes. Moreover, to validate CGB5′s role in immunotherapy response, future experiments could employ CRISPR/Cas9-mediated CGB5 knockout in cancer cell lines to evaluate changes in immune cell infiltration and response to ICIs.