RAD54B as a biomarker for prognosis and immunotherapy response in bladder cancer

Abstract

Background

RAD54B is a homologous recombination–related DNA repair protein involved in maintaining genomic stability. However, its expression profile, clinical relevance, and immunological role in bladder cancer remain unclear. This study systematically explored the clinical significance of RAD54B in bladder cancer.

Methods

We conducted a comprehensive, multi-cohort analysis to evaluate the clinical significance of RAD54B in bladder cancer. RAD54B expression was evaluated using paired and unpaired analyses, promoter methylation profiling, single-cell RNA sequencing, and data from bladder cancer cell lines. Tumor immune infiltration was estimated using CIBERSORT, and immunotherapy response was predicted using immunophenoscore (IPS). RAD54B protein expression was validated by immunohistochemistry in a bladder cancer tissue microarray.

Results

RAD54B was significantly upregulated in bladder cancer and multiple malignancies and was associated with promoter hypomethylation. Higher levels of RAD54B were associated with significantly improved patient outcomes across multiple independent cohorts. Functional enrichment analyses revealed that RAD54B was primarily involved in DNA damage repair, cell cycle regulation, RNA metabolism, and genomic stability, while immune-related pathways were relatively suppressed. High RAD54B expression was linked to elevated memory B cells, naïve CD4⁺ T cells, and activated dendritic cells, while low expression was associated with increased activated memory CD4⁺ T cells, M2 macrophages, and neutrophils. Notably, patients with low RAD54B expression exhibited higher IPS scores under PD-1– and CTLA4–positive conditions, suggesting enhanced sensitivity to immune checkpoint blockade.

Conclusions

RAD54B serves as both a prognostic biomarker and an immune modulator in bladder cancer, supporting its use in immunotherapeutic stratification.

Introduction

Bladder cancer is a common urinary tract malignancy, with both incidence and mortality rising globally in recent years [1, 2]. Notwithstanding progress in surgery, chemotherapy, and immunotherapy, effective treatment of advanced bladder cancer remains a major clinical challenge [3, 4]. Tumor recurrence and distant metastasis are the primary causes of treatment failure and poor prognosis [5]. Accordingly, there is a pressing need to identify robust prognostic biomarkers and potential therapeutic targets to improve clinical outcomes in bladder cancer.

RAD54B (RAD54 homolog B), a member of the RAD52 gene family, plays a critical role in the DNA repair pathway and is essential for maintaining genomic stability [6]. Emerging evidence suggests that RAD54B exhibits oncogenic potential in various malignancies and may contribute to tumor cell proliferation, metastasis, and resistance to chemotherapy [7–9]. Liu et al. [10] reported that in bladder cancer, AP1M2 enhances chemoresistance to gemcitabine-cisplatin by stabilizing RAD54B mRNA, thereby upregulating its expression and promoting homologous recombination-mediated DNA repair, ultimately leading to therapy resistance. Moreover, a prognostic risk model related to cell cycle progression, which includes RAD54B, KPNA2, and TPM1, has been shown to effectively predict clinical outcomes and patterns of immune cell infiltration in bladder cancer patients [11]. However, the expression profile, clinical relevance, and role of RAD54B within the tumor microenvironment (TME) remain inadequately characterized, limiting its potential as a therapeutic target.

The tumor microenvironment critically influences tumor development, progression, and treatment response [12, 13]. With the advancement of immunotherapeutic approaches such as immune checkpoint inhibitors (ICIs), increasing attention has been directed toward elucidating the interactions between key genes and immune cell infiltration to uncover potential immune-related targets and predictive biomarkers [14–16]. Nevertheless, the role of RAD54B in the bladder cancer TME, particularly its relationship with immune cell infiltration, remains poorly understood and warrants further investigation.

This study systematically investigates the expression and clinical significance of RAD54B in bladder cancer. Through integrated public dataset analyses and clinical validation, elevated RAD54B expression in bladder cancer was identified and associated with clinical outcomes. Additional analyses examine the potential involvement of RAD54B in disease progression through modulation of the tumor immune microenvironment and its possible influence on immunotherapy response. These results suggest RAD54B holds promise as a predictive biomarker for prognosis and immunotherapy responsiveness in bladder cancer, providing a framework for future therapeutic investigations.

Materials and methods

Data collection and analysis

We systematically acquired RNA-sequencing data and corresponding clinical information for bladder cancer from The Cancer Genome Atlas Program (TCGA, https://portal.gdc.cancer.gov) and Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/), including TCGA-BLCA [17], GSE52219 [18], GSE48075 [18], GSE39281 [19], GSE37815 [20], GSE31684 [21], GSE19423 [20], GSE13507 [22], and IMvigor210 cohort [23]. The expression landscape of RAD54B in bladder cancer cell lines was characterized using the Cancer Cell Line Encyclopedia (CCLE) [24]. Furthermore, its cell type-specific expression pattern within the tumor microenvironment was resolved at single-cell resolution through the Tumor Immune Single-cell Hub (TISCH) database [25, 26]. Across all bladder cancer cohorts examined, we systematically evaluated RAD54B expression patterns stratified by key clinical parameters. Subsequently, we integrated these molecular profiles with comprehensive survival data to assess potential prognostic associations.

Differential gene expression and functional enrichment analysis

TCGA-BLCA samples were stratified into high- and low-expression RAD54B groups according to the median, followed by DESeq2-based differential expression analysis. Significant differential expression was defined as an absolute log₂ fold change > 1.0 with an adjusted p value < 0.05. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the RAD54B-related differentially expressed genes were performed using the “clusterProfiler” R package [27]. Functional enrichment analyses were conducted using curated gene sets from MSigDB as reference backgrounds [28]. Statistical significance was defined as a P value below 0.05 and an FDR less than 0.25.

Analysis of immune infiltration associated with RAD54B expression

Using the median RAD54B expression as a threshold, TCGA-BLCA patients were classified into two groups to assess its immunological associations. The CIBERSORT algorithm, employing the LM22 leukocyte gene signature, was applied to quantify the relative fractions of 22 immune cell subtypes within the tumor microenvironment [29]. Spearman correlation analysis was conducted to explore the relationship between RAD54B expression and immune cell infiltration across the cohort.

Immune checkpoint therapy response prediction

We employed the immunophenoscore (IPS) to estimate tumor immunogenicity and potential sensitivity to immune checkpoint inhibitors, with scores ranging from 0 to 10. IPS data were sourced from The Cancer Immunome Atlas (TCIA; https://tcia.at/home) [30]. Patients were categorized into high- and low-expression groups based on the median RAD54B level.

Clinical sample and immunohistochemistry assay

Tissue samples were obtained from a commercial bladder cancer tissue microarray (TMA) (Cat. ZL-BlaU961; Shanghai Zhuoli Biotechnology Co., Ltd., China), consisting of 60 bladder cancer tissues and 36 adjacent normal tissues (Table 1). Immunohistochemical staining was conducted using a commercially available kit (ZSBG-BIO, China) following the manufacturer’s instructions. Tissue sections were deparaffinized, rehydrated, and underwent antigen retrieval as described previously, followed by incubation with a primary anti-RAD54B antibody (1:200; GeneTex, USA) [31]. After appropriate washing, the samples were treated with a secondary antibody and visualized using the detection system provided in the kit. Two experienced pathologists independently evaluated the immunostaining results in a blinded manner and assigned IHC scores.

Table 1.

The clinical characteristic data of patients with bladder cancer in the validation cohort

Variable	Number
Gender
Male	50
Female	10
Age
> 70	18
≤ 70	42
Grade
Low	5
High	55
T stage
Tis/T1	30
T2	10
T3	14
T4	6
N stage
N0	23
N1-N3	13
Nx	24
M stage
M0	55
M1	5

Statistical analysis

All analyses were carried out using R software (version 4.3.3) in combination with the BEST online analysis tool [32]. Continuous variables were compared using Student’s t-test for two groups or one-way ANOVA for multiple groups. When normality assumptions were not met or sample sizes were small, non-parametric methods were employed (Wilcoxon rank-sum test for two groups; Kruskal–Wallis test for three or more groups). Survival analyses were performed using Kaplan–Meier curves with log-rank tests to assess differences. Two-sided p-values < 0.05 were considered statistically significant.

Results

Multomics analysis of the expression pattern of RAD54B in bladder tumors

RAD54B expression was systematically evaluated across various cancer types using TCGA and GTEx datasets. RAD54B expression was markedly elevated in numerous cancers, including bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colorectal adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, stomach adenocarcinoma, and uterine corpus endometrial carcinoma, compared with normal tissues, while it was notably reduced in kidney chromophobe carcinoma (Fig. 1A). Both unpaired and paired analyses further confirmed a marked upregulation of RAD54B expression in the TCGA-BLCA cohort (Fig. 1B and C). Additionally, promoter methylation analysis revealed that RAD54B exhibited significantly higher methylation levels in normal bladder tissues compared with bladder cancer tissues (Fig. 1D). The CCLE dataset analysis indicated considerable heterogeneity in RAD54B expression among 35 bladder cancer cell lines, with UMUC13 showing the highest and UMUC6 the lowest expression (Fig. 1E). Furthermore, single-cell transcriptomic analysis using the GSE149652 dataset showed that RAD54B was broadly expressed across six immune cell subtypes within the bladder cancer microenvironment (Fig. 1F and G).

Fig. 1.

Expression and epigenetic regulation of RAD54B in pan‑cancer and bladder cancer. A Differential expression of RAD54B between tumor and normal tissues across multiple cancer types from TCGA and GTEx datasets. B–C Validation of RAD54B upregulation in the TCGA‑BLCA cohort using unpaired (B) and paired (C) sample analyses. D Promoter methylation levels of RAD54B in normal bladder tissues versus primary bladder tumor tissues. Methylation is significantly higher in normal tissues. E RAD54B expression levels across 35 bladder cancer cell lines from the CCLE database. UMUC13 shows the highest expression, while UMUC6 shows the lowest. F UMAP visualization of a single‑cell transcriptomic atlas of bladder cancer (dataset GSE149652), colored by six major immune cell subtypes. G Violin plots showing the expression distribution of RAD54B across the six immune cell subtypes identified in (F)

Clinical significance of RAD54B expression in bladder cancer patients

Based on the TCGA-BLCA cohort, we further explored RAD54B expression across various clinicopathological subgroups. RAD54B expression was consistently elevated in tumor tissues compared with adjacent normal tissues across all examined categories. Specifically, increased RAD54B expression was observed in tumors from both T1/T2 and T3/T4 stages (Fig. 2A), across all N stages (Fig. 2B), M stages (Fig. 2C), overall tumor stages (Fig. 2D), histological grades (Fig. 2E), age groups (Fig. 2F), and molecular subtypes (Fig. 2H). To validate these findings, independent datasets were analyzed. In the GSE13507 cohort, RAD54B expression was significantly higher in low-grade tumors than in high-grade tumors (Fig. 2I) and showed stage-dependent expression differences across T stages (Fig. 2J). Analysis of the GSE52219 cohort further confirmed significant differences in RAD54B expression among molecular subtypes (Fig. 2K). Importantly, in the IMvigor210 cohort of bladder cancer patients treated with the PD-L1 inhibitor atezolizumab, responders exhibited significantly higher baseline RAD54B expression than non-responders (Fig. 2L).

Fig. 2.

RAD54B expression across clinicopathological features and its association with immunotherapy response in bladder cancer. A–H Expression levels of RAD54B in tumor versus adjacent normal tissues from the TCGA-BLCA cohort, stratified by (A) T stage (T1/T2 vs. T3/T4), B N stage, C M stage, D pathological stage, E histological grade, F age group, and H subtype. RAD54B was consistently overexpressed in tumor tissues across all subgroups. I RAD54B expression is significantly higher in low-grade compared to high-grade tumors in the GSE13507 validation cohort. J Differential expression of RAD54B across T stages in the GSE13507 cohort. K Differential expression of RAD54B across molecular subtypes in the GSE52219 cohort. L In the IMvigor210 immunotherapy cohort, baseline RAD54B expression is significantly higher in patients who responded to Atezolizumab treatment compared to non-responders

Evaluation of the prognostic value of RAD54B expression in patients with bladder cancer

We evaluated the prognostic significance of RAD54B expression using Kaplan–Meier survival analysis. Across multiple bladder cancer cohorts, elevated RAD54B expression was consistently linked to improved overall survival, as observed in TCGA-BLCA, GSE31684, GSE37815, GSE39281, GSE48075, and IMvigor210 datasets (Fig. 3A–F). RAD54B upregulation in the TCGA-BLCA cohort was significantly linked to favorable disease-specific survival and progression-free interval (Fig. 3G and H). In the TCGA-BLCA cohort, univariate Cox regression revealed T stage, N stage, M stage, clinical stage, age, molecular subtype, and RAD54B expression as significant prognostic factors (Fig. 3I). Following multivariate Cox regression, only the N stage was independently predictive of patient prognosis (Fig. 3J).

Fig. 3.

Prognostic value of RAD54B expression in bladder cancer. A-F Kaplan-Meier curves showing overall survival (OS) in patients stratified by high vs. low RAD54B expression in the (A) TCGA-BLCA cohort, and validation cohorts (B) GSE31684, C GSE37815, D GSE39281, E GSE48075, and F IMvigor210. Higher RAD54B expression is associated with better OS. G-H Kaplan-Meier curves in the TCGA-BLCA cohort showing that high RAD54B expression is associated with improved (G) disease-specific survival (DSS) and (H) progression-free interval (PFI). I Forest plot of univariate Cox regression analysis for prognostic factors (including RAD54B expression) in the TCGA-BLCA cohort. J Forest plot of multivariate Cox regression analysis for the same variables, showing that only N stage remains an independent prognostic factor

Verification of RAD54B expression and its association with clinical features in bladder cancer

RAD54B protein expression was further validated using a commercial bladder cancer tissue microarray. Quantitative immunohistochemical analysis demonstrated that RAD54B protein expression was significantly higher in tumor tissues than in adjacent normal tissues (Fig. 4A and B). Stratified analysis showed no significant difference in RAD54B IHC scores between patients aged > 70 years and those aged ≤ 70 years, although tumor tissues consistently exhibited higher expression than adjacent tissues in both age groups (Fig. 4C). Notably, male patients displayed significantly higher RAD54B IHC scores in tumor tissues compared with female patients, while tumor-associated upregulation was observed in both sexes (Fig. 4D). No significant associations were detected between RAD54B IHC scores and M stage (Fig. 4E), T stage (Fig. 4F), or N stage (Fig. 4G).

Fig. 4.

Validation of RAD54B protein expression in bladder cancer tissues by immunohistochemistry. A Representative immunohistochemical images of RAD54B expression in paired adjacent normal and tumor tissues from bladder cancer patients with different T stages (T1N0M0, T2N0M0, T3N0M0, and T4N0M0). Scale bar, 500 μm. B Quantitative comparison of RAD54B protein expression using IHC scores (H-score) between adjacent normal tissues and bladder tumor tissues (**p < 0.01). C Comparison of RAD54B IHC scores in tumor tissues between patients aged > 70 years and ≤ 70 years. IHC scores in paired adjacent tissues are shown as reference (ns, not significant for the inter-group tumor comparison). D Comparison of RAD54B IHC scores in tumor tissues between male and female patients (**p < 0.01). IHC scores in paired adjacent tissues are shown as reference. E-G RAD54B IHC scores in tumor tissues stratified by (E) M stage, (F) T stage, and (G) N stage (ns, not significant for all comparisons)

RAD54B-associated differential gene expression and functional enrichment in bladder cancer

Differential expression analysis identified 314 upregulated and 660 downregulated genes, which are visualized in a volcano plot (Fig. 5A). A heatmap illustrates the expression patterns of the top 20 upregulated and downregulated genes between the two groups (Fig. 5B). Functional enrichment analyses were subsequently performed. Gene Ontology (GO) analysis revealed that differentially expressed genes were significantly enriched in biological processes such as humoral immune response, epidermis development, skin development, keratinocyte differentiation, and keratinization (Fig. 5C). Cellular component analysis highlighted enrichment in the collagen-containing extracellular matrix, external side of the plasma membrane, anchored membrane components, cornified envelope, and collagen trimer (Fig. 5D). Molecular function analysis identified enrichment in glycosaminoglycan binding, carbohydrate binding, extracellular matrix structural constituents, sulfur compound binding, and heparin binding (Fig. 5E). KEGG pathway analysis revealed significant enrichment in neuroactive ligand–receptor interaction, Staphylococcus aureus infection, complement and coagulation cascades, drug metabolism–cytochrome P450, and retinol metabolism (Fig. 5F).

Fig. 5.

Identification and functional enrichment of genes differentially expressed by RAD54B status in bladder cancer. A Volcano plot of differentially expressed genes (DEGs) between RAD54B-high and RAD54B-low groups in the TCGA-BLCA cohort. Red dots represent 314 significantly upregulated genes, and blue dots represent 660 downregulated genes. B Heatmap showing the expression z-scores of the top 20 upregulated and top 20 downregulated DEGs across samples stratified by RAD54B expression. C–F Bubble plots illustrating the results of functional enrichment analysis for all DEGs. Top enriched terms in (C) Biological Process (BP), D Cellular Component (CC), and (E) Molecular Function (MF) from GO analysis, as well as (F) significantly enriched KEGG pathways, are displayed. Bubble size represents the number of enriched genes, and color indicates the statistical significance

Gene set enrichment analysis (GSEA)

GO-based GSEA demonstrated significant enrichment (NES > 0) of pathways related to nucleic acid metabolism and cell cycle regulation, including ribonucleoprotein complex biogenesis, ribosome biogenesis, ncRNA processing, tRNA metabolic process, and rRNA metabolic process (Fig. 6). Notably, several pathways associated with genomic stability were also significantly enriched, such as DNA-dependent DNA replication, mitotic sister chromatid segregation, and chromosome segregation. In addition, pathways related to RNA modification and RNA methylation were activated, suggesting a potential role for RAD54B in epigenetic regulation. In contrast, multiple immune-related pathways were significantly downregulated (NES < 0), including leukocyte migration, myeloid leukocyte migration, monocyte chemotaxis, adaptive immune response, antigen processing and presentation, and complement activation. KEGG-based GSEA further revealed that RAD54B was involved in multiple critical biological processes (Fig. 7). These included DNA damage repair pathways such as homologous recombination, non-homologous end joining, base excision repair, nucleotide excision repair, and mismatch repair, highlighting its role in maintaining genomic stability. RAD54B was also enriched in cell cycle and p53 signaling pathways. Additionally, RAD54B was associated with diverse metabolic pathways, including amino acid metabolism, energy metabolism, and one-carbon metabolism. Pathways related to transcriptional and post-transcriptional regulation, protein processing, and ubiquitin-mediated proteolysis were also significantly enriched.

Fig. 6.

The results of gene ontology (GO) analysis based gene set enrichment analysis

Fig. 7.

The results of kyoto encyclopedia of genes and genomes (KEGG) pathway analysis based gene set enrichment analysis

RAD54B expression and tumor immune cell infiltration in bladder cancer

CIBERSORT was used to quantify 22 immune cell subtypes in TCGA-BLCA patients grouped by RAD54B expression. The distribution of immune cell subtypes across the groups is presented as a stacked bar chart (Fig. 8A). Comparative analysis revealed distinct immune infiltration patterns between the two groups. Tumors with high RAD54B expression exhibited significantly increased infiltration of memory B cells, naïve CD4⁺ T cells, and activated dendritic cells. In contrast, tumors with low RAD54B expression were characterized by higher levels of activated memory CD4⁺ T cells, M2 macrophages, and neutrophils (Fig. 8B). Spearman correlation analysis further supported these findings. RAD54B expression was negatively correlated with neutrophils, activated memory CD4⁺ T cells, and M2 macrophages, while showing positive correlations with naïve CD4⁺ T cells, activated dendritic cells, and memory B cells (Fig. 8C).

Fig. 8.

Landscape of immune cell infiltration associated with RAD54B expression in bladder cancer (TCGA-BLCA cohort). A Stacked bar plot showing the relative proportions of 22 immune cell subtypes estimated by the CIBERSORT algorithm, grouped by high and low RAD54B expression. B Box plots comparing the infiltration levels of six key immune cell subtypes that showed significant differences between RAD54B-high and RAD54B-low groups. Statistical significance was determined by the Mann-Whitney U test (*p < 0.05, **p < 0.01, **p < 0.001). C Spearman correlation analysis between RAD54B expression and the estimated abundance of immune cell subtypes. The bar graph shows the correlation coefficient (R) for each cell type, with asterisks indicating statistical significance (p < 0.05, **p < 0.01, ***p < 0.001)

RAD54B expression is associated with predicted immunotherapy response

In light of the widespread use of immune checkpoint blockade, the potential relationship between RAD54B expression and immunotherapy response was investigated using IPS. Under the CTLA4-negative/PD-1-negative condition, patients stratified by RAD54B expression showed no significant difference in IPS (Fig. 9A). However, under the CTLA4-negative/PD-1-positive condition, patients with low RAD54B expression exhibited significantly higher IPS values than those with high RAD54B expression (Fig. 9B). Similarly, no significant difference was detected under the CTLA4-positive/PD-1-negative condition (Fig. 9C). Notably, when both CTLA4 and PD-1 were positive, the RAD54B low-expression group demonstrated markedly higher IPS scores compared with the high-expression group (Fig. 9D).

Fig. 9.

Association between RAD54B expression and predicted response to immune checkpoint blockade in TCGA-BLCA. Comparison of immunophenoscore (IPS) values between RAD54B high- and low-expression groups under different immune checkpoint inhibitor conditions: A CTLA4-negative/PD-1-negative, B CTLA4-negative/PD-1-positive, C CTLA4-positive/PD-1-negative, and D CTLA4-positive/PD-1-positive

Discussion

Bladder cancer remains a major clinical challenge owing to its high rates of recurrence and metastasis [5]. Although immune checkpoint inhibitors have expanded therapeutic options, durable clinical benefits are achieved in only a subset of patients [16]. The lack of reliable biomarkers for predicting prognosis and immunotherapy response has further hampered precision oncology in this disease. The present study systematically explored the clinical, molecular, and immunological relevance of RAD54B, a DNA repair–associated protein, in bladder cancer.

RAD54B has been reported to be overexpressed and amplified in several solid malignancies, including breast and liver cancers, where it is frequently associated with unfavorable outcomes [9, 33]. In breast cancer, RAD54B amplification is particularly prominent in the luminal A subtype and correlates with poor prognosis [9]. Consistent with these observations, our analyses demonstrate that RAD54B is significantly upregulated in bladder cancer tissues. Importantly, this upregulation is accompanied by hypomethylation of the RAD54B promoter region, suggesting that epigenetic dysregulation may contribute to its aberrant activation in bladder cancer.

Bladder cancer is characterized by marked clinical and biological heterogeneity, reflected in variable disease progression, therapeutic response, and survival outcomes [34]. Identifying molecular markers that capture both tumor biology and clinical behavior is of particular importance [35, 36]. Our clinical relevance analyses revealed that RAD54B expression was consistently elevated across tumor stages, grades, molecular subtypes, and age groups, indicating that RAD54B upregulation represents a common molecular event rather than a feature restricted to specific subgroups. Interestingly, RAD54B expression was higher in low-grade than in high-grade tumors in an independent cohort. This finding suggests that RAD54B activation may occur early during tumorigenesis and undergo dynamic modulation throughout disease progression, rather than exhibiting a simple linear relationship with tumor aggressiveness. Survival analyses further showed that higher RAD54B expression was associated with improved overall survival, disease-specific survival, and progression-free survival across multiple independent bladder cancer cohorts. Multivariate Cox regression revealed that RAD54B lacked independent prognostic value, with its contribution partly attenuated by key clinical variables including lymph node stage. Collectively, these results suggest that RAD54B is more likely to function as a biomarker reflecting the biological state and immune context of the tumor, rather than acting as a solitary determinant of patient outcome.

Previous studies have shown that RAD54B participates in multiple DNA repair pathways and promotes tumor cell survival under genotoxic stress [6, 10]. In gastric cancer, RAD54B has been reported to facilitate tumor growth and angiogenesis through activation of the Wnt/β-catenin pathway [37]. Consistent with these observations, our functional enrichment and GSEA analyses revealed that RAD54B overexpression is tightly associated with fundamental cellular processes, including DNA damage repair, cell cycle regulation, chromosome segregation, and RNA metabolism. Specifically, RAD54B was significantly enriched in homologous recombination, non-homologous end joining, and the p53 signaling pathway, underscoring its central role in maintaining genomic integrity and supporting tumor cell proliferation at the transcriptomic level [6]. Notably, multiple immune-related pathways were concurrently suppressed in tumors with high RAD54B expression, suggesting that RAD54B may indirectly modulate antitumor immune responses by enhancing intrinsic genomic stability and attenuating DNA damage-associated immunogenic signaling. The activity of RAD54B can reduce the cell cycle arrest induced by DNA damage and enhance the survival ability of cancer cells in stressful environments [6, 8]. However, it should be emphasized that these mechanistic interpretations are primarily based on bioinformatic inference and pathway enrichment analyses, and direct experimental validation will be required to substantiate these hypotheses.

From an immunological perspective, RAD54B appears to act as a modulator of DNA damage response (DDR)–related pathways with potential downstream immunoregulatory consequences [38, 39]. Our findings reveal RAD54B as an important modulator of DDR checkpoint strength and a contributor to immune regulation in bladder cancer. Previous studies have reported that RAD54B can facilitate p53 degradation, thereby attenuating DDR checkpoint activation and potentially suppressing p53-dependent immunogenic signaling [6]. Such checkpoint attenuation may not only enable tumor cells to sustain proliferation under genotoxic stress but also dampen p53-mediated immune activation pathways, including the cGAS-STING-type I interferon axis, which is essential for dendritic cell activation and CD8⁺T cell recruitment [39].

Given the central role of RAD54B in genomic maintenance and tumor biology, we next sought to elucidate whether its expression is associated with distinct immune microenvironmental features [40]. The tumor immune microenvironment analysis revealed a strong link between RAD54B and immune cell infiltration. In tumors with high expression of RAD54B, the proportions of naive CD4⁺ T cells, memory B cells, and activated dendritic cells increased, while in tumors with low expression of RAD54B, M2-type macrophages and neutrophils and other immunosuppressive-related cells were enriched. This result suggests that the expression level of RAD54B may reflect different immune ecological states. It should be noted that the proportions of immune cells are mainly inferred based on the CIBERSORT algorithm, although the results show a consistent trend; further experimental verification is still needed. In the analysis related to immunotherapy, we observed that RAD54B exhibited context-dependent effects under different analytical frameworks. Responders to PD-L1 blockade in the IMvigor210 cohort had higher baseline RAD54B expression, indicating a potential link between RAD54B levels and clinical benefit. On the other hand, the IPS analysis revealed that tumors with low expression of RAD54B had higher theoretical immunogenicity under conditions of PD-1 and/or CTLA4 positivity. This difference may reflect the fundamental distinction between the theoretical immunostimulatory potential and the actual immunotherapy response: IPS mainly assesses the expression of immune-related genes, while clinical efficacy is also influenced by multiple factors such as DNA repair capacity, tumor immune-suppressive microenvironment, and the spatial distribution of immune cells [41].

Several limitations should be acknowledged. The study is largely retrospective and based on public databases, precluding causal inference. Moreover, the regulatory effects of RAD54B on DNA damage repair and the immune microenvironment have not been experimentally validated in vivo or in vitro. Finally, the assessment of immune cell infiltration is computational, warranting confirmation through complementary experimental methods.

Conclusion

In summary, RAD54B is associated with DNA damage response–related pathways and immune characteristics in bladder cancer. Its expression may have potential value in reflecting tumor immune states and informing immunotherapy stratification, although further validation is required.

Abbreviations

IPS

Immunophenoscore

RAD54B

RAD54 homolog B

TME

Tumor microenvironment

ICIs

Immune checkpoint inhibitors

TCGA

The cancer genome atlas program

GEO

Gene expression omnibus

CCLE

Cancer cell line encyclopedia

TISCH

Tumor immune single-cell hub

GO

Gene ontology

KEGG

Kyoto encyclopedia of genes and genomes

TCIA

The cancer immunome atlas

TMA

Tissue microarray

DDR

DNA damage response

BLCA

Bladder urothelial carcinoma

Author contributions

QL: Data interpretation, manuscript drafting. JG, YX: Literature research. YC, CH: Conceptual advice, technical support. QL: Study design. All authors reviewed and approved the final manuscript.

Funding

None.

Data availability

The datasets analyzed in this study are publicly available. Transcriptomic and clinical data were obtained from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov) and the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo) under the accession numbers GSE52219, GSE48075, GSE39281, GSE37815, GSE31684, GSE19423, GSE13507 and GSE149652. In addition, the IMvigor210 cohort was included. Processed data used in this study are accessible via the BEST platform (https://rookieutopia.com). The datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All tissue samples and clinical data were obtained with patients’ informed consent in accordance with the Helsinki Declaration and approved by the institutional ethics committee of Shanghai Zhuoli Biotechnology Co., Ltd. (No. LLS M-15-01). As all data analyzed were derived from public databases and no human subjects were directly recruited by our institution, additional ethical approval was not required.

Consent for publication

Not applicable.

Informed consent

Informed consent was obtained from all patients prior to clinical data collection.

Competing interests

The authors declare no competing interests.