Interferon-gamma (IFN-gamma) is a cytokine with a paradoxical role in cancer immunity. On one hand, it activates cytotoxic T cells and NK cells to kill tumor cells. On the other hand, sustained IFN-gamma signaling can upregulate immune checkpoint molecules like PD-L1, enabling tumors to evade immune destruction. Understanding which genes mediate these IFN-gamma effects in renal cell carcinoma (RCC) is critical for improving immunotherapy outcomes.
RCC encompasses multiple histological subtypes, with clear cell RCC (ccRCC) being the most common, followed by papillary RCC (pRCC) and chromophobe RCC (chRCC). These subtypes differ in their molecular profiles, prognosis, and likely response to immune-based treatments. A multi-subtype analysis of IFN-gamma response genes could reveal shared or subtype-specific vulnerabilities.
Identifying genes that specifically regulate the immunosuppressive tumor microenvironment (TME) of RCC could uncover both prognostic biomarkers and potential therapeutic targets. This study used multi-omics data from 944 RCC patients to systematically screen IFN-gamma response genes and identify those with the greatest prognostic and immunological significance.
This study analyzed 944 RCC patients from The Cancer Genome Atlas (TCGA), encompassing ccRCC (n=530), pRCC (n=323), and chRCC (n=91). Transcriptomic, copy number variation, methylation, and mutation data were integrated to comprehensively profile IFN-gamma response gene activity across all three subtypes.
A curated list of 200 IFN-gamma response genes was used as the starting point. For each gene, univariate Cox regression identified those significantly associated with overall survival (OS) or progression-free survival (PFS). LASSO (Least Absolute Shrinkage and Selection Operator) Cox regression was then applied to narrow the list to the most informative independent prognostic genes while accounting for multicollinearity.
RBCK1 (RING Finger and CCCH-Type Zinc Finger Domain Containing 1) emerged as the top-ranked gene. To validate its functional role, RBCK1 was knocked down in the 786-O ccRCC cell line using siRNA, and the effects on proliferation, migration, invasion, and apoptosis were measured using colony formation assays, wound healing assays, Transwell invasion assays, and flow cytometry.
Immune cell composition was estimated using TIMER, CIBERSORT, and ssGSEA algorithms applied to TCGA bulk RNA-seq data. TIDE (Tumor Immune Dysfunction and Exclusion) scores were computed to predict immunotherapy response. Analyses were stratified by RBCK1 expression level across the full RCC cohort and per subtype.
RBCK1 expression was significantly upregulated in RCC compared to normal kidney tissue across all three subtypes. High RBCK1 expression correlated strongly with worse overall survival, with a hazard ratio of 3.296 (95% CI: 2.1 to 5.2) in the overall RCC cohort. For progression-free survival, the hazard ratio was 2.541, confirming that RBCK1 is a robust independent prognostic marker.
Kaplan-Meier curves showed clear separation between RBCK1-high and RBCK1-low patients in all three RCC subtypes, with the high-expression group experiencing significantly shorter OS and PFS (log-rank p less than 0.001 in ccRCC and pRCC). The prognostic impact was most pronounced in ccRCC, which is the subtype with the highest clinical relevance and patient volume.
Multivariate Cox regression confirmed that RBCK1 expression remained an independent predictor of OS even after adjusting for tumor stage, grade, age, and sex. This independence from conventional staging variables strengthens RBCK1's potential value as an additive biomarker in clinical risk stratification.
Copy number amplification and promoter hypomethylation were identified as potential mechanisms driving RBCK1 overexpression in RCC, linking genomic and epigenomic alterations to the observed transcriptomic upregulation. These findings suggest that RBCK1 dysregulation is a primary, not secondary, event in RCC biology.
To test whether RBCK1 has a direct functional role in RCC, the researchers silenced RBCK1 expression in the 786-O ccRCC cell line using siRNA. Colony formation assays showed a marked reduction in proliferative capacity following RBCK1 knockdown, confirming its role in sustaining cancer cell growth.
Wound healing and Transwell invasion assays demonstrated that RBCK1 knockdown significantly impaired cell migration and invasion. These functional changes suggest that RBCK1 contributes not only to primary tumor growth but also to the metastatic potential of ccRCC cells, consistent with the poor prognosis associated with high RBCK1 in patient data.
Flow cytometry revealed a significant increase in apoptotic cell fraction following RBCK1 knockdown, with both early and late apoptosis rates elevated compared to control cells. This suggests RBCK1 normally protects tumor cells from programmed cell death, a key survival mechanism exploited by aggressive cancers.
Western blot analysis showed changes in apoptosis-related proteins following RBCK1 knockdown, including upregulation of cleaved caspase-3 and downregulation of Bcl-2, confirming activation of the intrinsic apoptotic pathway. These in vitro findings corroborate the clinical significance of RBCK1 observed in patient datasets.
Immune deconvolution using TIMER and CIBERSORT revealed that high RBCK1 expression was associated with increased infiltration of M2 macrophages and regulatory T cells (Tregs) in the TME. Both cell types are known to suppress anti-tumor immune responses, creating an environment that shields the tumor from cytotoxic T cell attack.
By contrast, RBCK1-high tumors showed reduced CD8+ T cell infiltration and lower expression of cytotoxic effector molecules such as perforin and granzyme B. This pattern represents a classic immunosuppressive TME phenotype associated with poor immunotherapy response and disease progression.
ssGSEA enrichment analysis further confirmed upregulation of immune suppression gene sets and downregulation of T cell activation and effector function pathways in RBCK1-high tumors. The convergent evidence from multiple computational tools strengthens the conclusion that RBCK1 actively promotes immune evasion in RCC.
TIDE score analysis showed that patients with high RBCK1 expression had significantly higher TIDE scores, indicating a greater likelihood of immune dysfunction and exclusion. Higher TIDE scores correlate with worse response to PD-1/PD-L1 checkpoint inhibition, suggesting that RBCK1-high patients may be less likely to benefit from current standard-of-care immunotherapy.
This study establishes RBCK1 as a multi-functional driver of poor outcomes in RCC. Its upregulation correlates with worse survival, promotes tumor cell growth and invasion in vitro, and shapes an immunosuppressive tumor microenvironment that may undermine immune checkpoint therapy efficacy.
The convergence of genomic, epigenomic, transcriptomic, and functional evidence positions RBCK1 as one of the most comprehensively supported IFN-gamma response genes in RCC. The finding that it operates through both cell-intrinsic oncogenic mechanisms and cell-extrinsic immune evasion mechanisms makes it a particularly attractive target for therapeutic intervention.
Clinically, RBCK1 expression could be measured from tumor biopsies or potentially from blood-based assays to stratify newly diagnosed RCC patients. High-RBCK1 patients might benefit from combination strategies that simultaneously target tumor cell proliferation and the immunosuppressive TME, such as pairing a RBCK1 inhibitor with anti-PD-1 therapy.
Future research should explore whether pharmacological inhibition of RBCK1 is feasible, assess its predictive value for immunotherapy outcomes in prospective cohorts, and determine whether the immune microenvironment changes observed are mechanistically driven by RBCK1 activity or are co-regulated by upstream signals.