Gemcitabine is a widely used intravesical chemotherapeutic agent for non-muscle-invasive bladder cancer (NMIBC), yet its efficacy remains suboptimal, with high recurrence rates. While traditionally considered a cytotoxic agent, emerging evidence suggests that certain chemotherapeutics may exert immunomodulatory effects. This study investigated whether gemcitabine induces immunogenic cell death (ICD) and explored the underlying mechanisms involving endoplasmic reticulum (ER) stress. A retrospective analysis was conducted to assess the association between gemcitabine-related complications and NMIBC recurrence. In vitro assays were used to evaluate ICD hallmarks (ATP, HMGB1, CALR) and pro-inflammatory cytokines. RNA sequencing and western blotting were performed to explore ER stress pathways. Functional assays included dendritic cell (DC) maturation, phagocytosis, and CD8⁺T cell proliferation and cytotoxicity. In vivo vaccination and T cell depletion models were used to evaluate antitumor immunity. Clinical data revealed an inverse correlation between gemcitabine-related complications and tumor recurrence. Gemcitabine induced classical ICD markers in bladder cancer cells and elicited CD8⁺T cell-mediated antitumor immunity in vivo. Mechanistically, gemcitabine activated the PERK branch of the ER stress response, promoting CALR translocation and DAMP release. PERK overexpression enhanced DC maturation, phagocytosis, and stimulation of CD8⁺T cell proliferation and cytotoxicity, whereas PERK knockdown impaired these effects. This study demonstrates that short-term, high-dose gemcitabine induces PERK-dependent ICD, which enhances antitumor immune responses via DC and CD8⁺T cell activation. These findings provide a mechanistic rationale for combining gemcitabine with immunotherapy and suggest PERK as a potential biomarker and therapeutic target for ICD enhancement in bladder cancer.

Background: Renal cell carcinoma (RCC) is a prevalent malignancy of the urinary system that presents significant health and economic burdens. Despite existing treatments such as surgery and targeted therapies, challenges remain due to suboptimal efficacy and high recurrence rates. Previous studies have indicated that metformin and everolimus individually exhibit inhibitory effects on RCC. However, their synergistic potential when combined has not been fully elucidated. Therefore, this paper identified the antiproliferative effect and the hub genes that undergo significant changes in 786-O cells when treated with the combination drugs and their underlying mechanisms to inform the search for kidney renal clear cell carcinoma (KIRC) therapeutic targets. Methods: The effects of the combination of metformin and everolimus on 786-O cells viability, migration and invasion were investigated. Differentially expressed genes (DEGs) among different drug treatment groups were identified through ribonucleic acid (RNA) sequencing, raw data processing and differential expression analysis. The target genes were obtained by taking the intersection of different DEGs, and hub genes were identified by Maximal Clique Centrality (MCC) and Molecular Complex Detection (MCODE) algorithms, expression validation, and Kaplan-Meier (K-M) survival curve plotting. Subsequently, transcription factors (TFs) regulating the hub genes were identified and drug-hub gene interactions were explored through molecular docking. In addition, gene set enrichment analysis (GSEA) demonstrated hub gene-related biological functions and pathways, and gene set variation analysis (GSVA) explored differential pathways between different drug treatment groups. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the expression difference of hub genes among four groups. Results: The combination of metformin and everolimus is more effective than monotherapy at inhibiting cell viability, migration, and invasion in 786-O cells. In total, 3,030 DEG1, 2,953 DEG2, 3,591 DEG3, 1,571 DEG4 and 4,064 DEG5 were identified, yielding five target genes. After MCC and MCODE algorithms, expression validation, and K-M survival curve plotting, target genes were all noted as hub genes (SPC25, NCAPH, MCM10, UHRF1, SMC4). Eleven TFs regulated more than two hub genes, and the binding energy of metformin with SPC25 and everolimus with SMC4 was the lowest. Hub genes were negatively correlated with lysosome and positively associated with cell cycle, and the P13K/Akt/mTOR signaling pathway was significantly positively correlated with hub genes. Conclusions: Metformin and everolimus are synergistic in anticancer effects on RCC. Based on transcriptomic data, this study obtained five hub genes associated with everolimus and metformin combination therapy in KIRC to inform KIRC-related research.

Acquired and intrinsic resistance to sunitinib is a major obstacle to improving the therapeutic efficacy of treatment for clear cell renal cell carcinoma (ccRCC). This study aimed to identify novel therapeutic targets and the potential molecular mechanisms to overcome sunitinib resistance in ccRCC. Utilizing genome-wide CRISPR/Cas9 screening and resistant transcriptomics, we identified that prostaglandin reductase 2 (PTGR2) is a novel therapeutic target to overcome sunitinib resistance in ccRCC. The silencing of PTGR2 enhanced the cytotoxic effects of sunitinib in ccRCC cells, as measured by cell viability assays, and suppressed tumor growth in xenograft models. Mechanistically, PTGR2 physically interacts with lysine specific demethylase 6A (KDM6A) via endogenous/exogenous co-immunoprecipitation. PTGR2 knockdown reduced KDM6A protein expression, while KDM6A overexpression partially reversed the sensitization effect of PTGR2 silencing, suggesting KDM6A is a major downstream effector. Our findings establish the PTGR2-KDM6A axis as a potential target for overcoming sunitinib resistance in ccRCC. Pharmacological inhibition of PTGR2 or targeted modulation of KDM6A activity represents a promising combination strategy to overcome sunitinib resistance and improve patient outcomes.