Multi-omics integration identifies HSPA4 as a therapeutic target and reveals DON-induced endoplasmic reticulum stress in bladder cancer

Abstract

Background The therapeutic efficacy against bladder cancer is frequently constrained by chemotherapy resistance and high recurrence rates, underscoring the urgent need to identify novel therapeutic targets and develop effective intervention strategies. To address this challenge, this study employs an integrated approach combining bioinformatics analysis with experimental validation to systematically identify promising therapeutic targets and corresponding targeted drugs for bladder cancer. Methods An integrated analytical approach was employed to identify core genes associated with bladder cancer. This involved cross-validation of multiple transcriptomic datasets using differential expression analysis, weighted gene co-expression network analysis (WGCNA), and summary data-based Mendelian randomization (SMR) analysis. Candidate drugs targeting these core genes were subsequently evaluated through a combination of database screening, molecular docking, and 100 ns molecular dynamics (MD) simulations to assess binding affinity. Finally, the expression of the targets, drug efficacy, and underlying mechanisms were experimentally validated in T24 and UMUC-3 cell lines using quantitative reverse transcription polymerase chain reaction (qRT-PCR), functional assays, and Western blot analysis. Results HSPA4 was consistently identified as a highly expressed core gene demonstrating a causal association with bladder cancer. The compound 6-Diazo-5-oxo-L-norleucine (DON) was predicted and subsequently validated to bind stably to HSPA4. In functional assays, DON significantly inhibited the proliferation, clonogenic formation, and migration capacity of bladder cancer cells. Mechanistic investigations revealed that DON downregulates HSPA4 expression, which induces IRE1α phosphorylation and upregulates the spliced form of XBP1 (XBP1s), thereby activating the endoplasmic reticulum stress (ERS) pathway. Furthermore, overexpression of HSPA4 partially reversed the antitumor effects of DON. Conclusions This study underscores the significance of HSPA4 as a potential therapeutic target in bladder cancer and demonstrates that DON exerts antitumor effects by targeting HSPA4 and activating the ERS pathway. These findings provide a novel candidate drug and a theoretical basis for targeted therapy against this malignancy.