A NEK2/ZWINT-NDC80 regulatory axis drives bladder cancer progression and chemoresistance.

Bladder cancer progression and chemoresistance remain major clinical challenges, yet the molecular determinants underlying these aggressive phenotypes are incompletely understood. Integrating TCGA transcriptomics, GEO single-cell transcriptomics, patient tissue profiling, flow cytometry-based cell sorting, functional perturbation assays, and xenograft models, we investigated the cooperative roles of NEK2, ZWINT, and the downstream effector NDC80 in bladder cancer biology. We identified a previously unrecognized NEK2/ZWINT-NDC80 regulatory axis that governs malignant cell behavior. NEK2 and ZWINT were markedly co-upregulated in high-grade tumors and strongly associated with EMT-related transcriptional programs. NEK2⁺ZWINT⁺ double-positive cells represented a stable and aggressive tumor subpopulation enriched in invasive lesions. Dual knockdown of NEK2 and ZWINT profoundly impaired proliferation, clonogenicity, motility, and in vivo tumorigenicity, whereas single-gene perturbation produced only partial effects. Mechanistically, NDC80 acted as a critical downstream effector, integrating NEK2-ZWINT signals and acting as a key downstream effector that integrates signals from NEK2 and ZWINT to drive malignant phenotypes. NDC80 depletion suppressed tumor growth and significantly increased gemcitabine sensitivity. Clinically, NDC80 expression correlated with advanced stage and chemoresistant bladder cancer tissues. Our findings reveal the NEK2-ZWINT-NDC80 axis as a central regulatory module driving bladder cancer progression and gemcitabine resistance. Targeting this axis may offer a therapeutic strategy for high-risk bladder cancer.