Abstract
This study investigates the role of ERBB2 mutations in promoting recurrence and metastasis of non-muscle-invasive bladder cancer (NMIBC). Analysis of whole exome sequencing (WES) data from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases revealed a significant association between ERBB2 mutations and immune cell infiltration. To validate these findings, formalin-fixed, paraffin-embedded tumor tissues from patients with recurrent NMIBC were analyzed, with a focus on ERBB2 mutations. In addition, bladder cancer cell lines carrying wild type or mutant ERBB2 were established using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) technology. Functional experiments, including Western blotting, protein stability assays, and ubiquitination analyses, demonstrated that ERBB2 mutations promote hypoxia-inducible factor-1 (HIF-1) phosphorylation, leading to its stabilization and enhancing the proliferative, migratory, and invasive capacities of tumor cells. Furthermore, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and Transwell assays confirmed the impact of these mutations on cellular behavior, while drug sensitivity assays indicated increased susceptibility of ERBB2-mutant cells to therapeutic agents. In vivo studies using mouse models further supported these findings, showing that ERBB2 mutations promote tumor growth, metastasis, and macrophage infiltration. Collectively, these results suggest that ERBB2 mutations drive NMIBC progression by stabilizing HIF-1 through phosphorylation, thereby facilitating tumor development and immune modulation, and underscore the potential of ERBB2 as a therapeutic target for preventing NMIBC recurrence and metastasis.