NOVA1 regulates alternative splicing of CD44 to suppress breast cancer invasion and metastasis

Background: Aberrant alternative splicing (AS) promotes breast cancer progression by enhancing cellular plasticity, invasion, and metastasis. NOVA1, a neuron-specific RNA-binding protein and splicing regulator, has been implicated in multiple diseases, but its role in breast cancer remains unclear. Methods: NOVA1 expression and clinicopathologic relevance were analyzed using TCGA and GSEA datasets and validated in patient samples by RT-qPCR, Western blotting, and immunohistochemistry. Functional assays (wound healing, transwell invasion, and mouse metastasis models) were performed following NOVA1 overexpression or knockdown. Transcriptomic sequencing and AS analyses identified NOVA1-regulated splicing events, followed by validation and rescue experiments focusing on CD44 isoforms. Results: Analysis of 89 paired primary breast carcinomas and matched adjacent normal tissues from the TCGA dataset revealed that NOVA1 is among the most differentially expressed splicing factors. NOVA1 expression was reduced in breast cancer and associated with favorable prognosis. NOVA1 overexpression inhibited migration, invasion, and metastasis and suppressed epithelial-mesenchymal transition (EMT). Transcriptome profiling indicated that NOVA1 regulates extracellular matrix organization and alternative splicing of CD44 , leading to reduced expression of the pro-metastatic isoform CD44v6. Conversely, NOVA1 silencing increased CD44v6 expression and enhanced metastatic potential. Rosiglitazone treatment restored NOVA1 expression and reduced cancer cell invasiveness. Conclusions: NOVA1 functions as a metastasis suppressor by repressing CD44v6-mediated EMT, establishing a novel NOVA1-CD44v6 splicing axis in breast cancer. Pharmacologic induction of NOVA1 may represent a promising strategy to limit metastatic progression.