The RNA-binding protein RBFOX2 suppresses colorectal cancer proliferation and metastasis by reducing FUBP1 mRNA stability to induce mitochondrial dysfunction and ferroptosis

Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Aberrant expression of RNA binding fox-1 homolog 2 (RBFOX2) has been implicated in tumorigenesis and progression; however, its biological functions and clinical significance in CRC remain poorly understood. This study aimed to elucidate the role of RBFOX2 in CRC.

Analysis of RBFOX2 expression levels in clinical CRC specimens was initially conducted utilizing the Human Protein Atlas database, with immunohistochemistry (IHC) data providing validation. The transcriptional and protein levels of RBFOX2 and far upstream element binding protein 1 (FUBP1) were quantitatively examined in 42 matched pairs of CRC and adjacent non-cancerous tissues using RT-qPCR and Western blot. The molecular interaction between RBFOX2 and FUBP1 was assessed via RNA immunoprecipitation and dual-luciferase reporter assays. FUBP1 transcript stability was determined following actinomycin D treatment. Functional assays, including MTT and colony formation, were performed in SW480 cells to assess proliferative capacity, whereas Transwell and wound-healing assays were used to evaluate migratory and invasive abilities. Mitochondrial ultrastructure was visualized by electron microscopy. Intracellular reactive oxygen species (ROS) were measured by flow cytometry, and concentrations of malondialdehyde (MDA), glutathione (GSH), and Fe²⁺ were determined using specific commercial assay kits. For in vivo studies, stable RBFOX2-overexpressing SW480 cells were inoculated into mouse models to establish subcutaneous xenografts and metastatic tumors. Tumor tissues were subsequently analyzed by IHC for Ki-67 expression and by hematoxylin-eosin (H&E) staining to evaluate metastatic nodules.

RBFOX2 was downregulated in CRC. Overexpression of RBFOX2 suppressed CRC cell proliferation and migration in vitro and inhibited tumor growth and metastasis in vivo. RBFOX2 interacted with FUBP1 and regulated its mRNA stability, contributing to ferroptosis induction. FUBP1 overexpression reversed RBFOX2-mediated suppression of CRC cell proliferation and migration.

RBFOX2 interacts with FUBP1, destabilizes its mRNA, and promotes ferroptosis, thereby inhibiting CRC proliferation and metastasis, whereas FUBP1 exerts oncogenic effects. These findings provide novel insights into CRC molecular mechanisms and establish a theoretical foundation for targeted therapeutic strategies.