Cancer-associated fibroblasts-derived exosomal circ_0067557 promotes colorectal cancer epithelial-mesenchymal transition via BHLHE40-mediated transcriptional activation of OTUB2

OBJECTIVE: This study aimed to clarify how cancer-associated fibroblast-derived exosomal circ_0067557 (CAF-exo circ_0067557) promotes epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC) through BHLHE40-mediated transcriptional activation of OTUB2. METHODS: CAF-derived exosomes were isolated and characterized from CRC tissues, while normal fibroblast-derived exosomes were obtained from adjacent normal tissues. The transfer of circ_0067557 was tracked using fluorescence labeling and endocytosis assays. Expression of circ_0067557, BHLHE40, OTUB2, and EMT-related markers was assessed by qRT-PCR, Western blotting, and immunofluorescence. Gain- and loss-of-function models were established to determine the biological role of circ_0067557. RNA sequencing identified OTUB2 as a key downstream target. ChIP-qPCR and dual-luciferase reporter assays were performed to verify BHLHE40 binding to the OTUB2 promoter. RIP and RNA pull-down assays confirmed the interaction between circ_0067557 and BHLHE40. Mouse xenograft and tail vein metastasis models were used to evaluate the oncogenic effects of circ_0067557 in vivo. RESULTS: CAF-exos successfully delivered circ_0067557 into CRC cells, significantly enhancing their proliferative, migratory, invasive, and EMT capabilities. Mechanistically, circ_0067557 interacted with and recruited BHLHE40, thereby increasing its binding to the OTUB2 promoter and promoting OTUB2 transcription. In vivo experiments further confirmed that circ_0067557 markedly enhanced tumor growth and distant metastasis through the BHLHE40/OTUB2 signaling axis. CONCLUSION: CAF-exo circ_0067557 promotes EMT, invasion, metastasis, and tumor progression in CRC by recruiting BHLHE40 and activating OTUB2 transcription. These findings reveal a novel mechanism by which the tumor microenvironment (TME) regulates cancer cell metastasis.