EV-derived miR-941 drives anlotinib resistance in non-small cell lung cancer via Keap1/Nrf2 axis

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality, with anlotinib serving as a crucial multitargeted tyrosine kinase inhibitor for advanced stages. However, its long-term efficacy is severely hampered by the development of acquired drug resistance, a significant clinical challenge. While elevated extracellular vesicle (EV)-derived miR-941 has been observed in resistant patients, its precise functional role in mediating this resistance and the underlying molecular mechanisms have remained largely unknown, representing a critical gap in understanding and overcoming treatment failure.

Researchers conducted functional gain- and loss-of-function experiments in A549 and H1299 non-small cell lung cancer cell lines to evaluate cell viability, apoptosis, and migration. The direct interaction between miR-941 and the Keap1 3'UTR was confirmed using dual-luciferase reporter assays. In vivo, anlotinib resistance was assessed in xenograft models, comparing wild-type mice to those with a Keap1 3'UTR-mutant. Clinical relevance was further investigated by analyzing tumor tissues from anlotinib-treated patients for miR-941 expression and survival outcomes.

EV-derived miR-941 was significantly upregulated in anlotinib-resistant patients. Mechanistically, miR-941 directly targeted the Keap1 3'UTR, leading to the suppression of Keap1 protein expression and subsequent activation of the Nrf2 signaling pathway. This Keap1/Nrf2 axis inhibited apoptosis and upregulated anti-apoptotic proteins, including Bcl-2 and Mcl-1, while promoting malignant phenotypes in vitro. > Crucially, in vivo anlotinib resistance induced by miR-941 overexpression was completely abrogated when the Keap1 binding site was mutated, demonstrating a direct causal link. Clinical samples from resistant tumors confirmed elevated miR-941 levels and a molecular signature consistent with Keap1 downregulation and Nrf2 activation. Furthermore, high expression of miR-941 was strongly associated with shorter progression-free survival and overall survival in patients.