Abstract

Background The acidic tumor microenvironment (TME) is a major driver of immunosuppression and tumor progression in breast cancer. Although myosin VI (MYO6), an actin-dependent motor protein, is frequently upregulated in malignancies, its function in immune remodeling remains poorly understood. Objectives To identify MYO6 as a regulator of acidic TME remodeling and macrophage polarization in breast cancer, elucidate the underlying mechanism, and evaluate its therapeutic relevance for anti-programmed death-ligand 1 (anti-PD-L1) therapy. Methods Integrated analyses of The Cancer Genome Atlas and Gene Expression Omnibus datasets were performed to evaluate the association of MYO6 with prognosis and M2 tumor-associated macrophage (TAM) infiltration in breast cancer. Transcriptomic, molecular, cellular, and pharmacological approaches were used to investigate MYO6-mediated acidic TME remodeling and macrophage polarization. In vivo studies assessed the effects of MYO6 inhibition by 2,4,6-triiodophenol (TIP) on tumor acidification, immunity, and anti-PD-L1 efficacy. Results MYO6 expression correlated with M2 TAM infiltration and poor prognosis in breast cancer. Mechanistically, nuclear MYO6 recruited SP1 and RNA polymerase II to the carbonic anhydrase 9 (CA9) promoter, upregulating CA9 and promoting acidification. The acidic TME activated the proton-sensing receptor GPR65 and the cAMP/PKA/CREB pathway in macrophages, driving M2 polarization. TIP-mediated MYO6 inhibition reduced acidification, restored antitumor immunity, and enhanced anti-PD-L1 efficacy in vivo. Conclusion MYO6 is a key molecule regulating breast cancer immune suppression and immunotherapy responsiveness. By promoting CA9-dependent extracellular acidification, MYO6 drives macrophage M2 polarization through the GPR65/cAMP/PKA/CREB pathway. Targeting MYO6 with TIP may represent a promising strategy to overcome resistance to anti-PD-L1 therapy.