Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab

Abstract

OBJECTIVE(S): Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. METHODS: Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. RESULTS: Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. CONCLUSION: Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs. In addition, the exosome secretion pathway possibly contributes to the HER2 trafficking to plasma membrane, since the blockade of exosome secretion decreased surface HER2 levels.