Exosome-Mediated miR-7-5p Delivery Enhances the Anticancer Effect of Everolimus via Blocking MNK/eIF4E Axis in Non-Small Cell Lung Cancer

Abstract

Background Everolimus is a kind of mTOR inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). Typically, eIF4E phosphorylation increased by mTOR inhibitors was mainly mediated by MNKs. But the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposing. Our previous studies have confirmed that tumor suppressor miR-7-5p was decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. Here, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. Methods miR-7-5p level and expression of main markers of MNK/eIF4E axis were evaluated by qRT-PCR, western blot, in situ hybridization, and immunohistochemistry on NSCLC cell lines and human NSCLC samples. Proliferation, migration and invasion of NSCLC cells in culture were explored by Colony formation, CCK-8, Wound healing and Transwell assays. NSCLC cell tumorigenicity was assessed by xenotransplants in nude mice. Targeted binding of miR-7-5p to MNK1 was confirmed by the Dual-luciferase reporter assay. And the isolation and identification of exosomes were investigated by Invitrogen™ Total Exosome RNA Isolation Kit, western blot, transmission electron microscopy and Zetasizer Nano ZS90 instrument. Results Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but supplement of extra exosomal miR-7-5p could reverse it. Of note, both lower expression of miR-7-5p and elevated MNK1 protein were associated with poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus through inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and vivo. Combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy via dual abrogation of MNK/eIF4E and mTOR in NSCLC. Conclusion Everolimus decreases the intracellular miR-7-5p levels through release of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy via targeting MNK/eIF4E axis and mTOR. Therefore, exosome-mediated miR-7-5p delivery combined with Everolimus may be considered as a promising novel combined therapeutic strategy for NSCLC.