Everolimus-Induced Loss of Exosomal miR-7-5p Activates 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 cells (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 found that tumor suppressor miR-7-5p was loss in A549 cells after treatment of Everolimus. Exactly, MNK1 is the target of miR-7-5p. Here, we analyzed levels and functions of miR-7-5p in the usage of Everolimus in NSCLC.Methods: miR-7-5p level and expression of main markers of MNK/eIF4E axis was evaluated by qRT-PCR, in situ hybridization, and immunohistochemistry on human NPC samples, and by RT-PCR, western blot on NPC cell lines. Proliferation, migration and invasion of NPC cells in culture were assessed by Colony formation, CCK-8, Wound healing and Transwell assays. NPC 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 invested by Invitrogen™ Total Exosome RNA Isolation Kit, western blot, transmission electron microscopy and Zetasizer Nano ZS90 instrument.Results: Everolimus stimulatedtherelease of miR-7-5p loaded exosomes from NSCLC cellsinRab27A and Rab27B dependent manners, thereby reducing the intracellular tumor suppressor miR-7-5p to attenuate the inhibition of MNK1 and promote MNK-dependent eIF4E phosphorylation. Of note, both lower miR-7-5p and positive MNK1 could act as independent poor prognostic biomarkers for NSCLC. And delivery of miR-7-5p would inhibit the poor prognosis of it. Bothendogenous miR-7-5p-5p or exo-miR-7-5p collaborated with Everolimus, not only inhibited the proliferation, migration, and metastasisof NSCLC in vitro and in vivo, but also promoted the apoptosis of NSCLC viatargeting MNK/eIF4E axis.Conclusion: Everolimus-induced loss of exosomal miR-7-5p activates MNK/eIF4E axis to blunt effectiveness of itself in NSCLC. Therefore, delivery of miR-7-5p could act as a combined therapeutic strategy for mTOR-targeted cancer therapy and prognosis.