Therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib or erlotinib) significantly prolongs survival time for patients with tumors harboring an activated mutation on EGFR; however, up to 40% of lung cancer patients exhibit acquired resistance to EGFR-TKIs with an unknown mechanism. FOXO3a, a transcription factor of the forkhead family, triggers apoptosis, but the mechanistic details involved in EGFR-TKI resistance and cancer stemness remain largely unclear. Here, we observed that a high level of FOXO3a was correlated with EGFR mutation-independent EGFR-TKI sensitivity, the suppression of cancer stemness, and better progression-free survival in lung cancer patients. The suppression of FOXO3a obviously increased gefitinib resistance and enhanced the stem-like properties of lung cancer cells; consistent overexpression of FOXO3a in gefitinib-resistant lung cancer cells reduced these effects. Moreover, we identified that miR-155 targeted the 3′UTR of FOXO3a and was transcriptionally regulated by NF-κB, leading to repressed FOXO3a expression and increased gefitinib resistance, as well as enhanced cancer stemness of lung cancer in vitro and in vivo. Our findings indicate that FOXO3a is a significant factor in EGFR mutation-independent gefitinib resistance and the stemness of lung cancer, and suggest that targeting the NF-κB/miR-155/FOXO3a pathway has potential therapeutic value in lung cancer with the acquisition of resistance to EGFR-TKIs.

Background: Although there are numerous investigations regarding the bene cial effects of exercise rehabilitation (ER) or astragaloside (AST) after amyloid-beta (Ab) pathology, the mechanisms are still not well understood. We aim to assess whether ER and/or AST counteract Ab pathology via diminishing brain-derived neurotrophic factor (BDNF) signaling de cits and mitochondrial dysfunction.Methods: Ab 1-42 were microinjected into the bilateral ventricles to induce Ab neuropathology in rats. The Alzet osmotic pump containing full of AST was implanted subcutaneously during surgery. The ER group of rats started at seven days post-surgery and lasted for four weeks. The ANA12 was administrated once per day to the endpoint of the experiments to antagonize the BDNF action. Neurobehavioral functions were evaluated by Y-maze, radial maze, and rotarod tests one day before surgery and 14 to 35 days postsurgery. Cortical and hippocampal expressions of both BDNF/TrkB and cathepsin D were determined by Western blotting method. The rat primary cultured cortical neurons were incubated with BDNF and/or AST and ANA12 followed by exposure to aggregated Ab for 24 hours. The cell viability (by MTT assay), mitochondrial membrane permeability and electrochemical potential (by JC-1 stain), DNA fragmentation (sub-G1 and DNA ladder assay), synaptic plasticity (by immuno uorescence stain), and pTrkB/pAkt/pGSK3b/b-catenin signaling (by Western blot) were determined.Results: ER and/or AST reversed neurobehavioral disorders, downregulation of cortical and hippocampal expression of both BDNF/TrkB and cathepsin D, neural pathology, Ab accumulation, and altered microglial polarization caused by Ab. In vitro studies also con rmed that topical application of BDNF and/or AST reversed the Ab-induced cytotoxicity, apoptosis, mitochondrial distress, synaptotoxicity, and decreased expression of p-TrkB, AKT, p-GSK3b, and b-catenin in altered rat cortical neurons. In particular, the bene cial effects of combined ER (or BDNF) and AST therapy in vivo and in vitro were superior to ER (or BDNF) or AST alone. Furthermore, we observed that any gains from ER (or BDNF) and/or AST could be signi cantly eliminated by ANA-12, a potent BDNF/TrkB antagonist. Conclusion:These results indicate that whereas ER (or BDNF) and/or AST attenuate Ab pathology by reversing BDNF/TrkB signaling de cits and mitochondrial dysfunction. In particular, AST can be an alternative therapy to replace ER.

Hair is a noninvasive valuable biospecimen for the long-term assessment of endogenous metabolic disturbance. Whether the hair is suitable for identifying biomarkers of the Alzheimer’s disease (AD) process remains unknown. We aim to investigate the metabolism changes in hair after β-amyloid (Aβ1-42) exposure in rats using ultra-high-performance liquid chromatography-high-resolution mass spectrometry–based untargeted and targeted methods. Thirty-five days after Aβ1-42 induction, rats displayed significant cognitive deficits, and forty metabolites were changed, of which twenty belonged to three perturbed pathways: (1) phenylalanine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis—l-phenylalanine, phenylpyruvate, ortho-hydroxyphenylacetic acid, and phenyllactic acid are up-regulated; (2) arachidonic acid (ARA) metabolism—leukotriene B4 (LTB4), arachidonyl carnitine, and 5(S)-HPETE are upregulation, but ARA, 14,15-DiHETrE, 5(S)-HETE, and PGB2 are opposite; and (3) unsaturated fatty acid biosynthesis— eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), FA 18:3 + 1O, and FA 18:3 + 2O are downregulated. Linoleic acid metabolism belonging to the biosynthesis of unsaturated fatty acid includes the upregulation of 8-hydroxy-9,10-epoxystearic acid, 13-oxoODE, and FA 18:2 + 4O, and downregulation of 9(S)-HPODE and dihomo-γ-linolenic acid. In addition, cortisone and dehydroepiandrosterone belonging to steroid hormone biosynthesis are upregulated. These three perturbed metabolic pathways also correlate with cognitive impairment after Aβ1-42 stimulation. Furthermore, ARA, DHA, EPA, l-phenylalanine, and cortisone have been previously implicated in the cerebrospinal fluid of AD patients and show a similar changing trend in Aβ1-42 rats’ hair. These data suggest hair can be a useful biospecimen that well reflects the expression of non-polar molecules under Aβ1-42 stimulation, and the five metabolites have the potential to serve as novel AD biomarkers.

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