Anti-proliferative Effect Of PI3K/mTOR Dualinhibitor,PO-1023,is Synergistically Enhanced By Inhibition Of Neddylation In Human NSCLC A549 Cells

PI3K-Akt-mTOR signal pathway and cascaded signaling molecule play a vital role in modulating cell proliferation and metabolism,the disfunction of which frequently occur in the progress of lung cancer.Besides,the overexpression of NEDD8(neural precursor cell expressed developmentally down-regulated 8)and NAE(NEDD8-activating enzyme inhibitor),the main molecule participating in protein neddylation process,can also be detected in parts of the lung cancer patients During the study on PO-1023,a new synthesized PI3K/mTOR dual inhibitor,the results were accidently found that MLN4924 synergistically enhances the PI3K/mTOR dual inhibitor-induced anti-cancer effect.The following study aims at verifying cytotoxicity of the combination of PO-1023 and MLN4924 and furtherly revealing the molecular mechanism contributing to the effectBy analyzing the gene expression of PI3K-Akt-mTOR signal pathway key molecules and NAE through the databasis of TCGA-LUAD?LUSC,PIK3CA gene,one component of PI3K subunit,presented large quantity of amplification.Akt and NAE gene showed up a certain ratio of both amplification and mutation while the main alteration of mTOR gene was mutation.Among these genes,the high expression of PIK3CA,NAE would significantly reduce the survival rate of patients.In the molecular experiment,PO-1023 and MLN4924 inhibited the cell vitality of A549 by a dose-depended manner.The combination of both compounds displayed a better effect,which was further proved by cell proliferation and apoptosis experiments.By western blotting tests,PO-1023 imposed a potent inhibition on p-S473-Akt while less impact on p-S2448-mTOR compared with MLN4924.The combination of both compounds revealed a synergistical inhibition on p-Akt and p-mTOR.By exploring the mechanism,MLN4924 could indirectly inhibit the the connection between Ubiquitin and its substrates to block the ubiquitination degradation of Deptor which consequently increasing the expression of Deptor.Deptor inhibited the activity of mTOR complex and reduced the expression of p-mTOR.Furthermore,the knockdown of Deptor was found to increase the expression of p-mtor in A549 cells and enhanced cell activity on normal both MLN4924 condition,confirming the role of Deptor in the pharmacology of MLN4924 and providing concrete evidence for the mechanism of better synergistical effect of PO-1023 and MLN4924This study verified the anti-cancer activity and molecular mechanism of new PI3K/mTOR dual inhibitor,PO-1023.At the same time,the results confirmed the PO-1023 and MLN4924 together displayed better cytotoxicity and revealed the role of Deptor in the enhancement of the pharmacological effect.The study provided a new inspiration of how to enhance the anticancer activity of the leading compound targeting at PI3K/mTOR.Furthermore,the results verified the experimental basis for the regulation of key proteins related to tumor pathology(such as mTOR)utilizing the natural interaction of molecular transduction pathways...