Abstract
AimsEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.MethodsWe selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.ResultsIn the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.ConclusionOur results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.
Highlights
The epidermal growth factor receptor (EGFR) signaling pathway plays a central role in the development and progression of lung cancer [1]
Understanding mTOR-associated signaling-pathway differences between EGFR tyrosine kinase inhibitors (TKIs)-sensitive and -resistant non-small cell lung cancer (NSCLC) cells is critical for targeting mTOR to overcome patient resistance after TKI treatment
We found that targeting mTOR with ku-0063794 produced better antitumor effects and reduced G1 cell cycle arrest in both sensitive and resistant cells through MTT assay and flow cytometry analysis which should be further strengthened by colony formation capacity (CFC) assays in the future; we did not find significant G1-cell cycle arrest cell proliferation was inhibited in H1975 cells by ku-0063794 which maybe primarily due to other forms of cell death like apoptosis or autophagy
Summary
The epidermal growth factor receptor (EGFR) signaling pathway plays a central role in the development and progression of lung cancer [1]. EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are effective clinical therapies for patients with advanced NSCLC who have EGFR-activated mutations, compared with standard first-line cytotoxic chemotherapy [2,3,4]. Despite these dramatic benefits of EGFR TKIs, all of these patients inevitably develop resistance to gefitinib and erlotinib, usually 6–12 months after initiation of TKI treatment [5]. Strategies to overcome resistance consider the resistance mechanism itself [7,9,10], whereas an alternative strategy is to identify new molecules or mechanisms that overcome the resistance, such as mTOR
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