Metformin synergistically enhances the antitumor activity of the third-generation EGFR-TKI CO-1686 in lung cancer cells through suppressing NF-κB signaling

Purpose Third-generation irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), rociletinib (CO-1686), is great efficacy against EGFR-mutated patients bearing the T790M resistance mutation. However, acquired resistance may emerge. There is a need to characterize acquired resistance mechanism(s) and to devise ways to overcome CO-1686 resistance. Experimental Design MTT assay, ki67 incorporation assay, transwell assay and TUNEL assay were employed to analyze the effects of metformin to reverse CO-1686 resistance in vitro. The NF-kappa B activity was measured by the antibody of p50, p65, p-IKB alpha, and p-IKK alpha/beta. Western blotting was used to analyze the proteins in cells. Results We have established CO-1686-resistant cell lines of PC-9GRCOR and H1975COR from two parental cell lines of PC-9GR and H1975 by long-term exposure to increasing doses of CO-1686. Compared with the parental cells, PC-9GRCOR cells and H1975COR cells showed 90-folds and 20-folds higher resistance to CO-1686, respectively. Critically, we showed that the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) signaling molecular proteins subunits of p50, p65 and its inhibitor proteins of IKB alpha, IKK alpha/beta in phosphorylation levels in resistant cells were higher than parental cells. Accordingly, inhibition of NF-kappa B activity used TPCA-1 effective in decreasing viability and inducing apoptosis of resistant cells. Moreover, metformin overcame the acquired resistance to CO-1686 by reducing cell proliferation and invasion. Metformin combined with CO-1686 synergistically inhibited the p-IKB alpha;, p-IKK alpha/beta, p50, and p65. Conclusions NF-kappa B signaling activation induced acquired resistance to CO-1686. Metformin sensitized resistant cells to CO-1686 via inhibiting NF-kappa B signaling.