Abstract 3554: The combined effect of afatinib and metformin on glycolytic regulation in EGFR-mutant non-small cell lung cancer
Authors: Barrios-Bernal Pedro , Hernandez-Pedro Norma Yanet , Soca-Chafre Giovanny , Orozco-Morales Mario , Arrieta Oscar 
Published: July 2017

Abstract
Background: The metabolic features of cancer cells allows their rapid proliferation and escape from apoptosis. Novel targeted treatments are being developed for metabolic dysregulation of malignant cells. There is a growing body of evidence regarding the effects of metformin on the multiple pathways that regulate carcinogenesis. Metformin interacts with insulin growth factor signaling to reduce proliferation and migration of cancer cells. This drug has profound effects on the mitochondrial respiration rate and ATP production. Metformin affects multiple cellular pathways via the activation of AMP-activated protein kinase (AMPK) by liver kinase 1 (LKB1), decreasing growth factor signaling and proliferation via mTOR inhibition. Metformin has been demonstrated to sensitize EGFR tyrosine kinase inhibitor (TKI)-resistant lung cancer cells by reversing the EMT and decreasing IL-6 signaling activation. Antiproliferative synergism between metformin and the multikinase inhibitor sorafenib via AMPK activation has also been demonstrated in NSCLC cells harboring KRAS mutations.
Hypothesis: The Metformin-Afatinib treatment has synergistic effect on cytotoxicity and reduces the Warburg effect by regulating the EGFR and AMPK signaling pathways in Non-Small Cell Lung Cancer (NSCLC).
Objective: Determine the effect of the combined Afatinib-Metformin treatment on NSCLC with different mutational status of EGFR.
Methods: Cell lines with different mutational status of EGFR: A549, H1975 and HCC827 (ATCC) were used. Once the IC50 of afatinib and metformin were found, different combinations were made to determine whether there was a synergistic, additive or antagonistic effect using the Compusyn software. Cell viability was measured by the MTT assay and apoptosis was evaluated by flow cytometry.
Results: The IC50 values of Afatinib for each cell line were as follows: 7 μM (A549), 2 μM (H1975) and 4nM (HCC827) and the IC50 values for Metformin were 15mM (A549) 5mM (H1975) and 8mM (HCC827). We found a synergistic effect in H1975 for the combination of 3µM Afatinib plus 15mM Metformin. In A549 we found a synergistic effect in all combinations. Finally, in HCC827 the combination of 5 nM and 4 nM Afatinib plus 8 nM metformin showed a synergistic effect. The apoptosis rate was increased up to 47% in all combinations for A549 cells. The combination of 3nM Afatinib and 8 mM Metformin induced 59% apoptosis in HCC827. Finally, H1975 induced 46% of apoptosis in the combination that showed synergistic effects.
Conclusions: Afatinib-Metformin showed synergistic effects in a dose-dependent manner in three different cell lines of NSCLC.
Citation Format: Pedro Barrios-Bernal, Norma Yanet Hernandez-Pedro, Giovanny Soca-Chafre, Mario Orozco-Morales, Oscar Arrieta. The combined effect of afatinib and metformin on glycolytic regulation in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3554. doi:10.1158/1538-7445.AM2017-3554