American Association for Cancer Research
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Supplementary Figures 1 - 18 from Metformin Sensitizes EGFR-TKI–Resistant Human Lung Cancer Cells In Vitro and In Vivo through Inhibition of IL-6 Signaling and EMT Reversal

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journal contribution
posted on 2023-03-31, 17:26 authored by Li Li, Rui Han, Hualiang Xiao, Caiyu Lin, Yubo Wang, Hao Liu, Kunlin Li, Hengyi Chen, Fenfen Sun, Zhenzhou Yang, Jianxin Jiang, Yong He

PDF file - 1625K, Supplementary Figure 1 IC50 values of different cell lines to TKIs under indicated conditions Supplementary Figure 2 BrdU incorporation assay for H1975 cells. Supplementary Figure 3 Expression of OCT-1 and LKB1 in different cell lines used in the current study. Supplementary Figure 4 Metformin decreases invasion and motility of erlotinib-resistant H1650-M3 cells. Supplementary Figure 5 Metformin decreases SNAIL expression in TKI-resistant human lung cancer cells. Supplementary Figure 6 Quantification of blots shown in Fig. 2E. Supplementary Figure 7 Metformin decreases IL-6 gene transcription in TKI-resistant cell lines. Supplementary Figure 8 Metformin reverses TKI resistance in IL-6 overexpressing PC-9psb cells. Supplementary Figure 9 Inhibition of IL-6 signaling is essential for metformin to overcome erlotinib resistance in H1650-M3 cells. Supplementary Figure 10 Body weight of PC-9GR xenografts Supplementary Figure 11 Serum insulin levels and glucose levels from xenografts established by PC-9GR cells. Supplementary Figure 12 Metformin in combination with gefitinib suppresses tumor growth in PC-9 xenografts Supplementary Figure 13 Metformin in combination with gefitinib increased the expression of E-cadherin and decreased the expression of Vimentin in tumor sections of PC-9 xenografts. Supplementary Figure 14 Quantification of blots in Fig. 6D. Supplementary Figure 15 Western blotting analysis of the expression of indicated markers on protein extracts obtained from harvested tumors of the gefitinib group and gefitinib+metformin group. Supplementary Figure 16 Metformin in combination with gefitinib decreases expression of phosphorylation of AKT and STAT3 in xenografts established by PC-9GR cells. Supplemental Figure 17 Metformin overcomes TKI resistance through EMT reversal and IL-6 signaling inhibition. Supplemental Figure 18 Metformin disrupts the evil axis of TGFbeta/IL-6, EMT, CSCs and TKI resistance.



Purpose: The EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have become a standard therapy in patients with EGFR-activating mutations. Unfortunately, acquired resistance eventually limits the clinical effects and application of EGFR-TKIs. Studies have shown that suppression of epithelial–mesenchymal transition (EMT) and the interleukin (IL)-6/STAT3 pathway may abrogate this acquired mechanism of drug resistance of TKIs. This study aims to investigate the effect of metformin on sensitizing EGFR-TKI–resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal.Experimental Design: The effect of metformin on reversing TKI resistance was examined in vitro and in vivo using MTT, BrdUrd incorporation assay, invasion assay, flow cytometry analysis, immunostaining, Western blot analysis, and xenograft implantation.Results: In this study, metformin, a widely used antidiabetic agent, effectively increased the sensitivity of TKI-resistant lung cancer cells to erlotinib or gefitinib. Metformin reversed EMT and decreased IL-6 signaling activation in TKI-resistant cells, while adding IL-6 to those cells bypassed the anti-TKI-resistance effect of metformin. Furthermore, overexpression or addition of IL-6 to TKI-sensitive cells induced TKI resistance, which could be overcome by metformin. Finally, metformin-based combinatorial therapy effectively blocked tumor growth in xenografts with TKI-resistant cancer cells, which was associated with decreased IL-6 secretion and expression, EMT reversal, and decreased IL-6–signaling activation in vivo.Conclusion: Metformin, generally considered nontoxic and remarkably inexpensive, might be used in combination with TKIs in patients with non–small cell lung cancer, harboring EGFR mutations to overcome TKI resistance and prolong survival. Clin Cancer Res; 20(10); 2714–26. ©2014 AACR.