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.

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Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.

Purpose: Preclinical and retrospective studies suggested a role for metformin in sensitizing patients who have diabetes with non-small cell lung cancer (NSCLC) to EGFR tyrosine kinase inhibitors (TKIs). We therefore examined its effects in combination with gefitinib in patients without diabetes harboring EGFR mutations (EGFRm).Patients and Methods: A total of 224 patients without diabetes with treatment-na€ ve stage IIIB-IV EGFRm NSCLC were randomly assigned in a 1:1 ratio to receive gefitinib plus either metformin or placebo. The primary endpoint was progression-free survival (PFS) rate at 1 year and secondary endpoints included overall survival (OS), PFS, objective response rate (ORR), and safety. Serum levels of IL6 were also examined in an exploratory analysis.Results: The median duration of follow-up was 19.15 months. The estimated 1-year PFS rates were 41.2%[95% confidence interval (CI), 30.0-52.2] with gefitinib plus metformin and 42.9% (95% CI, 32.6-52.7) with gefitinib plus placebo (P ¼ 0.6268). Median PFS (10.3 months vs. 11.4 months) and median OS (22.0 months vs. 27.5 months) were numerically lower in the metformin group, while ORRs were similar between the two arms (66% vs. 66.7%). No significant treatment group differences were detected across all subgroups with respect to PFS, including those with elevated levels of IL6. Metformin combined with gefitinib resulted in a remarkably higher incidence of diarrhea compared with the control arm (78.38% vs. 43.24%).Conclusions: Our study showed that addition of metformin resulted in nonsignificantly worse outcomes and increased toxicity and hence does not support its concurrent use with first-line EGFR-TKI therapy in patients without diabetes with EGFRm NSCLC.