Metformin increases antitumor activity of MEK inhibitors through GLI1 downregulation in LKB1 positive human NSCLC cancer cells

Abstract

Purpose: Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1-wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization.

Experimental design: Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene.

Results: The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters.

Conclusions: Metformin potentiates the antitumor activity of MEK-Is in human LKB1-wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

Keywords: MEK; NSCLC; metformin; pimasertib; selumetinib.

Figure 1. Effect of metformin alone and in combination with selumetinib on cell proliferation, on the induction of apoptosis and activation of GLI1 in CALU-3, H1299, H358 and H1975 cell lines

A. Effect of metformin alone and in combination with selumetinib on cell proliferation in CALU-3, H1299, H358 and H1975 cell lines. Cells were treated with metformin, selumetinib and combination of both. Cell proliferation was measured by BrdUrd incorporation assay. BrdUrd was added for 1 hour, and cells were processed for immunofluorescence with anti-BrdUrd. Cell nuclei were counterstained with Hoechst. The average results ± SD of 3 independent experiments in which at least 500 cells were counted are shown. B. Combination index (CI) values from CALU-3, H1299, H358 and H1975 cell lines treated with metformin alone and in combination with selumetinib obtained with CompuSyn Program for different doses. ED50, ED75 ED90 represent the doses effecting 50, 75, and 90%, respectively of growth inhibition compared to control. C. Apoptosis was evaluated as described in Materials and Methods with Annexin V staining in CALU-3, H1299, H358 and H1975 cancer cells, which were treated, in the absence or presence of recombinant Sonic Hedgehog, with metformin, selumetinib or both. Columns mean of 3 identical wells of a single representative experiment. Western Blot analysis for PARP, (89)-cleaved-PARP fragment were performed on protein lysates from cell after the indicated treatment.

Figure 2. GLI1-mediated effects of metformin, selumetinib or both in NSCLC cell lines

A. anchorage-independent colony formation in soft-agar; B. Invasion assay; in the absence or presence of recombinant Sonic Hedgehog; The results are the average ± SD of three independent experiments, each done in triplicate. C. GLI1-driven luciferase expression in H1299 cells before and after treatment with metformin, selumetinib or both. Western Blot analysis for EMT-related protein Vimentin and Snail and GLI1 were performed on protein lysates from cell after the indicated treatment.

Figure 3. Effects of metformin, selumetinib or both on the downstream pathway in NSCLC

A. Western blotting analysis of intracellular proteins MEK, MAPK, AMPK, Akt, S6, and their phosphorylated forms following treatment with the indicated concentration of metformin, selumetinib or both in H1299 and H1975 NSCLC cell line. β-Actin was included as a loading control. B. Western blotting analysis of Vimentin, GLI1, MAPK and its activated form on protein extracts from H1975 tumors harvested by mice treated with the indicated concentrations of metformin, selumetinib or their combination. β-Actin was included as a loading control.

Figure 4. Effects of metformin and MEK inhibitor on intracellular pathways and MMP-9, MMP2 and uPA expression and activity in NSCLC cells

A. Secretion of MMP-2, MMP-9 and uPA into the conditioned medium of H1299 and H1975 NSCLC cells, as measured in cell culture media by specific ELISAs. B. MMP-2 and MMP-9 activities determined by gelatin zymography in the conditioned media of H1299 and H1975 NSCLC cells. C. ChIP Assay evaluating the binding of NF-κB (p65) to the MMP9 and MMP2 promoters in H1299 cells.

Figure 5. In vivo effects of the combined treatment with metformin and selumetinib

A, B. Athymic nude mice were injected subcutaneously into the dorsal flank with 10⁷ NSCLC cancer cells. When the average tumor size was 75 mm³ in H1299 xenografts (A) and 150 mm³ in H1975 xenografts (B), mice were treated as indicated in Materials and Methods. Xenografted mice received only vehicle (control group), metformin (200 mg/mL metformin diluted in drinking water and present throughout the treatment period), selumetinib (25 mg/kg p.o.), or their combination. Data represent the average ± SD. Student t test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. C. Percentage of human Alu sequences in the lungs of mice after tail vein injection with H1299 and H1975 cells and the indicated treatments.