Metformin prevents tobacco carcinogen--induced lung tumorigenesis

Abstract

Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen-induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Metformin reduced lung tumor burden by up to 53% at steady-state plasma concentrations that are achievable in humans. mTOR was inhibited in lung tumors but only modestly. To test whether intraperitoneal administration of metformin might improve mTOR inhibition, we injected mice and assessed biomarkers in liver and lung tissues. Plasma levels of metformin were significantly higher after injection than oral administration. In liver tissue, metformin activated AMPK and inhibited mTOR. In lung tissue, metformin did not activate AMPK but inhibited phosphorylation of insulin-like growth factor-I receptor/insulin receptor (IGF-1R/IR), Akt, extracellular signal-regulated kinase (ERK), and mTOR. This suggested that metformin indirectly inhibited mTOR in lung tissue by decreasing activation of insulin-like growth factor-I receptor/insulin receptor and Akt upstream of mTOR. Based on these data, we repeated the NNK-induced lung tumorigenesis study using intraperitoneal administration of metformin. Metformin decreased tumor burden by 72%, which correlated with decreased cellular proliferation and marked inhibition of mTOR in tumors. These studies show that metformin prevents tobacco carcinogen-induced lung tumorigenesis and support clinical testing of metformin as a chemopreventive agent.

Figure 1. Oral administration of metformin prevents NNK-induced lung tumorigenesis in A/J mice and is associated with modest inhibition of the mTOR pathway

a) Schema for the prevention study with oral metformin in a tobacco carcinogen-driven mouse model of lung tumorigenesis. b) Mean plasma levels of metformin in A/J mice treated with 1 or 5 mg/mL metformin orally were determined using LC-MS/MS. c) Tumor multiplicity (left panel), tumor volume (middle panel) and tumor burden (right panel) were assessed after 13 weeks of oral administration of 1 mg/mL or 5 mg/mL metformin in A/J mice. For the dot plots, each point represents a mouse and the lines represent the median values. For the box and whisker graph, boxes indicate interquartile range, lines indicate median, and whiskers indicate minimal and maximal values. ‘n.s.’ is not significant. d) Representative images of IHC analysis for P-S6 in airway epithelium (AE) and lung tumors from control mice and from mice that received oral metformin (left panel), and quantification of IHC analysis (right panel). Graph shows mean staining indices and error bars represent SD. IHC analysis was performed on 5 mice/treatment group.

Figure 2. Intraperitoneal administration of metformin causes tissue-specific modulation of the AMPK and mTOR pathways in A/J mice

a–c) Immunoblotting analysis of components of the AMPK pathway and pathways downstream of IGF-1R/IR that converge on mTOR in liver (a and b) and lung tissues (c). Tissues were harvested from mice treated short-term with intraperitoneal injections of 250 mg/kg metformin (qd x 3) in two independent studies, and representative blots from these studies are shown. Densitometry was performed on immunoblots of liver lysates from both studies, which is presented in the right panel of (a). b) Liver tissue was also harvested from mice treated with 5 mg/mL metformin orally for 7 d, for comparison. d) OCT1 expression in liver and lung tissues from A/J mice was assessed by RT-PCR.

Figure 3. Administration of metformin by intraperitoneal injection prevents NNK-induced lung tumorigenesis

a) Schema for the prevention study with metformin administered by intraperitoneal injection in a tobacco carcinogen-driven mouse model of lung tumorigenesis. Tumor multiplicity (b), tumor volume (c) and tumor burden (d) were assessed after 13 weeks of administration of 250 mg/kg metformin qd by intraperitoneal injection in A/J mice. For the dot plots in (b) and (d), each point represents a mouse and the lines represent the median values. For the box and whisker graph shown in (c), boxes indicate interquartile range, lines indicate median, and whiskers indicate minimal and maximal values.

Figure 4. Intraperitoneal administration of metformin inhibits the mTOR pathway in lung tissue, decreases in tumor cell proliferation, and decreases lung- and tumor-associated Foxp3+ Treg

a) Representative images of IHC analysis for p-S6 in airway epithelium (AE) and lung tumors from vehicle-treated mice and from mice that received intraperitoneal metformin (left panel), and quantification of IHC analysis (right panel). Graph shows mean staining indices and error bars represent SD. b) Representative images of IHC analysis for Ki-67, a marker of cellular proliferation, in lung tumors from vehicle and metformin treated mice (upper panels), and quantification of IHC analysis (bottom panel). Graph shows mean number of Ki-67+ cells/tumor and error bars represent SD. c) Representative images of IHC for tumor-associated Foxp3+ and CD3+ cells in A/J mice treated with vehicle or intraperitoneal metformin (left panel), and quantification of IHC analysis for lung- and tumor-associated percent Foxp3+/CD3+ cells (upper and lower right panels, respectively). For (a), (b), and (c), IHC analysis was performed on 5 mice/treatment group.

Figure 5. The tumorigenic diet AIN-93G/M causes fatty liver disease in A/J mice, which is prevented by metformin, but does not cause hyperglycemia

a) H&E (right panel) or Oil Red O staining (left panel) of liver sections from A/J mice fed AIN-93G/M and treated with vehicle. b) H&E staining of liver sections from A/J mice fed AIN-93G/M and treated with 250 mg/kg metformin QD i.p. for 13 weeks. For (a) and (b), images were taken at 10× and 40× magnification. c) Blood glucose levels in A/J mice that were fed the cereal diet NIH-07 or the tumorigenic diet AIN-93G/M (n=5 mice/group). Graph shows mean values, and error bars represent SD.

Figure 6. Metformin-induced inhibition of the mTOR pathway in lung tissue is associated with decreases in circulating levels of IGF-1 and insulin

a–b) Plasma levels of insulin (a) or IGF-1 (b) were assessed by ELISA in A/J mice treated with oral or intraperitoneal administration of metformin at the end of the tumorigenesis studies. Graphs show mean values and error bars represent SD. This analysis was performed on 5 mice/treatment group. Plasma IGF-1 levels were also assessed in mice given intraperitoneal injections of saline or 250 mg/kg metformin qd x 3 (b, far right panel). (c) Immunoblotting analysis of IGF-1R/IR/Akt/mTOR pathway in liver and lung tissues harvested from A/J mice 0.5 h after administration of 0.5 mg/kg IGF-1 or 0.75 U of insulin.