PDF file - 653K, Supplemental Figure 1. Densitometry was peformed on the immunoblots from Figures 2C (A) and 2E (B) using Image J software. Supplemental Figure 2. A/J mice were treated with metformin (250 mg/kg ip qdX3) as in Figure 2C. Supplemental Figure 3. A/J mice were exposed to NNK (100 mg/kg ip qdX3) to induce lung tumorigenesis, and then treated with 5 mg/mL metformin in drinking water for 13 weeks. Supplemental Figure 4. A/J mice (no NNK) were treated with or without 5mg/mL metformin in their drinking water for 13 weeks, and lungs were harvested as previously described. Supplemental Figure 5. (A) Absolute levels of liver metformin uptake between the three routes of administration. (B) Absolute metformin uptake in tissues, converted to molarity.
ARTICLE ABSTRACTMetformin is the most commonly prescribed drug for type II diabetes and is associated with decreased cancer risk. Previously, we showed that metformin prevented tobacco carcinogen (NNK)–induced lung tumorigenesis in a non-diabetic mouse model, which was associated with decreased IGF-I/insulin receptor signaling but not activation of AMPK in lung tissues, as well as decreased circulating levels of IGF-I and insulin. Here, we used liver IGF-I–deficient (LID) mice to determine the importance of IGF-I in NNK-induced lung tumorigenesis and chemoprevention by metformin. LID mice had decreased lung tumor multiplicity and burden compared with wild-type (WT) mice. Metformin further decreased lung tumorigenesis in LID mice without affecting IGF-I levels, suggesting that metformin can act through IGF-I–independent mechanisms. In lung tissues, metformin decreased phosphorylation of multiple receptor tyrosine kinases (RTK) as well as levels of GTP-bound Ras independently of AMPK. Metformin also diminished plasma levels of several cognate ligands for these RTKs. Tissue distribution studies using [14C]-metformin showed that uptake of metformin was high in liver but four-fold lower in lungs, suggesting that the suppression of RTK activation by metformin occurs predominantly via systemic, indirect effects. Systemic inhibition of circulating growth factors and local RTK signaling are new AMPK-independent mechanisms of action of metformin that could underlie its ability to prevent tobacco carcinogen–induced lung tumorigenesis. Cancer Prev Res; 6(8); 801–10. ©2013 AACR.