Metformin prevents liver tumourigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma

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Abstract

Metformin is a hypoglycaemic agent used to treat type 2 diabetes mellitus (DM2) patients, with a broad safety profile. Since previous epidemiological studies had shown that the incidence of hepatocellular carcinoma (HCC) decreased significantly in metformin treated DM2 patients, we hypothesised that intervention with metformin could reduce the risk of neoplastic transformation of hepatocytes. HCC is the most common primary liver malignancy and it generally originates in a background of liver fibrosis and cirrhosis. In the present study, we took advantage of a transgenic mouse (TG221) characterized by microRNA-221 overexpression, with cirrhotic liver background induced by chronic administration of carbon tetrachloride (CCl4). This mouse model develops fibrosis, cirrhosis and liver tumours that become visible in 100% of mice at 5–6 months of age. Our results demonstrated that metformin intervention improves liver function, inhibits hepatic stellate cell (HSC) activation, reduces liver fibrosis, depletes lipid accumulation in hepatocytes, halts progression to decompensated cirrhosis and abrogates development HCC in CCl4 challenged transgenic mouse model. The study establishes the rationale for investigating metformin in cirrhotic patients regardless of concomitant DM2 status.

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References

  1. 1.

    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwidefor 36 cancers in 185 countries. CA: a cancer J Clin. 2018;68:394–424.

  2. 2.

    El-Serag HB. Hepatocellular carcinoma. New Engl J Med. 2011;365:1118–27.

  3. 3.

    Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al. Hepatocellular carcinoma. Nat Rev Dis Prim. 2016;2:16018.

  4. 4.

    Ioannou GN, Green PK, Berry K. HCV eradication induced by direct-acting antiviral agents reduces the risk of hepatocellular carcinoma. J. Hepatol. 2017;68:25–32.

  5. 5.

    Nordenstedt H, White DL, El-Serag HB. The changing pattern of epidemiology in hepatocellular carcinoma. Dig Liver Dis. 2010;42:S206–14.

  6. 6.

    Global Burden of Disease Liver Cancer C, Akinyemiju T, Abera S, Ahmed M, Alam N, Alemayohu MA, et al. The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: results from the global burden of disease study 2015. JAMA Oncol. 2017;3:1683–91.

  7. 7.

    Fukumura D, Incio J, Shankaraiah R, Jain RK. Obesity and cancer: an angiogenic and inflammatory link. Microcirculation. 2016;23:191–206.

  8. 8.

    Yoon K-H, Lee J-H, Kim J-W, Cho JH, Choi Y-H, Ko S-H, et al. Epidemic obesity and type 2 diabetes in Asia. Lancet. 2006;368:1681–8.

  9. 9.

    Bo S, Ciccone G, Rosato R, Villois P, Appendino G, Ghigo E, et al. Cancer mortality reduction and metformin: a retrospective cohort study in type 2 diabetic patients. Diabetes, Obes Metab. 2012;14:23–9.

  10. 10.

    Lee JH, Kim TI, Jeon SM, Hong SP, Cheon JH, Kim WH. The effects of metformin on the survival of colorectal cancer patients with diabetes mellitus. Int J Cancer. 2012;131:752–9.

  11. 11.

    Lee MS, Hsu CC, Wahlqvist ML, Tsai HN, Chang YH, Huang YC. Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese: a representative population prospective cohort study of 800,000 individuals. BMC Cancer. 2011;11:20.

  12. 12.

    Nkontchou G, Cosson E, Aout M, Mahmoudi A, Bourcier V, Charif I, et al. Impact of metformin on the prognosis of cirrhosis induced by viral hepatitis C in diabetic patients. J Clin Endocrinol Metab. 2011;96:2601–8.

  13. 13.

    Dasgupta B, Chhipa RR. Evolving lessons on the complex role of AMPK in normal physiology and cancer. Trends Pharmacol Sci. 2016;37:192–206.

  14. 14.

    Yang X, Liu Y, Li M, Wu H, Wang Y, You Y, et al. Predictive and preventive significance of AMPK activation on hepatocarcinogenesis in patients with liver cirrhosis. Cell Death Dis. 2018;9:264.

  15. 15.

    Incio J, Suboj P, Chin SM, Vardam-Kaur T, Liu H, Hato T, et al. Metformin reduces desmoplasia in pancreatic cancer by reprogramming stellate cells and tumor-associated macrophages. PLoS ONE. 2015;10:e0141392.

  16. 16.

    Qian W, Li J, Chen K, Jiang Z, Cheng L, Zhou C, et al. Metformin suppresses tumor angiogenesis and enhances the chemosensitivity of gemcitabine in a genetically engineered mouse model of pancreatic cancer. Life Sci. 2018;208:253–61.

  17. 17.

    DePeralta DK, Wei L, Ghoshal S, Schmidt B, Lauwers GY, Lanuti M, et al. Metformin prevents hepatocellular carcinoma development by suppressing hepatic progenitor cell activation in a rat model of cirrhosis. Cancer. 2016;122:1216–27.

  18. 18.

    Kim JH, Lee KJ, Seo Y, Kwon JH, Yoon JP, Kang JY, et al. Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism. Sci Rep. 2018;8:409.

  19. 19.

    Casadei Gardini A, Marisi G, Scarpi E, Scartozzi M, Faloppi L, Silvestris N, et al. Effects of metformin on clinical outcome in diabetic patients with advanced HCC receiving sorafenib. Expert Opin Pharmacother. 2015;16:2719–25.

  20. 20.

    Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008;22:659–61.

  21. 21.

    Kodack DP, Askoxylakis V, Ferraro GB, Sheng Q, Badeaux M, Goel S, et al. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation.Sci Transl Med. 2017;9:eaal4682.

  22. 22.

    Deutsch MJ, Schriever SC, Roscher AA, Ensenauer R. Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells. Anal Biochem. 2014;445:87–9.

  23. 23.

    Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73.

  24. 24.

    Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102:15545–50.

  25. 25.

    Liu W, Wang X. Prediction of functional microRNA targets by integrative modeling of microRNA binding and target expression data. Genome Biol. 2019;20:18.

  26. 26.

    Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods. 2009;41:1149–60.

  27. 27.

    Callegari E, Elamin BK, Giannone F, Milazzo M, Altavilla G, Fornari F, et al. Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model. Hepatology. 2012;56:1025–33.

  28. 28.

    European Association For The Study Of The L, European Organisation For R, Treatment Of C. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol. 2012;56:908–43.

  29. 29.

    Muir AJ. Understanding the complexities of cirrhosis. Clin Ther. 2015;37:1822–36.

  30. 30.

    Gluchowski NL, Becuwe M, Walther TC, Farese RV Jr. Lipid droplets and liver disease: from basic biology to clinical implications. Nat Rev Gastroenterol Hepatol. 2017;14:343–55.

  31. 31.

    Tandra S, Yeh MM, Brunt EM, Vuppalanchi R, Cummings OW, Unalp-Arida A, et al. Presence and significance of microvesicular steatosis in nonalcoholic fatty liver disease. J Hepatol. 2011;55:654–9.

  32. 32.

    Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and physiological activities. Exp Mol Med. 2016;48:e224.

  33. 33.

    Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev. 2011;25:1895–908.

  34. 34.

    Fullerton MD, Galic S, Marcinko K, Sikkema S, Pulinilkunnil T, Chen ZP, et al. Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin. Nat Med. 2013;19:1649–54.

  35. 35.

    Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2:489–501.

  36. 36.

    Wong FW, Chan WY, Lee SS. Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. Toxicol Appl Pharmacol. 1998;153:109–18.

  37. 37.

    Kao J-H, Chen D-S. Global control of hepatitis B virus infection. Lancet Infect Dis. 2002;2:395–403.

  38. 38.

    Casadei Gardini A, Faloppi L, De Matteis S, Foschi FG, Silvestris N, Tovoli F, et al. Metformin and insulin impact on clinical outcome in patients with advanced hepatocellular carcinoma receiving sorafenib: Validation study and biological rationale. Eur J Cancer. 2017;86:106–14.

  39. 39.

    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

  40. 40.

    Tennant DA, Duran RV, Boulahbel H, Gottlieb E. Metabolic transformation in cancer. Carcinogenesis. 2009;30:1269–80.

  41. 41.

    Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8.

  42. 42.

    Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol. 2017;14:397–411.

  43. 43.

    Chaube B, Bhat MK. AMPK, a key regulator of metabolic/energy homeostasis and mitochondrial biogenesis in cancer cells. Cell Death Dis. 2016;7:e2044.

  44. 44.

    Kluwe J, Wongsiriroj N, Troeger JS, Gwak GY, Dapito DH, Pradere JP, et al. Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis. Gut. 2011;60:1260–8.

  45. 45.

    Hollander MC, Maier CR, Hobbs EA, Ashmore AR, Linnoila RI, Dennis PA. Akt1 deletion prevents lung tumorigenesis by mutant K-ras. Oncogene. 2011;30:1812–21.

  46. 46.

    Linnerth-Petrik NM, Santry LA, Petrik JJ, Wootton SK. Opposing functions of Akt isoforms in lung tumor initiation and progression. PLoS ONE. 2014;9:e94595.

  47. 47.

    Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA. Metformin prevents tobacco carcinogen—induced lung tumorigenesis. Cancer Prev Res. 2010;3:1066–76.

  48. 48.

    Singh S, Singh PP, Singh AG, Murad MH, Sanchez W. Anti-diabetic medications and the risk of hepatocellular cancer: a systematic review and meta-analysis. Am J Gastroenterol. 2013;108:881–91. quiz 892.

  49. 49.

    Schulte L, Scheiner B, Voigtlander T, Koch S, Schweitzer N, Marhenke S, et al. Treatment with metformin is associated with a prolonged survival in patients with hepatocellular carcinoma. Liver Int. 2019;39:714–26.

  50. 50.

    Murff HJ, Roumie CL, Greevy RA, Hackstadt AJ, McÿGowan LED, Hung AM, et al. Metformin use and incidence cancer risk: evidence for a selective protective effect against liver cancer. Cancer Causes Control. 2018;29:823–32.

  51. 51.

    Kasmari AJ, Welch A, Liu G, Leslie D, McGarrity T, Riley T. Independent of cirrhosis, hepatocellular carcinoma risk is increased with diabetes and metabolicsyndrome. Am J Med. 2017;130:746 e741–7.

  52. 52.

    Chen HP, Shieh JJ, Chang CC, Chen TT, Lin JT, Wu MS, et al. Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies. Gut. 2013;62:606–15.

  53. 53.

    Hassan MM, Curley SA, Li D, Kaseb A, Davila M, Abdalla EK, et al. Association of diabetes duration and diabetes treatment with the risk of hepatocellular carcinoma. Cancer. 2010;116:1938–46.

  54. 54.

    Donadon V, Balbi M, Ghersetti M, Grazioli S, Perciaccante A, Valentina GD, et al. Antidiabetic therapy and increased risk of hepatocellular carcinoma in chronic liver disease. World J Gastroenterol. 2009;15:2506.

  55. 55.

    Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013;58:593–608.

  56. 56.

    Scaglione S, Kliethermes S, Cao G, Shoham D, Durazo R, Luke A, et al. The Epidemiology of cirrhosis in the United States: a population-based Study. J Clin Gastroenterol. 2015;49:690–6.

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Acknowledgements

This work was supported by the Italian Association for Cancer Research (AIRG IG-15615, AIRC IG-20055) to Massimo Negrini, PhD.

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Correspondence to Massimo Negrini.

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