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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  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.

    Google Scholar 

  2. 2.

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

    CAS  PubMed  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    Google Scholar 

  5. 5.

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

    PubMed  PubMed Central  Google Scholar 

  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.

    Google Scholar 

  7. 7.

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

    PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  20. 20.

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

    CAS  PubMed  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Google Scholar 

  29. 29.

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

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

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

    CAS  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  37. 37.

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

    PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  39. 39.

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

    CAS  Google Scholar 

  40. 40.

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

    CAS  PubMed  Google Scholar 

  41. 41.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

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

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  PubMed Central  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    PubMed  Google Scholar 

Download references


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

Author information



Corresponding author

Correspondence to Massimo Negrini.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shankaraiah, R.C., Callegari, E., Guerriero, P. et al. Metformin prevents liver tumourigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma. Oncogene 38, 7035–7045 (2019).

Download citation

Further reading


Quick links