Prevention and treatment options for hepatocellular carcinoma (HCC) are presently limited, underscoring the necessity for further elucidating molecular mechanisms underlying HCC development and identifying new prevention and therapeutic targets. Here, we demonstrate a unique protumorigenic niche in the livers of Ncoa5+/− mouse model of HCC, which is characterized by altered expression of a subset of genes including p21WAF1/CIP1 and proinflammatory cytokine genes, increased putative hepatic progenitors, and expansions of activated and tissue-resident memory (TRM) CD8+ T lymphocytes, myeloid-derived suppressor cells (MDSCs), and alternatively activated M2 macrophages. Importantly, prophylactic metformin treatment reversed these characteristics including aberrant p21WAF1/CIP1 expression and subsequently reduced HCC incidence in Ncoa5+/− male mice. Heterozygous deletion of the p21WAF1/CIP1 gene alleviated the key features associated with the protumorigenic niche in the livers of Ncoa5+/− male mice. Moreover, transcriptomic analysis reveals that preneoplastic livers of Ncoa5+/− mice are similar to the livers of nonalcoholic steatohepatitis patients as well as the adjacent noncancerous liver tissues of a subset of HCC patients with a relatively poor prognosis. Together, our results suggest that p21WAF1/CIP1 overexpression is essential in the development of protumorigenic microenvironment induced by NCOA5 deficiency and metformin prevents HCC development via alleviating p21WAF1/CIP1 overexpression and protumorigenic microenvironment.
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The RNA sequencing data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE110524. All other data that support the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request.
Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends–an update. Cancer Epidemiol Biomark Prev. 2016;25:16–27.
Gerbes A, Zoulim F, Tilg H, Dufour JF, Bruix J, Paradis V, et al. Gut roundtable meeting paper: selected recent advances in hepatocellular carcinoma. Gut. 2018;67:380–8.
Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al. Hepatocellular carcinoma. Nat Rev Dis Prim. 2016;2:16018.
Sia D, Jiao Y, Martinez-Quetglas I, Kuchuk O, Villacorta-Martin C, Castro de Moura M, et al. Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features. Gastroenterology. 2017;153:812–26.
Nakagawa H, Maeda S. Inflammation- and stress-related signaling pathways in hepatocarcinogenesis. World J Gastroenterol. 2012;18:4071–81.
Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science. 2007;317:121–4.
Park EJ, Lee JH, Yu GY, He G, Ali SR, Holzer RG, et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell. 2010;140:197–208.
Dhar D, Seki E, Karin M. NCOA5, IL-6, type 2 diabetes, and HCC: The deadly quartet. Cell Metab. 2014;19:6–7.
Gao S, Li A, Liu F, Chen F, Williams M, Zhang C, et al. NCOA5 haploinsufficiency results in glucose intolerance and subsequent hepatocellular carcinoma. Cancer Cell. 2013;24:725–37.
Bhalla K, Hwang BJ, Dewi RE, Twaddel W, Goloubeva OG, Wong KK, et al. Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis. Cancer Prev Res. 2012;5:544–52.
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.
Tajima K, Nakamura A, Shirakawa J, Togashi Y, Orime K, Sato K, et al. Metformin prevents liver tumorigenesis induced by high-fat diet in C57Bl/6 mice. Am J Physiol Endocrinol Metab. 2013;305:E987–998.
el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993;75:817–25.
He GB, Dhar D, Nakagawa H, Font-Burgada J, Ogata H, Jiang YH, et al. Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling. Cell. 2013;155:384–96.
Luo X, Liao R, Hanley KL, Zhu HH, Malo KN, Hernandez C, et al. Dual Shp2 and Pten deficiencies promote non-alcoholic steatohepatitis and genesis of liver tumor-initiating cells. Cell Rep. 2016;17:2979–93.
Finkin S, Yuan D, Stein I, Taniguchi K, Weber A, Unger K, et al. Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nat Immunol. 2015;16:1235–44.
Yu LX, Ling Y, Wang HY. Role of nonresolving inflammation in hepatocellular carcinoma development and progression. NPJ Precis Oncol. 2018;2:6.
Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27:670–84.
Zheng C, Zheng L, Yoo JK, Guo H, Zhang Y, Guo X, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell. 2017;169:1342–56 e1316.
Pauken KE, Sammons MA, Odorizzi PM, Manne S, Godec J, Khan O, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science. 2016;354:1160–5.
Lee SY, Lee SH, Yang EJ, Kim EK, Kim JK, Shin DY, et al. Metformin ameliorates inflammatory bowel disease by suppression of the STAT3 signaling pathway and regulation of the between Th17/Treg balance. PLoS ONE. 2015;10:e0135858.
Tan XL, Bhattacharyya KK, Dutta SK, Bamlet WR, Rabe KG, Wang E, et al. Metformin suppresses pancreatic tumor growth with inhibition of NFkappaB/STAT3 inflammatory signaling. Pancreas. 2015;44:636–47.
Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011;27:1739–40.
Roessler S, Jia HL, Budhu A, Forgues M, Ye QH, Lee JS, et al. A unique metastasis gene signature enables prediction of tumor relapse in early-stage hepatocellular carcinoma patients. Cancer Res. 2010;70:10202–12.
Villanueva A, Portela A, Sayols S, Battiston C, Hoshida Y, Mendez-Gonzalez J, et al. DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma. Hepatology. 2015;61:1945–56.
Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA. Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia. JAMA. 2010;304:2706–15.
Arendt BM, Comelli EM, Ma DW, Lou W, Teterina A, Kim T, et al. Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids. Hepatology. 2015;61:1565–78.
Warfel NA, El-Deiry WS. p21WAF1 and tumourigenesis: 20 years after. Curr Opin Oncol. 2013;25:52–58.
Gartel AL, Tyner AL. Transcriptional regulation of the p21((WAF1/CIP1)) gene. Exp Cell Res. 1999;246:280–9.
Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer. 2009;9:400–14.
Cmielova J, Rezacova M. p21Cip1/Waf1 protein and its function based on a subcellular localization [corrected]. J Cell Biochem. 2011;112:3502–6.
Nguyen P, Valanejad L, Cast A, Wright M, Garcia JM, El-Serag HB, et al. Elimination of age-associated hepatic steatosis and correction of aging phenotype by inhibition of cdk4-C/EBPalpha-p300 axis. Cell Rep. 2018;24:1597–609.
Tomita K, Teratani T, Suzuki T, Oshikawa T, Yokoyama H, Shimamura K, et al. p53/p66Shc-mediated signaling contributes to the progression of non-alcoholic steatohepatitis in humans and mice. J Hepatol. 2012;57:837–43.
Wu H, Wade M, Krall L, Grisham J, Xiong Y, Van Dyke T. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev. 1996;10:245–60.
Yano M, Ohkoshi S, Aoki YH, Takahashi H, Kurita S, Yamazaki K, et al. Hepatitis B virus X induces cell proliferation in the hepatocarcinogenesis via up-regulation of cytoplasmic p21 expression. Liver Int. 2013;33:1218–29.
Marhenke S, Buitrago-Molina LE, Endig J, Orlik J, Schweitzer N, Klett S, et al. p21 promotes sustained liver regeneration and hepatocarcinogenesis in chronic cholestatic liver injury. Gut. 2014;63:1501–12.
Ehedego H, Boekschoten MV, Hu W, Doler C, Haybaeck J, Gabetaler N, et al. p21 ablation in liver enhances DNA damage, cholestasis, and carcinogenesis. Cancer Res. 2015;75:1144–55.
Nicolae CM, O’Connor MJ, Constantin D, Moldovan GL. NFkappaB regulates p21 expression and controls DNA damage-induced leukemic differentiation. Oncogene. 2018;37:3647–56.
Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA, et al. Diabetes and cancer: a consensus report. Diabetes Care. 2010;33:1674–85.
Donadon V, Balbi M, Mas MD, Casarin P, Zanette G. Metformin and reduced risk of hepatocellular carcinoma in diabetic patients with chronic liver disease. Liver Int. 2010;30:750–8.
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.
Kalender A, Selvaraj A, Kim SY, Gulati P, Brule S, Viollet B, et al. Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab. 2010;11:390–401.
Algire C, Moiseeva O, Deschenes-Simard X, Amrein L, Petruccelli L, Birman E, et al. Metformin reduces endogenous reactive oxygen species and associated DNA damage. Cancer Prev Res. 2012;5:536–43.
Hirsch HA, Iliopoulos D, Struhl K. Metformin inhibits the inflammatory response associated with cellular transformation and cancer stem cell growth. Proc Natl Acad Sci USA. 2013;110:972–7.
Fujita K, Iwama H, Miyoshi H, Tani J, Oura K, Tadokoro T, et al. Diabetes mellitus and metformin in hepatocellular carcinoma. World J Gastroenterol. 2016;22:6100–13.
Molnar Z, Millward AB, Tse W, Demaine AG. p21(WAF1/CIP1) expression is differentially regulated by metformin and rapamycin. Int J Chronic Dis. 2014;2014:327640.
Noren Hooten N, Martin-Montalvo A, Dluzen DF, Zhang Y, Bernier M, Zonderman AB, et al. Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence. Aging Cell. 2016;15:572–81.
Rohr-Udilova N, Klinglmuller F, Schulte-Hermann R, Stift J, Herac M, Salzmann M, et al. Deviations of the immune cell landscape between healthy liver and hepatocellular carcinoma. Sci Rep. 2018;8:6220.
Eikawa S, Nishida M, Mizukami S, Yamazaki C, Nakayama E, Udono H. Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Proc Natl Acad Sci USA. 2015;112:1809–14.
Shalapour S, Lin XJ, Bastian IN, Brain J, Burt AD, Aksenov AA, et al. Erratum: Inflammation-induced IgA(+) cells dismantle anti-liver cancer immunity. Nature. 2017;551:340–5.
Verdura S, Cuyas E, Martin-Castillo B, Menendez JA. Metformin as an archetype immuno-metabolic adjuvant for cancer immunotherapy. Oncoimmunology. 2019;8:e1633235.
Liu X, Liu F, Gao S, Reske J, Li A, Wu C-L, et al. A single non-synonymous NCOA5 variation in type 2 diabetic patients with hepatocellular carcinoma impairs the function of NCOA5 in cell cycle regulation. Cancer Lett. 2017;391:152–61.
This work was supported by the grants (R01 CA188305 and R21 CA185021) of National Cancer Institute to HX and a NIH-NCI K22 award (5K22CA18814802) to RD. MW was supported by a NIH F32 student training fellowship (5 T32 GM 92715-3). XL was partially supported by a fellowship from Integrated Hospital of Traditional Chinese Medicine, Southern Medical University. The authors thank J. Rennhack and E. Andrechek for sharing expertise in RNA-seq data analysis; E. Andrechek for manuscript comments; and members of the HX lab for helpful discussions. The results reported here are partially based on data generated by the TCGA Research Network: http://cancergenome.nih.gov, which we also gratefully acknowledge.
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Williams, M., Liu, X., Zhang, Y. et al. NCOA5 deficiency promotes a unique liver protumorigenic microenvironment through p21WAF1/CIP1 overexpression, which is reversed by metformin. Oncogene 39, 3821–3836 (2020). https://doi.org/10.1038/s41388-020-1256-x