MASLD-mimicking microenvironment drives an aggressive phenotype and represses IDH2 expression in hepatocellular carcinoma

Abstract

Aim: Hepatocellular carcinoma (HCC) in patients with Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD) is expected to be a significant public health issue in the near future. Therefore, understanding the tumor microenvironment interactions in MASLD-induced HCC is crucial, and the development of relevant preclinical models is needed. Hence, we aimed to determine the effects of a MASLD-mimicking microenvironment (ME) on the aggressiveness of HCC cells and identify target genes that drive HCC by developing a 3D-in vitro co-culture system.

Methods: A 3D co-culture system mimicking the MASLD-ME was created with LX-2 liver stellate cells embedded in 3D collagen gel in the lower and SNU-449 HCC cells on the upper parts of Boyden chambers, and cells were grown in an optimized metabolic medium (MM). The effects of MASLD-ME on motility, sphere formation, proliferation, and cell cycle of SNU-449 cells were tested by Boyden chamber, 3D sphere formation, XTT, and Flow cytometry, respectively. The protein expression/activation profiles of motile SNU-449 cells that passed the membrane toward MASLD-ME or control condition were investigated using a multiplex protein profiling system DigiWest and confirmed with RT-PCR, WB, and Flow cytometry. IDH2 levels were examined in primary human HCC and adjacent liver tissues by IHC and in TCGA and CPTAC cohorts by bioinformatics tools.

Results: MM treatment increased fat accumulation, motility, and spheroid formation of both SNU-449 and LX-2 cells. MASLD-ME induced activation of LX2 cells, leading to the formation of bigger colonies with many intrusions compared to related controls. DigiWest analysis showed that metabolism-related proteins such as IDH2 were the most affected molecules in SNU-449 cells that migrated toward the MASLD-ME compared to those that migrated toward the control condition. Downregulation of IDH2 expression was confirmed in SNU-449 cells grown in MASLD-ME, in primary HCC tumor samples by IHC, and in HCC patient cohorts by bioinformatics analysis.

Conclusion: This study reports the potential involvement of MASLD-ME in the downregulation of IDH2 expression and promoted motility and colonization capacity of HCC cells. The 3D MASLD model presented in this study may be useful in investigating the mechanistic roles of MASLD-ME in HCC.

Keywords

NAFLD / HCC / IDH2 / motility / aggressive phenotype / 3D-co-culture / MASLD

References

[1] Riazi K,Charette JH.The prevalence and incidence of NAFLD worldwide: a systematic review and meta-analysis.Lancet Gastroenterol Hepatol2022;7:851-61

[2] Teng ML,Huang DQ.Global incidence and prevalence of nonalcoholic fatty liver disease.Clin Mol Hepatol2023;29:S32-42 PMCID:PMC10029957

[3] Younossi ZM,Abdelatif D,Henry L.Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology2016;64:73-84

[4] Tan DJH,Ng CH.Global burden of liver cancer in males and females: Changing etiological basis and the growing contribution of NASH.Hepatology2023;77:1150-63

[5] Rinella ME,Ratziu V.NAFLD Nomenclature consensus groupA multisociety Delphi consensus statement on new fatty liver disease nomenclature.J Hepatol2023;78:1966-86 PMCID:PMC10653297

[6] Rinella ME,Siddiqui MS.AASLD practice guidance on the clinical assessment and management of nonalcoholic fatty liver disease.Hepatology2023;77:1797-835 PMCID:PMC10735173

[7] Chalasani N,Lavine JE.The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American association for the study of liver diseases.Hepatology2018;67:328-57

[8] European Association for the Study of the Liver (EASL)European Association for the Study of Diabetes (EASD)European Association for the Study of Obesity (EASO)EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease.J Hepatol2016;59:1121-40

[9] Bessone F,Roma MG.Molecular pathways of nonalcoholic fatty liver disease development and progression.Cell Mol Life Sci2019;76:99-128

[10] Day CP.Steatohepatitis: a tale of two "hits"?.Gastroenterology1998;114:842-5

[11] Buzzetti E,Tsochatzis EA.The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD).Metabolism2016;65:1038-48

[12] Musso G,De Michieli F.Dietary habits and their relations to insulin resistance and postprandial lipemia in nonalcoholic steatohepatitis.Hepatology2003;37:909-16

[13] Leoni S,Napoli L,Ferri S.Current guidelines for the management of non-alcoholic fatty liver disease: a systematic review with comparative analysis.World J Gastroenterol2018;24:3361-73 PMCID:PMC6092580

[14] Donnelly KL,Schwarzenberg SJ,Boldt MD.Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.J Clin Invest2005;115:1343-51 PMCID:PMC1087172

[15] Mota M,Cazanave SC.Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease.Metabolism2016;65:1049-61 PMCID:PMC4931958

[16] Kamm DR.Hepatic stellate cells in physiology and pathology.J Physiol2022;600:1825-37 PMCID:PMC9012702

[17] Brunt EM.Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond.Hepatology2000;31:241-6

[18] Piscaglia F,Barchetti A.HCC-NAFLD Italian study groupclinical patterns of hepatocellular carcinoma in nonalcoholic fatty liver disease: a multicenter prospective study.Hepatology2016;63:827-38

[19] Siddiqui RA,Neff K,Kovacs RJ.Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.Biochim Biophys Acta2001;1499:265-75

[20] Atabey N,Yao ZJ.Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.J Biol Chem2001;276:14308-14

[21] Bozkaya G,Cokaklı M.Cooperative interaction of MUC1 with the HGF/c-Met pathway during hepatocarcinogenesis.Mol Cancer2012;11:64 PMCID:PMC3542123

[22] UCSC in-silico PCR. Available from: https://genome.ucsc.edu/cgi-bin/hgPcr [Last accessed on 17 May 2024]

[23] Bağırsakçı E,Atabey N,Guerra V.Role of albumin in growth inhibition in hepatocellular carcinoma.Oncology2017;93:136-42

[24] Chandrashekar DS,Balasubramanya SAH.UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses.Neoplasia2017;19:649-58 PMCID:PMC5516091

[25] Chandrashekar DS,Korla PK.UALCAN: an update to the integrated cancer data analysis platform.Neoplasia2022;25:18-27 PMCID:PMC8788199

[26] Cerami E,Dogrusoz U.The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.Cancer Discov2012;2:401-4 PMCID:PMC3956037

[27] Gao J,Dogrusoz U.Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.Sci Signal2013;6:pl1 PMCID:PMC4160307

[28] de Bruijn I,Mastrogiacomo B.Analysis and visualization of longitudinal genomic and clinical data from the AACR project GENIE biopharma collaborative in cBioPortal.Cancer Res2023;83:3861-7 PMCID:PMC10690089

[29] TCGA-PanCanAtlas publications. Available from: https://gdc.cancer.gov/about-data/publications/pancanatlas [Last accessed on 17 May 2024]

[30] Kanehisa M.KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res2000;28:27-30 PMCID:PMC102409

[31] Clough E. The gene expression omnibus database. In: Mathé E, Davis S, editors. Statistical Genomics. New York: Springer; 2016. pp. 93-110.

[32] Sarkans U,Athar A.The biostudies database-one stop shop for all data supporting a life sciences study.Nucleic Acids Res2018;46:D1266-70 PMCID:PMC5753238

[33] Lake AD,Fisher CD.Analysis of global and absorption, distribution, metabolism, and elimination gene expression in the progressive stages of human nonalcoholic fatty liver disease.Drug Metab Dispos2011;39:1954-60 PMCID:PMC3186211

[34] Yang W,Zhou J.A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma.Sci Transl Med2021;13:eaaz6804

[35] Spector AA,Fletcher JE.Binding of long-chain fatty acids to bovine serum albumin.J Lipid Res1969;10:56-67.

[36] Jensen T,Sullivan S.Fructose and sugar: a major mediator of non-alcoholic fatty liver disease.J Hepatol2018;68:1063-75 PMCID:PMC5893377

[37] Treindl F,Beiter Y.A bead-based western for high-throughput cellular signal transduction analyses.Nat Commun2016;7:12852 PMCID:PMC5036152

[38] Korhan P,Atabey N.MiR-181a-5p is downregulated in hepatocellular carcinoma and suppresses motility, invasion and branching-morphogenesis by directly targeting c-Met.Biochem Biophys Res Commun2014;450:1304-12

[39] Korhan P,Kandemiş E.Reciprocal activating crosstalk between c-Met and caveolin 1 promotes invasive phenotype in hepatocellular carcinoma.PLoS One2014;9:e105278 PMCID:PMC4141763

[40] Kohrman AQ.Divide or conquer: cell cycle regulation of invasive behavior.Trends Cell Biol2017;27:12-25 PMCID:PMC5186408

[41] Yamaguchi K,McCall S.Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.Hepatology2007;45:1366-74

[42] Feldstein AE,Canbay A.Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway.Hepatology2004;40:185-94

[43] Cheng B,Wu L,Lin W.Activation of human hepatic stellate cells enhances the metastatic ability of hepatocellular carcinoma cells via up-regulation of interleukin-1β.J BUON2021;26:435-43

[44] Hong F,Lee TF.Hepatic stellate cells express functional CXCR4: role in stromal cell-derived factor-1alpha-mediated stellate cell activation.Hepatology2009;49:2055-67 PMCID:PMC2893547

[45] Cubero FJ,Zoubek ME,Hatting M.Disruption of the FasL/Fas axis protects against inflammation-derived tumorigenesis in chronic liver disease.Cell Death Dis2019;10:115 PMCID:PMC6368573

[46] Peterová E,Podmolíková L,Kanta J.The role of cytokines TGF-beta1 and FGF-1 in the expression of characteristic markers of rat liver myofibroblasts cultured in three-dimensional collagen gel.Physiol Res2016;65:661-72

[47] Bronsard J,Massart J.3D multi-cell-type liver organoids: a new model of non-alcoholic fatty liver disease for drug safety assessments.Toxicol In Vitro2024;94:105728

[48] Guo J,Yang Z,Yin D.Biological roles and therapeutic applications of IDH2 mutations in human cancer.Front Oncol2021;11:644857 PMCID:PMC8107474

[49] Liu WR,Jin L.High expression of 5-hydroxymethylcytosine and isocitrate dehydrogenase 2 is associated with favorable prognosis after curative resection of hepatocellular carcinoma.J Exp Clin Cancer Res2014;33:32 PMCID:PMC4081660

[50] Tian GY,Wang L,Shi JP.Isocitrate dehydrogenase 2 suppresses the invasion of hepatocellular carcinoma cells via matrix metalloproteinase 9.Cell Physiol Biochem2015;37:2405-14

[51] Pan JH,Beane KE.IDH2 deficiency aggravates fructose-induced NAFLD by modulating hepatic fatty acid metabolism and activating inflammatory signaling in female mice.Nutrients2018;10:679 PMCID:PMC6024877

[52] Lukow DA,Suri P.Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies.Dev Cell2021;56:2427-39.e4 PMCID:PMC8933054

[53] Tang M,O'Byrne KJ.Tumor hypoxia drives genomic instability.Front Cell Dev Biol2021;9:626229 PMCID:PMC8007910

[54] Zhang H,Dai J.Genetic variations in IDH gene as prognosis predictors in TACE-treated hepatocellular carcinoma patients.Med Oncol2014;31:278