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The balance of protein farnesylation and geranylgeranylation during the progression of nonalcoholic fatty liver disease

  • Yue Zhao
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China

    MOE Key Laboratory of Model Animal for Disease Study, Model Animals Research Center, Nanjing University, Nanjing 210093, China
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  • Tian-Yu Wu
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China
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  • Meng-Fei Zhao
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China
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  • Chao-Jun Li
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China

    MOE Key Laboratory of Model Animal for Disease Study, Model Animals Research Center, Nanjing University, Nanjing 210093, China
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Open AccessPublished:March 05, 2020DOI:https://doi.org/10.1074/jbc.REV119.008897
      Protein prenylation is an essential posttranslational modification and includes protein farnesylation and geranylgeranylation using farnesyl diphosphate or geranylgeranyl diphosphate as substrates, respectively. Geranylgeranyl diphosphate synthase is a branch point enzyme in the mevalonate pathway that affects the ratio of farnesyl diphosphate to geranylgeranyl diphosphate. Abnormal geranylgeranyl diphosphate synthase expression and activity can therefore disrupt the balance of farnesylation and geranylgeranylation and alter the ratio between farnesylated and geranylgeranylated proteins. This change is associated with the progression of nonalcoholic fatty liver disease (NAFLD), a condition characterized by hepatic fat overload. Of note, differential accumulation of farnesylated and geranylgeranylated proteins has been associated with differential stages of NAFLD and NAFLD-associated liver fibrosis. In this review, we summarize key aspects of protein prenylation as well as advances that have uncovered the regulation of associated metabolic patterns and signaling pathways, such as Ras GTPase signaling, involved in NAFLD progression. Additionally, we discuss unique opportunities for targeting prenylation in NAFLD/hepatocellular carcinoma with agents such as statins and bisphosphonates to improve clinical outcomes.

      Introduction

      Prenylation is a type of lipid modification wherein a farnesyl (15-carbon) or a geranylgeranyl (20-carbon) side chain is added to a C-terminal cysteine residue of a CaaX or CaaX-like motif, dependent on the characteristics of X (where C is cysteine, a is any aliphatic amino acid, and X is another amino acid); these modifications are called farnesylation and geranylgeranylation, respectively (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ). Given the hydrophobicity of the lipids involved, prenylated proteins are anchored to cellular membranes in proximity to downstream signaling pathways involved in numerous cellular processes, including cell proliferation and differentiation, cell metabolism, and intracellular protein trafficking (
      • Wang M.
      • Casey P.J.
      Protein prenylation: unique fats make their mark on biology.
      ). Geranylgeranyl diphosphate synthase (GGPPS)
      The abbreviations used are: GGPPS
      geranylgeranyl diphosphate synthase
      NAFLD
      non alcoholic fatty liver disease
      NASH
      non alcoholic steatohepatitis
      HCC
      hepatocellular carcinoma
      FPPS
      farnesyl diphosphate synthase
      GGPP
      geranylgeranyl diphosphate
      FPP
      farnesyl diphosphate
      MVA
      mevalonate
      FTase
      farnesyltransferase
      GGTase
      geranylgeranyltransferase
      FTI
      farnesyltransferase inhibitor
      DNL
      de novo lipogenesis
      GGTI
      geranylgeranyltransferase inhibitor
      HMGCR
      HMG-CoA reductase
      DGBP
      digeranyl bisphosphate
      IRS
      insulin receptor substrate
      SREBP
      sterol-regulatory element–binding protein
      NBP
      nitrogenous bisphosphate
      QC
      quality control
      NAFL
      nonalcoholic fatty liver
      ERK
      extracellular signal–regulated kinase
      PI3K
      phosphatidylinositol 3-kinase
      AMPK
      AMP-activated protein kinase
      LKB1
      liver kinase B1
      MAPK
      mitogen-activated protein kinase
      HFD
      high-fat diet
      YAP
      Yes-associated protein
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.
      is the branch point enzyme in the mevalonate (MVA) pathway that is responsible for synthesizing GGPP from its substrate FPP, and abnormal expression of this enzyme affects the ratio of FPP to GGPP, disrupting the balance of protein farnesylation and geranylgeranylation (
      • Wang X.X.
      • Ying P.
      • Diao F.
      • Wang Q.
      • Ye D.
      • Jiang C.
      • Shen N.
      • Xu N.
      • Chen W.B.
      • Lai S.S.
      • Jiang S.
      • Miao X.L.
      • Feng J.
      • Tao W.W.
      • Zhao N.W.
      • et al.
      Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection.
      ,
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ,
      • Chen Z.
      • Xu N.
      • Chong D.
      • Guan S.
      • Jiang C.
      • Yang Z.
      • Li C.
      Geranylgeranyl pyrophosphate synthase facilitates the organization of cardiomyocytes during mid-gestation through modulating protein geranylgeranylation in mouse heart.
      ).
      The existence of imbalances in this system has a high correlation with the development of many diseases, including nonalcoholic fatty liver disease (NAFLD) and NAFLD-associated fibrosis. NAFLD refers to a clinical condition characterized by hepatic fat overload without alcoholism (
      • Masuoka H.C.
      • Chalasani N.
      Nonalcoholic fatty liver disease: an emerging threat to obese and diabetic individuals.
      ). It is strongly associated with obesity, diabetes, and insulin resistance and is considered a metabolic syndrome (
      • Marengo A.
      • Rosso C.
      • Bugianesi E.
      Liver cancer: connections with obesity, fatty liver, and cirrhosis.
      ). NAFLD is classified into nonalcoholic fatty liver (NAFL, simple steatosis) and nonalcoholic steatohepatitis (NASH) (
      • Takahashi Y.
      • Soejima Y.
      • Fukusato T.
      Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.
      ). The simple steatosis in NAFL represents a state of imbalance where triglyceride deposition overwhelms its consumption. Prolonged lipid accumulation and inflammation can progress to NASH, advanced liver fibrosis, cirrhosis, and, ultimately, hepatocellular carcinoma (HCC).
      Although the pathogenesis of NAFLD has been investigated through extensive research and clinical studies, the molecular mechanism involved in the progression from NAFLD to HCC remains to be elucidated. Several central molecules/pathways related to the MVA pathway, including Ras-ERK1/2, PI3K-Akt, sterol regulatory element–binding protein 1 (SREBP), Rac, and AMPK, are activated during the progression of NAFLD to HCC. These changes give the cell features of proliferation, genomic instability, and immortalization, eventually promoting progression to HCC (Fig. 1).
      Figure thumbnail gr1
      Figure 1Several signaling pathways affected by metabolites in the MVA pathway involved in the progression from NAFLD to HCC. The progression of NAFLD to HCC is classified into four phases: normal liver, NAFL (simple steatosis), NASH, and HCC. When NAFLD develops, insulin resistance occurs as PI3K-Akt is activated in the liver. Simultaneously, LXR-α and AMPK, sensors of metabolic state dysfunction, promote DNL and glucose uptake. Activation of Rac1 and Ras-FasL is involved in the development of NASH by promoting cirrhosis and apoptosis. Then the Ras-ERK1/2 axis mediates proliferation, leading to the onset of HCC. All of the above pathways are regulated by metabolites in the MVA pathway, and corresponding targeted therapies have been developed.
      Interestingly, the accumulation of differential amounts of farnesylated and geranylgeranylated proteins regulated by GGPPS has been associated with differential stages of NAFLD and NAFLD-associated fibrosis (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ,
      • Zhao Y.
      • Zhao M.F.
      • Jiang S.
      • Wu J.
      • Liu J.
      • Yuan X.W.
      • Shen D.
      • Zhang J.Z.
      • Zhou N.
      • He J.
      • Fang L.
      • Sun X.T.
      • Xue B.
      • Li C.J.
      Liver governs adipose remodelling via extracellular vesicles in response to lipid overload.
      ). Statins, a class of compounds widely used to lower cholesterol, are inhibitors of HMG-CoA reductase (HMGCR, the upstream enzyme in the MVA pathway) and consequently alter the ratio of FPP/GGPP followed by the balance of protein prenylation (
      • Wang M.
      • Casey P.J.
      Protein prenylation: unique fats make their mark on biology.
      ). Considering the effects of several inhibitors targeting MVA pathway enzymes on immune control (
      • Clark E.A.
      • Golub T.R.
      • Lander E.S.
      • Hynes R.O.
      Genomic analysis of metastasis reveals an essential role for RhoC.
      ), metabolic disease (
      • Miersch S.
      • Sliskovic I.
      • Raturi A.
      • Mutus B.
      Antioxidant and antiplatelet effects of rosuvastatin in a hamster model of prediabetes.
      ), and cancer progression (
      • Swanson K.M.
      • Hohl R.J.
      Anti-cancer therapy: targeting the mevalonate pathway.
      ), protein prenylation can also affect the progression of NAFLD through processes such as metabolic reprogramming and signaling pathway activation. More importantly, identifying a drug targeting the prenylation balance can provide insights for prospective therapeutic strategies for NAFLD and HCC.

      Protein prenylation

      Anchorage to cellular membranes is a prerequisite for the biological function of many regulatory proteins, which can be located on the membrane surface or embedded in the lipid bilayer. Many peripheral proteins are targeted to membranes as a result of posttranslational modification with lipid moieties. Two types of isoprenoid lipids, FPP and GGPP, which are intermediates in the MVA pathway for cholesterol, terpene and terpenoid synthesis, are utilized for such modification (Fig. 2, left). Proteins with cysteine residues typically found in the CaaX motif can be farnesylated with FPP or geranylgeranylated with GGPP. Either of these biochemical reactions depends upon the nature of the X residue. If X is serine, methionine, alanine, or glutamine, the protein is farnesylated; if X refers to leucine or isoleucine, the protein is geranylgeranylated (
      • Perez-Sala D.
      Protein isoprenylation in biology and disease: general overview and perspectives from studies with genetically engineered animals.
      ).
      Figure thumbnail gr2
      Figure 2Two functions of FPP and GGPP. Left, FPP and GGPP can be covalently attached to proteins, especially GTPases, by the isoprenyltransferases FTase and GGTase in processes called farnesylation and geranylgeranylation, respectively. Right, FPP and GGPP can directly interact with other proteins as ligands via noncovalent binding to regulate their activities. For example, GGPP can interact with Skp2 and PPARγ, whereas FPP interacts with FXR.
      Protein prenylation depends on the activity of prenyltransferases. There are three prenyltransferases, all of which are heterodimeric enzymes containing α and β subunits. Farnesyltransferase (FTase) and geranylgeranyltransferase 1 (GGTase1) share the same α subunit but contain different β subunits. Both transferases recognize substrates with a CaaX sequence, the site where lipid modification occurs. Another prenyltransferase, GGTase2, is formed by RabGGTA (the α subunit) and RabGGTB (the β subunit). GGTase2 prenylates sites in additional C-terminal motifs, including CCXXX, CCXX, XCCX, XXCC, and XCXC. Unlike FTase and GGTase1, the prenylation by GGTase2 requires the participation of the Rab escort protein, an accessory protein involved in the recognition of Rab by GGTase2 (
      • Perez-Sala D.
      Protein isoprenylation in biology and disease: general overview and perspectives from studies with genetically engineered animals.
      ). Distinct substrates have been identified for FTase (H-Ras, K-Ras, N-Ras, Ras2, Rap2, pre-Lamin A, Lamin B, RhoB, RhoE, and Rheb), GGTase1 (RhoA, RhoB, RhoC, Rab8, Rab11, Rab13, Rac1, Rac2, RalA, Rap1B, and Cdc42), and GGTase2 (Rab GTPases) (
      • James G.L.
      • Goldstein J.L.
      • Brown M.S.
      Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro.
      ,
      • Baron R.
      • Fourcade E.
      • Lajoie-Mazenc I.
      • Allal C.
      • Couderc B.
      • Barbaras R.
      • Favre G.
      • Faye J.C.
      • Pradines A.
      RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.
      ,
      • Carboni J.M.
      • Yan N.
      • Cox A.D.
      • Bustelo X.
      • Graham S.M.
      • Lynch M.J.
      • Weinmann R.
      • Seizinger B.R.
      • Der C.J.
      • Barbacid M.
      Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.
      ,
      • Rowell C.A.
      • Kowalczyk J.J.
      • Lewis M.D.
      • Garcia A.M.
      Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo.
      ,
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • Bishop W.R.
      • Pai J.K.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ). In addition, recently, a new prenyltransferase, GGTase3, which consists of the α subunit PTAR1 and the β subunit of GGTase2 (RabGGTB), was identified. This enzyme geranylgeranylates FBXL2, a ubiquitin ligase, allowing it to associate with cell membranes (
      • Kuchay S.
      • Wang H.
      • Marzio A.
      • Jain K.
      • Homer H.
      • Fehrenbacher N.
      • Philips M.R.
      • Zheng N.
      • Pagano M.
      GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase.
      ) (Fig. 3). After recognition, the aaX residues at the C terminus can be further removed by Ras-converting CaaX endopeptidase 1 (RCE1), and isoprenylcysteine carboxylmethyltransferase adds a methyl group to the isoprenoid-modified cysteine residue (
      • Perez-Sala D.
      Protein isoprenylation in biology and disease: general overview and perspectives from studies with genetically engineered animals.
      ).
      Figure thumbnail gr3
      Figure 3Prenyltransferases and their substrates. Shown are the four classes of human prenyltransferases with α (PTAR1, FNTA (PTAR2), and RabGGTA (PTAR3)) and β (FNTB, PGGT1B, and RabGGTB) subunits and their substrates, recognition sequences, and accessory proteins. N/A, not applicable.
      A large number of prenylated peptides in living cells without metabolic perturbation have been reported in a newly developed proteome-scale analysis (
      • Storck E.M.
      • Morales-Sanfrutos J.
      • Serwa R.A.
      • Panyain N.
      • Lanyon-Hogg T.
      • Tolmachova T.
      • Ventimiglia L.N.
      • Martin-Serrano J.
      • Seabra M.C.
      • Wojciak-Stothard B.
      • Tate E.W.
      Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics.
      ). Hundreds of prenylated candidates have been identified by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics, such as Ganab, K-Ras, N-Ras, Nos2, Nos3, Rab, Rac, and Rheb. Among these candidates, Ganab, Nos2, and Nos3 are involved in metabolic pathways, and K-Ras and N-Ras participate in thermogenesis, the insulin signaling pathway, and choline metabolism in cancer. In addition, Ras, Rheb, Rab, Rac, and liver kinase B1 (LKB1) are involved in the MAPK, PI3K/Akt, AMPK, and other signaling pathways, which are also engaged in metabolic regulation. These metabolism-related candidates give a hint that protein prenylation may influence metabolic state.

      The balance of protein farnesylation and geranylgeranylation and its effect on altered metabolic states

      The balance of protein farnesylation and geranylgeranylation is highly related to the activation state of GGPPS, the branch point enzyme in the MVA pathway. When GGPPS is activated, the GGPP/FPP ratio increases; consequently, protein geranylgeranylation is enhanced or vice versa, because the expression level and activity of FTase and GGTase normally do not change significantly. However, some proteins can alternatively undergo either type of prenylation under extreme conditions; for example, when either GGPP or FPP is unavailable, or X is phenylalanine, the proteins can be either farnesylated or geranylgeranylated (
      • Carboni J.M.
      • Yan N.
      • Cox A.D.
      • Bustelo X.
      • Graham S.M.
      • Lynch M.J.
      • Weinmann R.
      • Seizinger B.R.
      • Der C.J.
      • Barbacid M.
      Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.
      ). In addition, both geranylgeranylation and farnesylation of H-Ras can occur when GGPPS is knocked out (
      • Wang X.X.
      • Ying P.
      • Diao F.
      • Wang Q.
      • Ye D.
      • Jiang C.
      • Shen N.
      • Xu N.
      • Chen W.B.
      • Lai S.S.
      • Jiang S.
      • Miao X.L.
      • Feng J.
      • Tao W.W.
      • Zhao N.W.
      • et al.
      Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection.
      ), and K-Ras can be geranylgeranylated in cells treated with farnesyltransferase inhibitors (FTIs) (
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • Bishop W.R.
      • Pai J.K.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ). Furthermore, several studies about these alternative prenylation patterns have been published (
      • James G.L.
      • Goldstein J.L.
      • Brown M.S.
      Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro.
      ,
      • Baron R.
      • Fourcade E.
      • Lajoie-Mazenc I.
      • Allal C.
      • Couderc B.
      • Barbaras R.
      • Favre G.
      • Faye J.C.
      • Pradines A.
      RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.
      ,
      • Carboni J.M.
      • Yan N.
      • Cox A.D.
      • Bustelo X.
      • Graham S.M.
      • Lynch M.J.
      • Weinmann R.
      • Seizinger B.R.
      • Der C.J.
      • Barbacid M.
      Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.
      ,
      • Rowell C.A.
      • Kowalczyk J.J.
      • Lewis M.D.
      • Garcia A.M.
      Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo.
      ,
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • Bishop W.R.
      • Pai J.K.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ), although the molecular mechanisms remain unclear.
      Another possible effect by alteration of FPP/GPP ratio is the direct change of metabolic pathways in the cell. FPP is a key intermediate in cholesterol metabolism (
      • Wang M.
      • Casey P.J.
      Protein prenylation: unique fats make their mark on biology.
      ), whereas GGPP is crucial in the metabolism of dolichols (
      • Sakaihara T.
      • Honda A.
      • Tateyama S.
      • Sagami H.
      Subcellular fractionation of polyprenyl diphosphate synthase activities responsible for the syntheses of polyprenols and dolichols in spinach leaves.
      ), which are essential for protein glycosylation (
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ,
      • Elharar Y.
      • Podilapu A.R.
      • Guan Z.
      • Kulkarni S.S.
      • Eichler J.
      Assembling glycan-charged dolichol phosphates: chemoenzymatic synthesis of a Haloferax volcanii N-glycosylation pathway intermediate.
      ). Considering the different Km of the enzymes for FPP or GGPP destination, the FPP/GGPP ratio change might also influence the synthesis of cholesterol, heme A, dolichol, etc. by FPP and/or ubiquinone, etc. by GGPP. Therefore, FPP and GGPP, as metabolites, may regulate cell functions with a different mechanism to maintain the metabolic homeostasis apart from protein prenylation.
      GGPPS is highly abundant in the liver, adipose tissue, and muscle of mice with obesity and insulin resistance but is expressed at a relatively low level under normal conditions (
      • Vicent D.
      • Maratos-Flier E.
      • Kahn C.R.
      The branch point enzyme of the mevalonate pathway for protein prenylation is overexpressed in the ob/ob mouse and induced by adipogenesis.
      ). Previous studies have shown that insulin can induce Rab geranylgeranylation by activating GGTase enzymes in 3T3-L1 preadipocytes and that abrogation of GGTase activity inhibits the phosphorylation of MAPK pathway components in 3T3-L1 preadipocytes (
      • Solomon C.S.
      • Leitner J.W.
      • Goalstone M.L.
      Dominant negative α-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes.
      ). GGPP stimulates PPARγ expression and adipogenesis (
      • Weivoda M.M.
      • Hohl R.J.
      Geranylgeranyl pyrophosphate stimulates PPARγ expression and adipogenesis through the inhibition of osteoblast differentiation.
      ). Inhibition of GGPPS by digeranyl bisphosphonate (DGBP) leads to reduced GGPP levels but accumulation of FPP, impairing protein geranylgeranylation in MC3T3-E1 preosteoblast cells (
      • Weivoda M.M.
      • Hohl R.J.
      The effects of direct inhibition of geranylgeranyl pyrophosphate synthase on osteoblast differentiation.
      ). Our studies have also revealed that the MAPK/Egr-1/GGPPS/Ras axis plays an essential role in certain metabolic states, such as type 2 diabetes. Long-term insulin stimulation activates GGPPS and further activates K-Ras by enhancing its geranylgeranylation. Ras/MAPK/Erk1/2 signaling results in insulin receptor substrate-1 (IRS-1) phosphorylation, contributing to insulin resistance (
      • Shen N.
      • Yu X.
      • Pan F.Y.
      • Gao X.
      • Xue B.
      • Li C.J.
      An early response transcription factor, Egr-1, enhances insulin resistance in type 2 diabetes with chronic hyperinsulinism.
      ). Furthermore, we observed increased GGPPS expression in the skeletal muscles of mice with insulin resistance, and specific knockout of GGPPS in skeletal muscle improved systemic insulin sensitivity and glucose homeostasis by enhancing glucose uptake in skeletal muscle. These metabolic alterations mediated by ggpps knockout were achieved through decreased geranylgeranylation of RhoA, which further induced the phosphorylation of IRS-1 (
      • Chen M.C.
      • Tsai Y.C.
      • Tseng J.H.
      • Liou J.J.
      • Horng S.
      • Wen H.C.
      • Fan Y.C.
      • Zhong W.B.
      • Hsu S.P.
      Simvastatin inhibits cell proliferation and migration in human anaplastic thyroid cancer.
      ). Thus, the GGPPS/RhoA/Rho kinase/IRS-1 pathway mediates lipid-induced systemic insulin resistance in obese mice (
      • Tao W.
      • Wu J.
      • Xie B.X.
      • Zhao Y.Y.
      • Shen N.
      • Jiang S.
      • Wang X.X.
      • Xu N.
      • Jiang C.
      • Chen S.
      • Gao X.
      • Xue B.
      • Li C.J.
      Lipid-induced muscle insulin resistance is mediated by GGPPS via modulation of the RhoA/Rho kinase signaling pathway.
      ). In summary, GGPPS is crucial for the balance of protein farnesylation and geranylgeranylation in the regulation of metabolic states, which may influence other metabolic diseases, such as NAFLD/HCC.

      Metabolic states mediated by the prenylation balance in NAFLD/HCC progression

      Proliferative cancer cells often exploit nutrients, such as glucose, to support their energy demand and biomass synthesis. They tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), referred to as the Warburg effect (
      • Harrison S.A.
      • Torgerson S.
      • Hayashi P.H.
      The natural history of nonalcoholic fatty liver disease: a clinical histopathological study.
      ,
      • Cohen J.C.
      • Horton J.D.
      • Hobbs H.H.
      Human fatty liver disease: old questions and new insights.
      ). Although compared with fatty acid β-oxidation, glycolysis is an ineffective way to generate energy from glucose, hepatocytes rely more on glycolysis than on β-oxidation for energy during NAFLD/HCC progression (
      • Cohen J.C.
      • Horton J.D.
      • Hobbs H.H.
      Human fatty liver disease: old questions and new insights.
      ). This alteration in metabolic control may eventually alter existing cell metabolism in a way that supports cell growth (
      • Vander Heiden M.G.
      • Cantley L.C.
      • Thompson C.B.
      Understanding the Warburg effect: the metabolic requirements of cell proliferation.
      ).
      Our previous work revealed that GGPPS was highly expressed in the livers of NAFLD patients but down-regulated in HCC patients (
      • Yu D.C.
      • Liu J.
      • Chen J.
      • Shao J.J.
      • Shen X.
      • Xia H.G.
      • Li C.J.
      • Xue B.
      • Ding Y.T.
      GGPPS1 predicts the biological character of hepatocellular carcinoma in patients with cirrhosis.
      ). Additionally, GGPPS was first up-regulated in the livers of mice with high-fat diet (HFD)-induced NAFLD and was then down-regulated after long-term HFD overload in NAFLD, which was associated with fibrosis, suggesting that the GGPPS-dependent protein prenylation balance mediates metabolic alterations during the development of NAFLD-associated fibrosis (Fig. 4). The balance of prenylation regulated by the two-phase change in GGPPS expression is associated with differential stages of NAFLD progression to HCC by influencing the activity of metabolic enzymes and signal transduction through the related signaling pathways.
      Figure thumbnail gr4
      Figure 4GGPPS regulates NAFLD-HCC progression by determining the hepatic glucose/fatty acid preference under fat overload. Short-term HFD overload increases GGPPS expression and triggers a change in the prenylation pattern favoring GGPP synthesis (
      • Zhao Y.
      • Zhao M.F.
      • Jiang S.
      • Wu J.
      • Liu J.
      • Yuan X.W.
      • Shen D.
      • Zhang J.Z.
      • Zhou N.
      • He J.
      • Fang L.
      • Sun X.T.
      • Xue B.
      • Li C.J.
      Liver governs adipose remodelling via extracellular vesicles in response to lipid overload.
      ), leading to increased glycolysis and DNL. However, long-term HFD overload decreases GGPPS expression and disrupts the FPP and GGPP balance so as to unbalance the ratio of farnesylated proteins and geranylgeranylated proteins, which drives the Warburg effect through metabolic reprogramming favoring glycolysis to exacerbate fibrosis (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ).
      During NAFLD progression, an HFD enhances hepatic lipid oxidation and glycolysis. Additionally, an HFD accelerates lipid accumulation by up-regulating the expression of GGPPS (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ,
      • Zhao Y.
      • Zhao M.F.
      • Jiang S.
      • Wu J.
      • Liu J.
      • Yuan X.W.
      • Shen D.
      • Zhang J.Z.
      • Zhou N.
      • He J.
      • Fang L.
      • Sun X.T.
      • Xue B.
      • Li C.J.
      Liver governs adipose remodelling via extracellular vesicles in response to lipid overload.
      ), which alters the relative ratio of FPP to GGPP, thereby influencing SREBP activation. However, GGPPS is down-regulated during the progression of NAFLD to HCC, which drives NAFLD-associated fibrosis by promoting glycolysis and suppressing oxidative phosphorylation via the LKB1/AMPK axis (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ). Hence, these factors promote the Warburg effect to support glycolysis and result in metabolic reprogramming during NAFLD progression, thus driving HCC progression.

      FPP/GGPP and SREBP activation

      SREBP is a critical regulator for maintaining lipid homeostasis (
      • Gong Y.
      • Lee J.N.
      • Lee P.C.
      • Goldstein J.L.
      • Brown M.S.
      • Ye J.
      Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake.
      ). SREBP precursors form a complex with SREBP cleavage–activating protein, which is retained by the insulin-induced gene-1/2 proteins in the endoplasmic reticulum in the presence of increased cellular sterol levels. Downstream effectors of SREBP are primarily encoded by genes involved in regulating lipid metabolism, particularly those associated with de novo lipogenesis (DNL), including FAS (
      • Wang S.
      • Moustaid-Moussa N.
      • Chen L.
      • Mo H.
      • Shastri A.
      • Su R.
      • Bapat P.
      • Kwun I.
      • Shen C.L.
      Novel insights of dietary polyphenols and obesity.
      ), ACC1 (
      • Wang S.
      • Moustaid-Moussa N.
      • Chen L.
      • Mo H.
      • Shastri A.
      • Su R.
      • Bapat P.
      • Kwun I.
      • Shen C.L.
      Novel insights of dietary polyphenols and obesity.
      ), and SCD1 (
      • Yao D.
      • Luo J.
      • He Q.
      • Shi H.
      • Li J.
      • Wang H.
      • Xu H.
      • Chen Z.
      • Yi Y.
      • Loor J.J.
      SCD1 alters long-chain fatty acid (LCFA) composition and its expression is directly regulated by SREBP-1 and PPARγ 1 in dairy goat mammary cells.
      ). Moreover, these genes can mediate the inflammatory reaction in NAFLD, which is essential for HCC progression. Hence, SREBP down-regulation can reduce the inflammatory reaction and prevent the progression from NAFLD to NASH, cirrhosis, and even HCC. A recent study revealed that geranylgeranylated RhoA-dependent actomyosin contraction inhibits SREBP1 activation. FPP accumulation resulting from GGPPS deficiency inhibits DNL in hepatocytes, suppressing SREBP-1 expression and LXR activation by activating FXR/SHP signaling (
      • Watanabe M.
      • Houten S.M.
      • Wang L.
      • Moschetta A.
      • Mangelsdorf D.J.
      • Heyman R.A.
      • Moore D.D.
      • Auwerx J.
      Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c.
      ). Moreover, a recent report indicated that SREBP-1 couples mechanical cues and lipid metabolism via protein geranylgeranylation, indicating the role of isoprenoids in regulating SREBP activation in lipid metabolism (
      • Bertolio R.
      • Napoletano F.
      • Mano M.
      • Maurer-Stroh S.
      • Fantuz M.
      • Zannini A.
      • Bicciato S.
      • Sorrentino G.
      • Del Sal G.
      Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism.
      ). Moreover, our study showed that zoledronic acid, an inhibitor of FPPS, inhibits hepatic DNL and liver steatosis by suppressing RhoA prenylation-dependent SREBP-1c activation (
      • Tang Q.
      • Jiang S.
      • Jia W.
      • Shen D.
      • Qiu Y.
      • Zhao Y.
      • Xue B.
      • Li C.
      Zoledronic acid, an FPPS inhibitor, ameliorates liver steatosis through inhibiting hepatic de novo lipogenesis.
      ). Considering the alteration of the prenylation balance by GGPPS in NAFLD, this finding indicates that up-regulation of SREBP mediates inflammatory reactions and increases hepatic total cholesterol and triglyceride levels, consequently correlating with HCC progression (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ).

      LKB farnesylation and AMPK activation

      As the LKB1-AMPK pathway is important for cells to maintain metabolic homeostasis by sensing the AMP/ATP levels (
      • Shackelford D.B.
      • Shaw R.J.
      The LKB1-AMPK pathway: metabolism and growth control in tumour suppression.
      ), it is thought to control the GGPPS-regulated metabolic reprogramming process. Mechanistically, as we reported, Ggpps deficiency enhances the farnesylation of LKB1 and promotes metabolic reprogramming by regulating AMPK activity (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ). AMPK activation turns off ATP-consuming pathways and switches on ATP-producing pathways. Such metabolic alterations further induce hepatic inflammation through elevated macrophage infiltration and proinflammatory cytokine production. In addition, insulin resistance is frequently detected in patients with NAFLD; this state decreases AMPK activity and produces a hyperuricemic environment, resulting in hepatic ATP depletion and further favoring glycolysis (
      • Abdelmalek M.F.
      • Suzuki A.
      • Guy C.
      • Unalp-Arida A.
      • Colvin R.
      • Johnson R.J.
      • Diehl A.M.
      • Nonalcoholic Steatohepatitis Clinical Research Network
      Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease.
      ). Thus, the GGPPS-regulated protein prenylation balance is a metabolic controller of fat overload–induced NAFLD and fibrosis development (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ).

      Signaling pathways mediated by prenylation in NAFLD/HCC progression

      The progression from NAFLD to HCC is related to several signaling pathways involved in steatogenic, fibrogenic, proliferative, and proinflammatory signaling (
      • Polyzos S.A.
      • Kountouras J.
      • Mantzoros C.S.
      Obesity and nonalcoholic fatty liver disease: from pathophysiology to therapeutics.
      ), such as the Ras/PI3K/AKT and Hippo-Yes–associated protein (YAP)/YAZ pathways. These signaling pathways have been reported to be regulated by the prenylation balance. Thus, an abnormal balance of protein prenylation may contribute to HCC progression from NAFLD via signal transduction through related signaling pathways.

      Ras signaling pathway

      The intracellular GTP-binding proteins involved in signal transduction comprise the largest family of prenylated proteins. The Ras protein is the most extensively studied small GTPase (
      • Swanson K.M.
      • Hohl R.J.
      Anti-cancer therapy: targeting the mevalonate pathway.
      ). As malignancy is associated with Ras mutation, Ras is a potential target for cancer therapy. For example, Ras mutations have been found in pancreatic cancer (90%) (
      • Almoguera C.
      • Shibata D.
      • Forrester K.
      • Martin J.
      • Arnheim N.
      • Perucho M.
      Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.
      ), thyroid cancer (50%) (
      • Lemoine N.R.
      • Mayall E.S.
      • Wyllie F.S.
      • Williams E.D.
      • Goyns M.
      • Stringer B.
      • Wynford-Thomas D.
      High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis.
      ), acute myeloid leukemia (44%) (
      • Janssen J.W.
      • Steenvoorden A.C.
      • Lyons J.
      • Anger B.
      • Böhlke J.U.
      • Bos J.L.
      • Seliger H.
      • Bartram C.R.
      RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes.
      ), colon cancer (47%) (
      • Vogelstein B.
      • Fearon E.R.
      • Hamilton S.R.
      • Kern S.E.
      • Preisinger A.C.
      • Leppert M.
      • Nakamura Y.
      • White R.
      • Smits A.M.
      • Bos J.L.
      Genetic alterations during colorectal-tumor development.
      ), melanoma (36%) (
      • Ball N.J.
      • Yohn J.J.
      • Morelli J.G.
      • Norris D.A.
      • Golitz L.E.
      • Hoeffler J.P.
      Ras mutations in human melanoma: a marker of malignant progression.
      ), and lung cancer (30%) (
      • Rodenhuis S.
      • Slebos R.J.
      • Boot A.J.
      • Evers S.G.
      • Mooi W.J.
      • Wagenaar S.S.
      • van Bodegom P.C.
      • Bos J.L.
      Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung.
      ). To date, three forms of mutated Ras have been identified: H-Ras, K-Ras, and N-Ras. K-Ras mutations occur more commonly in cancer than do N-Ras mutations, which are usually found in hematologic malignancies (
      • Bos J.L.
      ras oncogenes in human cancer: a review.
      ). These three different types of mutated Ras share over 90% sequence homology but vary in their association with the inner plasma membrane (
      • Niv H.
      • Gutman O.
      • Kloog Y.
      • Henis Y.I.
      Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells.
      ), which accounts for their differing oncogenic potential (
      • Drosten M.
      • Simón-Carrasco L.
      • Hernández-Porras I.
      • Lechuga C.G.
      • Blasco M.T.
      • Jacob H.K.C.
      • Fabbiano S.
      • Potenza N.
      • Bustelo X.R.
      • Guerra C.
      • Barbacid M.
      H-Ras and K-Ras oncoproteins induce different tumor spectra when driven by the same regulatory sequences.
      ).
      Differences in the balance of prenylation and membrane-anchored Ras isoforms can explain differences in the activation of K-Ras, N-Ras, and H-Ras. K-Ras and N-Ras can be geranylgeranylated when FTase is inhibited, whereas H-Ras can only be farnesylated by FTase (
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • Bishop W.R.
      • Pai J.K.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ). After prenylation, K-Ras bypasses the Golgi complex, yet N-Ras and H-Ras encounter palmitoyl acyltransferases on the cytoplasmic surface of the Golgi (
      • Apolloni A.
      • Prior I.A.
      • Lindsay M.
      • Parton R.G.
      • Hancock J.F.
      H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway.
      ). The locations of the three isoforms at the cell membrane also differ; K-Ras and N-Ras are not associated with lipid rafts, although the GDP-bound form of H-Ras binds to lipid rafts (
      • Prior I.A.
      • Harding A.
      • Yan J.
      • Sluimer J.
      • Parton R.G.
      • Hancock J.F.
      GTP-dependent segregation of H-ras from lipid rafts is required for biological activity.
      ).
      Once activated by prenylation, Ras acts as an upstream master regulator, directly activating downstream pathways involved in various cellular functions. Two pathways, the PI3K/AKT pathway and the Raf/MEK/MAPK/ERK pathway, mediate tumor cell proliferation, migration, and metastasis. Activation of PI3K/AKT signaling by growth factors increases the expression of SREBP1 and SREBP2 (
      • Demoulin J.B.
      • Ericsson J.
      • Kallin A.
      • Rorsman C.
      • Rönnstrand L.
      • Heldin C.H.
      Platelet-derived growth factor stimulates membrane lipid synthesis through activation of phosphatidylinositol 3-kinase and sterol regulatory element-binding proteins.
      ,
      • Zhou R.H.
      • Yao M.
      • Lee T.S.
      • Zhu Y.
      • Martins-Green M.
      • Shyy J.Y.
      Vascular endothelial growth factor activation of sterol regulatory element binding protein: a potential role in angiogenesis.
      ,
      • Fleischmann M.
      • Iynedjian P.B.
      Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt.
      ,
      • Luu W.
      • Sharpe L.J.
      • Stevenson J.
      • Brown A.J.
      Akt acutely activates the cholesterogenic transcription factor SREBP-2.
      ,
      • Porstmann T.
      • Griffiths B.
      • Chung Y.L.
      • Delpuech O.
      • Griffiths J.R.
      • Downward J.
      • Schulze A.
      PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP.
      ), which results in elevated lipid and cholesterol production and progression of NAFLD (
      • Ricoult S.J.
      • Yecies J.L.
      • Ben-Sahra I.
      • Manning B.D.
      Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP.
      ,
      • Yamauchi Y.
      • Furukawa K.
      • Hamamura K.
      • Furukawa K.
      Positive feedback loop between PI3K-Akt-mTORC1 signaling and the lipogenic pathway boosts Akt signaling: induction of the lipogenic pathway by a melanoma antigen.
      ,
      • Calvisi D.F.
      • Wang C.
      • Ho C.
      • Ladu S.
      • Lee S.A.
      • Mattu S.
      • Destefanis G.
      • Delogu S.
      • Zimmermann A.
      • Ericsson J.
      • Brozzetti S.
      • Staniscia T.
      • Chen X.
      • Dombrowski F.
      • Evert M.
      Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.
      ). Moreover, PI3K activity may be decreased with inhibition of the MVA pathway, possibly through decreased Ras prenylation (
      • Kusama T.
      • Mukai M.
      • Iwasaki T.
      • Tatsuta M.
      • Matsumoto Y.
      • Akedo H.
      • Inoue M.
      • Nakamura H.
      3-Hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis.
      ).
      Several studies have revealed that prenylation inhibition may be an efficient therapeutic strategy for cancer via inhibition of the Ras signaling pathway. Simultaneous knockout of prenyltransferase β subunits (both Fntb and Pggt1b) suppresses K-Ras–induced lung tumor progression more efficiently than deletion of either subunit alone (
      • Liu M.
      • Sjogren A.K.
      • Karlsson C.
      • Ibrahim M.X.
      • Andersson K.M.
      • Olofsson F.J.
      • Wahlstrom A.M.
      • Dalin M.
      • Yu H.
      • Chen Z.
      • Yang S.H.
      • Young S.G.
      • Bergo M.O.
      Targeting the protein prenyltransferases efficiently reduces tumor development in mice with K-RAS-induced lung cancer.
      ). In addition, conditional Fntb or Pggt1b deficiency reduces K-Ras-G12D–induced lung cancer formation in mice (
      • Liu M.
      • Sjogren A.K.
      • Karlsson C.
      • Ibrahim M.X.
      • Andersson K.M.
      • Olofsson F.J.
      • Wahlstrom A.M.
      • Dalin M.
      • Yu H.
      • Chen Z.
      • Yang S.H.
      • Young S.G.
      • Bergo M.O.
      Targeting the protein prenyltransferases efficiently reduces tumor development in mice with K-RAS-induced lung cancer.
      ), suggesting that FTase and GGTase1 are targets for cancer therapy.

      Other Ras GTPase superfamily signaling pathways

      All proteins in the Ras GTPase superfamily are localized to the membrane by prenylation, and several studies have shown that statin treatment decreases the prenylated and membrane-associated forms of Ras, Rho, Rac, Rap, and Rab subfamily proteins (
      • Tsubaki M.
      • Mashimo K.
      • Takeda T.
      • Kino T.
      • Fujita A.
      • Itoh T.
      • Imano M.
      • Sakaguchi K.
      • Satou T.
      • Nishida S.
      Statins inhibited the MIP-1α expression via inhibition of Ras/ERK and Ras/Akt pathways in myeloma cells.
      ). In pancreatic cancer with K-Ras mutation (90%) (
      • Bos J.L.
      ras oncogenes in human cancer: a review.
      ), pathways mediated by geranylgeranylated RalA and RalB correlate much more strongly with malignancy onset than do MEK or AKT pathways (
      • Hamad N.M.
      • Elconin J.H.
      • Karnoub A.E.
      • Bai W.
      • Rich J.N.
      • Abraham R.T.
      • Der C.J.
      • Counter C.M.
      Distinct requirements for Ras oncogenesis in human versus mouse cells.
      ). In addition, geranylgeranylated RhoC performs an essential function in tumor metastasis (
      • Hamad N.M.
      • Elconin J.H.
      • Karnoub A.E.
      • Bai W.
      • Rich J.N.
      • Abraham R.T.
      • Der C.J.
      • Counter C.M.
      Distinct requirements for Ras oncogenesis in human versus mouse cells.
      ,
      • Clark E.A.
      • Golub T.R.
      • Lander E.S.
      • Hynes R.O.
      Genomic analysis of metastasis reveals an essential role for RhoC.
      ). A recent study reported that GGPPS inhibition therapy can be a novel strategy for the treatment of pancreatic ductal adenocarcinoma by disrupting Rab geranylgeranylation to induce the unfolded protein response pathway (
      • Haney S.L.
      • Varney M.L.
      • Chhonker Y.S.
      • Shin S.
      • Mehla K.
      • Crawford A.J.
      • Smith H.J.
      • Smith L.M.
      • Murry D.J.
      • Hollingsworth M.A.
      • Holstein S.A.
      Inhibition of geranylgeranyl diphosphate synthase is a novel therapeutic strategy for pancreatic ductal adenocarcinoma.
      ), which also occurs in HCC (
      • Wu H.
      • Wei L.
      • Fan F.
      • Ji S.
      • Zhang S.
      • Geng J.
      • Hong L.
      • Fan X.
      • Chen Q.
      • Tian J.
      • Jiang M.
      • Sun X.
      • Jin C.
      • Yin Z.-Y.
      • Liu Q.
      • et al.
      Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.
      ).
      Furthermore, the Rho subfamily GTPase Rac1 is required for the induction of K-Ras–driven lung cancer in mice (
      • Sjogren A.K.
      • Andersson K.M.
      • Liu M.
      • Cutts B.A.
      • Karlsson C.
      • Wahlstrom A.M.
      • Dalin M.
      • Weinbaum C.
      • Casey P.J.
      • Tarkowski A.
      • Swolin B.
      • Young S.G.
      • Bergo M.O.
      GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.
      ). Importantly, geranylgeranylated cell division cycle 42 (Cdc42) and Rac are downstream targets of Ras in mediating fibroblast transformation (
      • Qiu R.G.
      • Abo A.
      • McCormick F.
      • Symons M.
      Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation.
      ). Atorvastatin, which blocks FPP and GGPP production by inhibiting HMGCR, prevents HCC development by decreasing Rac1 prenylation to inhibit MYC phosphorylation (
      • Cao Z.
      • Fan-Minogue H.
      • Bellovin D.I.
      • Yevtodiyenko A.
      • Arzeno J.
      • Yang Q.
      • Gambhir S.S.
      • Felsher D.W.
      MYC phosphorylation, activation, and tumorigenic potential in hepatocellular carcinoma are regulated by HMG-CoA reductase.
      ). As MYC is a potent oncogene that causes transformation in various cancer types, inhibition of MYC phosphorylation can induce sustained regression of HCC (
      • Meyer N.
      • Penn L.Z.
      Reflecting on 25 years with MYC.
      ). Thus, the MVA pathway is important in this MYC-driven HCC model.
      Another Ras GTPase superfamily protein, RhoB, is both geranylgeranylated (70%) and farnesylated (30%) under physiological conditions. It has been shown that geranylgeranylated RhoB suppresses Ras-induced transformation and that inhibition of RhoB farnesylation contributes to FTI-induced apoptosis (
      • Mazières J.
      • Tillement V.
      • Allal C.
      • Clanet C.
      • Bobin L.
      • Chen Z.
      • Sebti S.M.
      • Favre G.
      • Pradines A.
      Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells.
      ). However, both geranylgeranylated RhoB and farnesylated RhoB suppress tumor activity in several human epithelial cancer cells (
      • Chen Z.
      • Sun J.
      • Pradines A.
      • Favre G.
      • Adnane J.
      • Sebti S.M.
      Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumor growth in nude mice.
      ). A previous study also showed that the oncogene YAP and transcriptional coactivator with PDZ-binding motif (TAZ) require GGPP-mediated RhoA prenylation to be functional (
      • Sorrentino G.
      • Ruggeri N.
      • Specchia V.
      • Cordenonsi M.
      • Mano M.
      • Dupont S.
      • Manfrin A.
      • Ingallina E.
      • Sommaggio R.
      • Piazza S.
      • Rosato A.
      • Piccolo S.
      • Del Sal G.
      Metabolic control of YAP and TAZ by the mevalonate pathway.
      ). Mechanistically, the balance of prenylation maintains GGPP levels, regulating the nuclear localization of YAP and TAZ through RhoA prenylation (
      • Santinon G.
      • Pocaterra A.
      • Dupont S.
      Control of YAP/TAZ activity by metabolic and nutrient-sensing pathways.
      ). Another study demonstrated that treatment with atorvastatin or geranylgeranyltransferase inhibitors (GGTIs) increased the levels of Lats1 and Lats2, two important tumor suppressors involved in the Hippo-YAP/YAZ pathway, which further controls cell proliferation and metabolism (
      • Aylon Y.
      • Gershoni A.
      • Rotkopf R.
      • Biton I.E.
      • Porat Z.
      • Koh A.P.
      • Sun X.
      • Lee Y.
      • Fiel M.I.
      • Hoshida Y.
      • Friedman S.L.
      • Johnson R.L.
      • Oren M.
      The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation.
      ). Therefore, the balance of prenylation participates in signal transduction through pathways involved in NAFLD/HCC progression.

      Other factors involved in protein prenylation in NAFLD/HCC progression

      Proinflammatory cytokine release regulated by protein prenylation

      Inflammation plays a key role in extracellular matrix deposition and fibrosis, which further leads to HCC (
      • Wang X.
      • Zheng Z.
      • Caviglia J.M.
      • Corey K.E.
      • Herfel T.M.
      • Cai B.
      • Masia R.
      • Chung R.T.
      • Lefkowitch J.H.
      • Schwabe R.F.
      • Tabas I.
      Hepatocyte TAZ/WWTR1 promotes inflammation and fibrosis in nonalcoholic steatohepatitis.
      ). The progression from NAFLD to fibrosis requires glucose, lipid, and amino acid metabolic reprogramming in response to a stressful microenvironment (
      • Sookoian S.
      • Pirola C.J.
      NAFLD. Metabolic make-up of NASH: from fat and sugar to amino acids.
      ). Such metabolic changes further contribute to hepatic inflammation via enhanced proinflammatory cytokine production and macrophage infiltration. In a previous study, we illustrated a glycolysis-inflammation regulatory network in hepatocytes, referring to reprogrammed metabolism and inflammation in hepatocytes, with macrophages collectively accelerating progression from NAFLD to fibrosis (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ). Further evidence indicates that proinflammatory cytokines, such as interleukin-6, C-reactive protein, C-peptide, and adiponectin, are essential for the development of fibrosis from NASH (
      • Cohen J.C.
      • Horton J.D.
      • Hobbs H.H.
      Human fatty liver disease: old questions and new insights.
      ). Previous work has shown that statins can reduce C-reactive protein production stimulated by interleukin-6 (
      • Arnaud C.
      • Burger F.
      • Steffens S.
      • Veillard N.R.
      • Nguyen T.H.
      • Trono D.
      • Mach F.
      Statins reduce interleukin-6-induced C-reactive protein in human hepatocytes: new evidence for direct antiinflammatory effects of statins.
      ), further inhibiting inflammatory reactions and hepatic tissue fibrosis. Overall, proinflammatory cytokine release regulated by altered protein prenylation is essential for the progression of NAFLD to fibrosis.

      Rab geranylgeranylation and mitochondrial function

      Mitochondrial quality control (QC) plays an important role in HCC progression, as mitochondrial dysfunction is commonly found in HCC (
      • Begriche K.
      • Massart J.
      • Robin M.A.
      • Bonnet F.
      • Fromenty B.
      Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease.
      ); moreover, by eliminating damaged mitochondria, mitophagy is required for mitochondrial QC (
      • Scherz-Shouval R.
      • Elazar Z.
      Regulation of autophagy by ROS: physiology and pathology.
      ). Therefore, accumulation of damaged mitochondria is attributed to defective mitophagy, a metabolic defect in patients with NASH (
      • Sanyal A.J.
      Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.
      ). Defective mitophagy and mitochondrial biosynthesis can promote fibrosis, which is one of the most important factors during HCC progression. Rab has a crucial role in membrane trafficking, particularly vesicle formation, transport, fusion, cargo selection, and sorting, which are regulated by prenylation. Indeed, as a mitophagy effector downstream of the ubiquitin E3 ligase Parkin, Rab7 is fundamental in mitophagy (
      • Wong Y.C.
      • Ysselstein D.
      • Krainc D.
      Mitochondria-lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis.
      ). In our study, Ggpps deletion impaired Rab7 geranylgeranylation and mitophagy, consequently causing defects in mitophagy in hepatocytes, suggesting that dysregulation of Rab7 geranylgeranylation is a susceptibility factor for NAFLD/fibrosis progression. Thus, GGPPS-mediated prenylation in mitochondrial QC is important during HCC progression (
      • Liu J.
      • Jiang S.
      • Zhao Y.
      • Sun Q.
      • Zhang J.
      • Shen D.
      • Wu J.
      • Shen N.
      • Fu X.
      • Sun X.
      • Yu D.
      • Chen J.
      • He J.
      • Shi T.
      • Ding Y.
      • et al.
      Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.
      ). Furthermore, we analyzed the CaaX motifs in mitochondria-related proteins of mice and humans and found several potential mitochondrial prenyltransferase substrates, including Rab24 (
      • Ortiz Sandoval C.
      • Simmen T.
      Rab proteins of the endoplasmic reticulum: functions and interactors.
      ), Rab32 (
      • Haile Y.
      • Deng X.
      • Ortiz-Sandoval C.
      • Tahbaz N.
      • Janowicz A.
      • Lu J.Q.
      • Kerr B.J.
      • Gutowski N.J.
      • Holley J.E.
      • Eggleton P.
      • Giuliani F.
      • Simmen T.
      Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.
      ), and Rab35 (
      • Minowa-Nozawa A.
      • Nozawa T.
      • Okamoto-Furuta K.
      • Kohda H.
      • Nakagawa I.
      Rab35 GTPase recruits NDP52 to autophagy targets.
      ), which may also influence mitochondrial function.

      FPP/GGPP as ligands promoting the progression of NAFLD to HCC

      The metabolic changes that occur in tumors not only provide energy for cell division but also generate metabolites for downstream signaling. The isoprenoid and sterol metabolites produced by the MVA pathway also perform signaling functions.
      FPP and GGPP, as metabolites of the MVA pathway, can act as ligands and directly bind to proteins, thus acting as individual molecules rather than prenylation agents. FPP can directly interact with FXR to control the activity of downstream pathways (
      • Weinberger C.
      A model for farnesoid feedback control in the mevalonate pathway.
      ), whereas GGPP binds with Skp2 (
      • Chen M.C.
      • Tsai Y.C.
      • Tseng J.H.
      • Liou J.J.
      • Horng S.
      • Wen H.C.
      • Fan Y.C.
      • Zhong W.B.
      • Hsu S.P.
      Simvastatin inhibits cell proliferation and migration in human anaplastic thyroid cancer.
      ) and PPARγ (
      • Weivoda M.M.
      • Hohl R.J.
      Geranylgeranyl pyrophosphate stimulates PPARγ expression and adipogenesis through the inhibition of osteoblast differentiation.
      ) (Fig. 2, right). Currently, we are working to identify some proteins to which GGPP/FPP bind, which may predict metabolic properties and NAFLD progression.

      Drugs targeting the MVA pathway in NAFLD/HCC progression

      As mentioned above, the balance of prenylation determines the progression from NAFLD to HCC. Thus, identifying a drug-targeting mediator of the prenylation balance involved in the MVA pathway can provide insight into prospective targeted therapies for NAFLD and HCC.
      The MVA pathway is one of the most commonly clinically targeted biochemical pathways in human diseases, and inhibitors of the MVA pathway, such as statins and bisphosphonates, are extensively used in clinical trials (
      • Buhaescu I.
      • Izzedine H.
      Mevalonate pathway: a review of clinical and therapeutical implications.
      ). Statins, including lovastatin and atorvastatin, inhibit HMGCR, lowering the plasma cholesterol level in the treatment of hypercholesterolemia and atherosclerosis (
      • Maron D.J.
      • Fazio S.
      • Linton M.F.
      Current perspectives on statins.
      ,
      • Rosenson R.S.
      Statins in atherosclerosis: lipid-lowering agents with antioxidant capabilities.
      ). HCC is also responsive to statins (
      • Graf H.
      • Jüngst C.
      • Straub G.
      • Dogan S.
      • Hoffmann R.T.
      • Jakobs T.
      • Reiser M.
      • Waggershauser T.
      • Helmberger T.
      • Walter A.
      • Walli A.
      • Seidel D.
      • Goke B.
      • Jüngst D.
      Chemoembolization combined with pravastatin improves survival in patients with hepatocellular carcinoma.
      ), perhaps due to the hepatotropic pharmacology of these drugs (
      • Mullen P.J.
      • Yu R.
      • Longo J.
      • Archer M.C.
      • Penn L.Z.
      The interplay between cell signalling and the mevalonate pathway in cancer.
      ). In addition, reports indicate that statins can act as adjuvants to stimulate the immune system and boost immunity as potent cancer therapeutics targeting immune activation (
      • Robert C.
      • Vagner S.
      Boosting immunity by targeting post-translational prenylation of small GTPases.
      ,
      • Xia Y.
      • Xie Y.
      • Yu Z.
      • Xiao H.
      • Jiang G.
      • Zhou X.
      • Yang Y.
      • Li X.
      • Zhao M.
      • Li L.
      • Zheng M.
      • Han S.
      • Zong Z.
      • Meng X.
      • Deng H.
      • et al.
      The mevalonate pathway is a druggable target for vaccine adjuvant discovery.
      ). Statins can also reduce cancer stemness, targeting cancer stem cells without impairing the function of innate immunity. Mechanistically, statins attenuate the prenylation of Ras family proteins, which is also highly clinically significant in HCC (
      • Gruenbacher G.
      • Thurnher M.
      Mevalonate metabolism in cancer stemness and trained immunity.
      ,
      • Likus W.
      • Siemianowicz K.
      • Bieńk K.
      • Pakuła M.
      • Pathak H.
      • Dutta C.
      • Wang Q.
      • Shojaei S.
      • Assaraf Y.G.
      • Ghavami S.
      • Cieślar-Pobuda A.
      • Łos M.J.
      Could drugs inhibiting the mevalonate pathway also target cancer stem cells?.
      ). In addition, simvastatin decreases the synthesis of FPP and GGPP and exhibits anticancer effects against HCC by inducing G0/G1 arrest through up-regulation of p27 (
      • Chen Q.
      • Rong P.
      • Xu D.
      • Zhu S.
      • Chen L.
      • Xie B.
      • Du Q.
      • Quan C.
      • Sheng Y.
      • Zhao T.J.
      • Li P.
      • Wang H.Y.
      • Chen S.
      Rab8a deficiency in skeletal muscle causes hyperlipidemia and hepatosteatosis by impairing muscle lipid uptake and storage.
      ).
      On the other hand, GGTase1 and FTase are required for tumor progression and maintenance (
      • Sjogren A.K.
      • Andersson K.M.
      • Liu M.
      • Cutts B.A.
      • Karlsson C.
      • Wahlstrom A.M.
      • Dalin M.
      • Weinbaum C.
      • Casey P.J.
      • Tarkowski A.
      • Swolin B.
      • Young S.G.
      • Bergo M.O.
      GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.
      ,
      • Mijimolle N.
      • Velasco J.
      • Dubus P.
      • Guerra C.
      • Weinbaum C.A.
      • Casey P.J.
      • Campuzano V.
      • Barbacid M.
      Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development.
      ). GGTIs have been shown to be efficient in treating several cancers and inflammatory diseases (
      • Khan O.M.
      • Ibrahim M.X.
      • Jonsson I.M.
      • Karlsson C.
      • Liu M.
      • Sjogren A.K.
      • Olofsson F.J.
      • Brisslert M.
      • Andersson S.
      • Ohlsson C.
      • Hultén L.M.
      • Bokarewa M.
      • Bergo M.O.
      Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice.
      ), and one such agent, GGTI-2418, has been assessed in Phase I clinical trials (
      • Berndt N.
      • Hamilton A.D.
      • Sebti S.M.
      Targeting protein prenylation for cancer therapy.
      ). Two FTIs that have advanced to Phase III clinical trials are lonafarnib (
      • Berndt N.
      • Hamilton A.D.
      • Sebti S.M.
      Targeting protein prenylation for cancer therapy.
      ) and tipifarnib (
      • Van Cutsem E.
      • van de Velde H.
      • Karasek P.
      • Oettle H.
      • Vervenne W.L.
      • Szawlowski A.
      • Schoffski P.
      • Post S.
      • Verslype C.
      • Neumann H.
      • Safran H.
      • Humblet Y.
      • Perez Ruixo J.
      • Ma Y.
      • Von Hoff D.
      Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer.
      ,
      • Rao S.
      • Cunningham D.
      • de Gramont A.
      • Scheithauer W.
      • Smakal M.
      • Humblet Y.
      • Kourteva G.
      • Iveson T.
      • Andre T.
      • Dostalova J.
      • Illes A.
      • Belly R.
      • Perez-Ruixo J.J.
      • Park Y.C.
      • Palmer P.A.
      Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer.
      ,
      • Harousseau J.L.
      • Martinelli G.
      • Jedrzejczak W.W.
      • Brandwein J.M.
      • Bordessoule D.
      • Masszi T.
      • Ossenkoppele G.J.
      • Alexeeva J.A.
      • Beutel G.
      • Maertens J.
      • Vidriales M.B.
      • Dombret H.
      • Thomas X.
      • Burnett A.K.
      • Robak T.
      • et al.
      A randomized phase 3 study of tipifarnib compared with best supportive care, including hydroxyurea, in the treatment of newly diagnosed acute myeloid leukemia in patients 70 years or older.
      ). However, these drugs could not improve the outcomes of advanced pancreatic cancer (
      • Van Cutsem E.
      • van de Velde H.
      • Karasek P.
      • Oettle H.
      • Vervenne W.L.
      • Szawlowski A.
      • Schoffski P.
      • Post S.
      • Verslype C.
      • Neumann H.
      • Safran H.
      • Humblet Y.
      • Perez Ruixo J.
      • Ma Y.
      • Von Hoff D.
      Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer.
      ), advanced colon cancer (
      • Rao S.
      • Cunningham D.
      • de Gramont A.
      • Scheithauer W.
      • Smakal M.
      • Humblet Y.
      • Kourteva G.
      • Iveson T.
      • Andre T.
      • Dostalova J.
      • Illes A.
      • Belly R.
      • Perez-Ruixo J.J.
      • Park Y.C.
      • Palmer P.A.
      Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer.
      ), advanced non-small-cell lung cancer (
      • Berndt N.
      • Hamilton A.D.
      • Sebti S.M.
      Targeting protein prenylation for cancer therapy.
      ), or acute myeloid leukemia (
      • Harousseau J.L.
      • Martinelli G.
      • Jedrzejczak W.W.
      • Brandwein J.M.
      • Bordessoule D.
      • Masszi T.
      • Ossenkoppele G.J.
      • Alexeeva J.A.
      • Beutel G.
      • Maertens J.
      • Vidriales M.B.
      • Dombret H.
      • Thomas X.
      • Burnett A.K.
      • Robak T.
      • et al.
      A randomized phase 3 study of tipifarnib compared with best supportive care, including hydroxyurea, in the treatment of newly diagnosed acute myeloid leukemia in patients 70 years or older.
      ). More importantly, as H-, N-, and K-Ras require prenylation for their transforming activities (
      • Wright L.P.
      • Philips M.R.
      Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras.
      ), inhibition of prenylation exhibits significant therapeutic efficacy in ∼30% of human cancers driven by Ras mutations (
      • Berndt N.
      • Hamilton A.D.
      • Sebti S.M.
      Targeting protein prenylation for cancer therapy.
      ,
      • Cox A.D.
      • Der C.J.
      • Philips M.R.
      Targeting RAS membrane association: back to the future for anti-RAS drug discovery?.
      ). The results of these collective drug studies indicate that the MVA pathway can be considered a druggable target for HCC therapy.

      Prospective applications

      Although statins exhibit therapeutic efficacy in NAFLD/HCC, long-term use of statins is associated with adverse effects, such as myopathy (
      • Thompson P.D.
      • Clarkson P.
      • Karas R.H.
      Statin-associated myopathy.
      ) and liver injury (
      • Chalasani N.
      Statins and hepatotoxicity: focus on patients with fatty liver.
      ). These effects might develop because statins suppress the MVA pathway from its initiation, thus leading to isoprenoid depletion, which was later demonstrated to be essential for cellular functions. Approximately 300 proteins related to cellular functions, such as membrane trafficking and signal transduction, in the human proteome are modified by prenyltransferases (
      • Hougland J.L.
      • Fierke C.A.
      Getting a handle on protein prenylation.
      ). Therefore, to prevent these side effects, other therapies combining different types and doses of MVA pathway inhibitors should be evaluated to achieve the best treatment effect without influencing protein prenylation. In addition, other inhibitors, such as nitrogenous bisphosphonates (NBPs) and DGBP, hold great promise for the treatment of NAFLD by targeting the balance of protein farnesylation and geranylgeranylation.
      NBPs, including zoledronic acid, as potent inhibitors of FPPS, are widely used to treat osteoporosis and metastatic bone disease and may also be applied in cancer (
      • Fritz G.
      Targeting the mevalonate pathway for improved anticancer therapy.
      ). Similar to statins, zoledronic acid reduces the intracellular levels of both FPP and GGPP, leading to inhibition of protein prenylation (
      • Reszka A.A.
      • Rodan G.A.
      Nitrogen-containing bisphosphonate mechanism of action.
      ). Intriguingly, it depletes GGPP more efficiently than FPP (
      • Goffinet M.
      • Thoulouzan M.
      • Pradines A.
      • Lajoie-Mazenc I.
      • Weinbaum C.
      • Faye J.C.
      • Séronie-Vivien S.
      Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells.
      ). This effect might be explained by the observation that GGPPS contains three NBP-binding sites that result in its inhibition by NBP treatment (
      • Amin D.
      • Cornell S.A.
      • Gustafson S.K.
      • Needle S.J.
      • Ullrich J.W.
      • Bilder G.E.
      • Perrone M.H.
      Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.
      ); alternatively, it might arise due to the suppression of squalene synthase by NBPs (
      • Amin D.
      • Cornell S.A.
      • Gustafson S.K.
      • Needle S.J.
      • Ullrich J.W.
      • Bilder G.E.
      • Perrone M.H.
      Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.
      ,
      • Amin D.
      • Cornell S.A.
      • Perrone M.H.
      • Bilder G.E.
      1-Hydroxy-3-(methylpentylamino)-propylidene-1,1-bisphosphonic acid as a potent inhibitor of squalene synthase.
      ), which may ultimately restore the FPP level. Researchers have also demonstrated the cellular effects of NBPs in promoting apoptosis and autophagy, which are mainly mediated via downstream GGPP depletion and decreased protein geranylgeranylation (
      • Amin D.
      • Cornell S.A.
      • Gustafson S.K.
      • Needle S.J.
      • Ullrich J.W.
      • Bilder G.E.
      • Perrone M.H.
      Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.
      ,
      • Wasko B.M.
      • Dudakovic A.
      • Hohl R.J.
      Bisphosphonates induce autophagy by depleting geranylgeranyl diphosphate.
      ). In our previous study, zoledronic acid, an NBP, inhibited FPPS and reduced lipid deposition in the liver, as demonstrated in both mice with HFD-induced hepatic steatosis and ob/ob mice (
      • Tang Q.
      • Jiang S.
      • Jia W.
      • Shen D.
      • Qiu Y.
      • Zhao Y.
      • Xue B.
      • Li C.
      Zoledronic acid, an FPPS inhibitor, ameliorates liver steatosis through inhibiting hepatic de novo lipogenesis.
      ). Considering the changes observed in NAFLD progression, such inhibitors can be used in clinical trials on HCC prevention and treatment.
      In addition to developing inhibitors of HMGCR and FPPS, some research groups have recently developed several dialkyl bisphosphonate inhibitors of GGPPS, including DGBP, a potent inhibitor of GGPP synthesis (IC50 = 200 nm) (
      • Shull L.W.
      • Wiemer A.J.
      • Hohl R.J.
      • Wiemer D.F.
      Synthesis and biological activity of isoprenoid bisphosphonates.
      ). Through depletion of intracellular GGPP, DGBP specifically impairs protein geranylgeranylation without changing farnesylation (
      • Wiemer A.J.
      • Tong H.
      • Swanson K.M.
      • Hohl R.J.
      Digeranyl bisphosphonate inhibits geranylgeranyl pyrophosphate synthase.
      ). However, other studies have shown that DGBP administration causes intracellular FPP accumulation (
      • Weivoda M.M.
      • Hohl R.J.
      The effects of direct inhibition of geranylgeranyl pyrophosphate synthase on osteoblast differentiation.
      ,
      • Weivoda M.M.
      • Hohl R.J.
      Effects of farnesyl pyrophosphate accumulation on calvarial osteoblast differentiation.
      ). In addition, another study demonstrated that RAM2061, a potent GGPPS inhibitor, is efficient against pancreatic ductal adenocarcinoma (
      • Haney S.L.
      • Varney M.L.
      • Chhonker Y.S.
      • Shin S.
      • Mehla K.
      • Crawford A.J.
      • Smith H.J.
      • Smith L.M.
      • Murry D.J.
      • Hollingsworth M.A.
      • Holstein S.A.
      Inhibition of geranylgeranyl diphosphate synthase is a novel therapeutic strategy for pancreatic ductal adenocarcinoma.
      ). In summary, invention with GGPPS inhibitors provides a NAFLD-HCC therapy with improved selectivity. More studies investigating the balance of farnesylation and geranylgeranylation in NAFLD progression are required to promote the development of NAFLD-HCC therapies.

      Author contributions

      Y. Z. and C.-J. L. conceptualization; Y. Z., T.-Y. W., and M.-F. Z. resources; Y. Z. data curation; Y. Z. and T.-Y. W. software; Y. Z. formal analysis; Y. Z. supervision; Y. Z. funding acquisition; Y. Z. validation; Y. Z. investigation; Y. Z. visualization; Y. Z. methodology; Y. Z. writing-original draft; Y. Z. and C.-J. L. project administration; Y. Z. and C.-J. L. writing-review and editing.

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