decreased expression of fructose-1,6- bisphosphatase ...altering glucose metabolism, and they...

Molecular and Cellular Pathobiology

Decreased Expression of Fructose-1,6-bisphosphatase Associates with GlucoseMetabolism and Tumor Progression inHepatocellular CarcinomaHidenari Hirata1,2,3, Keishi Sugimachi1, Hisateru Komatsu1, Masami Ueda1,Takaaki Masuda1,Ryutaro Uchi1, Shotaro Sakimura1, Sho Nambara1, Tomoko Saito1, Yoshiaki Shinden1,Tomohiro Iguchi1, Hidetoshi Eguchi1, Shuhei Ito1, Kotaro Terashima2, Katsumi Sakamoto2,Masakazu Hirakawa2, Hiroshi Honda3, and Koshi Mimori1

Abstract

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzymein gluconeogenesis, is reduced in expression in certain cancerswhere it has been hypothesized to act as a tumor suppressor,including in hepatocellular carcinoma (HCC). Here, we reportfunctional evidence supporting this hypothesis, providing a pre-clinical rationale to develop FBP1 as a therapeutic target for HCCtreatment. Three independent cohorts totaling 594 cases of HCCwere analyzed to address clinical significance. Lower FBP1 expres-sion associated with advanced tumor stage, poor overall survival,and higher tumor recurrence rates. In HCC cell lines, whereendogenous FBP1 expression is low, engineering its ectopic over-expression inhibited tumor growth and intracellular glucoseuptake by reducing aerobic glycolysis. In patient specimens,

promoter methylation and copy-number loss of FBP1 were inde-pendently associated with decreased FBP1 expression. Similarly,FBP1 downregulation in HCC cell lines was also associated withcopy-number loss. HCC specimens exhibiting low expression ofFBP1 had a highly malignant phenotype, including large tumorsize, poor differentiation, impairedgluconeogenesis, and enhancedaerobic glycolysis. The effects of FBP1 expression on prognosis andglucose metabolism were confirmed by gene set enrichment anal-ysis. Overall, our findings established that FBP1 downregulation inHCC contributed to tumor progression and poor prognosis byaltering glucose metabolism, and they rationalize further study ofFBP1 as a prognostic biomarker and therapeutic target in HCCpatients. Cancer Res; 76(11); 3265–76. �2016 AACR.

IntroductionHepatocellular carcinoma (HCC) is one of the most prevalent

cancers worldwide and is associatedwith a highmortality rate (1).Therefore, identification of useful biomarkers predicting clinicaloutcomes andmolecular targets for treatment is required. Alteredglucose metabolism, particularly aerobic glycolysis, is one of thehallmarks of cancers, including HCC (2), supporting the notionthat themolecules involved in altered glucosemetabolismmaybepotential biomarkers and therapeutic targets. Many studies haveexamined aerobic glycolysis in various cancers; however, gluco-neogenesis has not been studied extensively. Gluconeogenesis isalso thought to play an important role in tumor progression inHCC because gluconeogenesis mainly occurs in normal hepato-

cytes (3). A recent study reported the loss of gluconeogeniccapacity inHCC (4).Moreover, the switch from aerobic glycolysisto gluconeogenesis may be an effective treatment in patients withHCC (5). Nevertheless, the role of impaired gluconeogenesis inHCC progression is still poorly understood.

The tumor-suppressive function of the fructose-1,6-bisphospha-tase (FBP1) gene, which encodes a rate-limiting gluconeogenicenzyme, has been reported in certain cancers. Loss of FBP1expression has been shown to promote tumor progression byenhancing aerobic glycolysis, thereby resulting in poor prognosisin patients with clear cell renal cell carcinoma (ccRCC; ref. 6) andbreast cancer (7). With regard to HCC, in vitro experiments haveshown that FBP1 expression is silenced by promoter methylationand that ectopic FBP1 expression inhibits anchorage-dependentgrowthof a cell line through cell-cycle arrest (8).However, the roleof FBP1 in altered glucose metabolism has not been assessed inHCC. Furthermore, no studies have investigatedwhether aberrantFBP1 expression affects clinical outcomes in patients with HCC.

Therefore, the objective of this study was to examine thefunction and clinical significance of FBP1 expression in HCC.Through analysis of three independent large cohorts of HCCcases, the present study evaluated whether aberrant FBP1 expres-sion was associated with tumor progression and prognosis inpatients with HCC. We also elucidated the genetic and epigeneticmechanisms regulating FBP1 expression and investigated the roleof FBP1 expression in altered glucose metabolism.

1Department of Surgery, Kyushu University Beppu Hospital, Beppu,Japan. 2Department of Radiology, Kyushu University Beppu Hospital,Beppu, Japan. 3Department of Clinical Radiology, Graduate School ofMedical Sciences, Kyushu University, Fukuoka, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Koshi Mimori, Kyushu University Beppu Hospital, 4546Tsurumihara, Beppu 8740838, Japan. Phone: 81-977-27-1650; Fax: 81-977-27-1651, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2601

�2016 American Association for Cancer Research.

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Materials and MethodsPatients and sample collection

One hundred eighteen patients with HCC who underwenthepatic resection at Kyushu University Beppu Hospital and affil-iated hospitals between 2000 and 2004 were enrolled in thisstudy. Resected tumor tissues and paired normal liver tissues wereimmediately stored in RNAlater (Ambion), frozen in liquidnitrogen, and kept at –80�C until RNA extraction. A 5-yearfollow-up was conducted after operation. The median follow-upfor the 118 patients was 59.5 months (range, 3.0–60 months).Patients were staged according to the seventh edition of theInternational Union against Cancer TNM classification system.Of the 118 HCC patients, paired normal liver tissues were avail-able for 115 patients. All protocols were approved by the Ethicsand Indications Committee of Kyushu University. Writteninformed consent was obtained from all patients.

Cell cultureHuH7 and HepG2 cells authenticated by short tandem repeat

profiling using the PowerPlex 1.2 System (Promega) wereobtained from the cell bank of RIKEN BioResource Center (Tsu-kuba, Japan) in 2015. Cells were used within 6 months ofculturing after resuscitation. Details are provided in the Supple-mentary Materials and Methods.

Construction of an FBP1 expression lentiviral vectorA full-length cDNA insert of human FBP1 was amplified by

PCR. Lentiviruseswere generatedby transfectionofHEK293T cellswith pCMV-VSV-G-RSV-Rev, pCAG-HIVgp, and either CSII-CMV-FBP1 or CSII-CMV-MCS (empty) plasmid DNAs (50 NheI and 30

XbaI sites) using Lipofectamine 2000 (Invitrogen) following themanufacturer's protocol. Forty-eight hours after transfection, thelentivirus-containing supernatant was collected and passedthrough a 0.45-mm filter. Infections were then carried out byincubating cells with medium containing the lentiviral superna-tant for 48 hours.

RNA preparation and quantitative real-time RT-PCRDetailedmethods are provided in the SupplementaryMaterials

and Methods.

Analysis of public clinical datasets and gene set enrichmentanalysis

We obtained paired gene-expression and survival profiles of242 HCC samples from the National Cancer for BiotechnologyInformation Gene Expression Omnibus (GEO) database (acces-sion codes GSE14520; ref. 9). Expression profiles of 228 pairednormal liver samples were available.

Moreover, we obtained paired RNA sequencing and survivaldata of 234HCC cases in The Cancer Genome Atlas (TCGA) fromthe Broad Institute's Firehose (http://gdac.broadinstitute.org/runs/stddata__2014_10_17/data/LIHC/20141017/). Expressionprofiles of 48 paired normal liver samples were available. Datafrommethylation arrays, SNP arrays, and whole-exome sequenc-ingwere also obtained.Details are provided in the SupplementaryMaterials and Methods.

Analysis of the Cancer Cell Line Encyclopedia databaseWe obtained normalized mRNA expression and DNA copy-

number data of human cancer cell lines in the Cancer Cell Line

Encyclopedia (CCLE; ref. 10) from cBioPortal For Cancer Geno-mics (http://www.cbioportal.org/index.do; ref. 11). Of the 967human cancer cell lines in the CCLE database, FBP1 expressionprofiles were available for 27 liver cancer cell lines. A z-scorethreshold of 2 was used as the cutoff value. For the analysis ofcopy-number alterations, data were available for 23 liver cancercell lines (Supplementary Table S1). Moreover, FBP1 promotermethylation status was determined using the results of methyl-ation-specific PCR, as described in a previous report (8), ormethylation array data from GEO database accession numbersGSE60753 (12) and GSE67485 (13). Data on promoter methyl-ation profiles were available for 7 liver cancer cell lines (Supple-mentary Table S2).

Western blotting analysisProteins were detected using antibodies against FBP1

(SAB1405798; Sigma-Aldrich), thioredoxin-interacting protein(TXNIP; K0204-3;Medical & Biological Laboratories), and b-actin(Santa Cruz Biotechnology). The methods are described in detailin the Supplementary Materials and Methods.

Sphere formation assaysDetailedmethods are provided in the SupplementaryMaterials

and Methods.

Xenograft studiesFive-week-old female BALB/c nu/nu mice were obtained from

SLC, Inc. and maintained under specific pathogen-free condi-tions. All animal procedures were performed in compliance withthe Guidelines for the Care and Use of Experimental Animalsestablished by the Committee for Animal Experimentation ofKyushu University. A total of 5 � 106 cells (empty vector controlcells or HuH7 cells stably expressing FBP1) in 150 mL of phos-phate-buffered saline were injected subcutaneously into bothflanks of the mice. Tumor size was measured every 3 to 4 dayswith a digital caliper and calculated using the following formula:tumor volume ¼ length � width2 � 0.5.

Quantification of metabolitesIntracellular glucose uptake was determined by 2-(N-[7-nitro-

benz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy-D-glucose (2-NBDG),a fluorescently-tagged glucose derivative, using a Glucose UptakeCell-Based Assay Kit (Cayman Chemical). Lactate secretion wasexamined using a Lactate Assay Kit (BioVision). Isotopomer dis-tribution analysis was performed by F-SCOPE package of HumanMetabolome Technologies using capillary electrophoresis time-of-flight mass spectrometry (14–16). Cells were incubated in DMEMwith glucose replaced by 0.1% [U-13C]glucose (Sigma-Aldrich) for4 hours. All methods are described in detail in the SupplementaryMaterials and Methods.

Statistical analysisFor continuous variables, statistical analyses were performed

using Student t tests orWelch t tests, and thedegree of linearitywasestimated by Pearson's correlation coefficient. Differences amongmultiple groups were examined by one-way ANOVA followed byTukey–Kramer tests. Categorical variables were compared usingc2 tests or Fisher's exact tests. Overall survival (OS) and recur-rence-free survival (RFS) were estimated using the Kaplan–Meiermethod, and survival curves were compared using log-rank tests.Multivariable analysis was performed using the Cox proportional

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hazards model to determine the prognostic value of FBP1 expres-sion. Differences or correlations with P values of less than 0.05were considered significant.Data analyseswereperformedusingRsoftware version 3.1.1 (The R Foundation).

ResultsFBP1 repressed tumor progression and improved alteredglucose metabolism in HCC cells

Initially, we performed functional analysis usingHCC cell linesto elucidate whether FBP1 suppressed tumor progression andaffected glucose metabolism in HCC. By estimating the FBP1expression profile across 967 cancer cell lines from theCCLE (10),we found that none of the 27 available liver cancer cell linesshowed high expression of FBP1 (Fig. 1A). Notably, 35% of livercancer cell lines from the CCLE database harbored FBP1 copy-number loss, with markedly reduced expression of FBP1 (Fig. 1B;Supplementary Table S1). Copy-number loss, promoter methyl-ation, or both contributed to downregulation of FBP1 expression(Supplementary Table S2). Therefore, because FBP1 expressionwas low in liver cancer cell lines, we used overexpression experi-ments rather than knockdown experiments. We established HCCcell lines (HuH7 and HepG2) stably expressing FBP1 or emptyvector control. Overexpression of FBP1 was confirmed by RT-PCRand Western blotting (Fig. 1C). Consistent with previous reportsin ccRCC cells (6) and breast cancer cells (7), FBP1 inhibitedanchorage-independent tumor cell growth in vitro (Fig. 1D) andxenograft tumor growth (Fig. 1E) in HCC cells.

Because the FBP1 gene encodes a rate-limiting gluconeogenicenzyme, we further investigated the effects of FBP1 on glucosemetabolism in HCC cells. Intracellular glucose uptake and lactatesecretionwere significantly decreased in FBP1-overexpressingHCCcells as compared with those in vector control cells (Fig. 2A and B).We also examined glucose-induced TXNIP expression, which iscommonly used as an intracellular glucose sensor (7, 17–19).TXNIP was robustly induced in vector control cells followingglucose stimulation. In contrast, the induction of TXNIP wassuppressed in FBP1-overexpressingHCCcells (Fig. 2C), supportingthe results showing that FBP1 inhibited intracellular glucoseuptake in HCC cells. Interestingly, the expression levels of hexoki-nase2 (HK2; refs. 20, 21) and phosphofructokinase-1 (PFK1, alsoknownasPFKM; refs. 22, 23), key regulators of aerobic glycolysis incancer cells, were significantly decreased in FBP1-overexpressingHCC cells as compared with those in vector control cells afterglucose stimulation (Fig. 2D). To further estimate the effect of FBP1on aerobic glycolysis, we performed isotopomer distribution anal-ysis using [U-13C]glucose, which directly produces glycolytic inter-mediates containing six or three 13C atoms (M6/M3 species), andthe first turn of the tricarboxylic acid (TCA) cycle intermediatescontaining two 13C atoms (M2 species; Supplementary Fig. S1A).M3 enrichment of lactate was significantly inhibited whenFBP1wasoverexpressed (Fig. 2E), andglycolytic intermediates suchas fructose-1,6-biphosphate (F-1,6-BP; M6 species; Fig. 2F), dihy-droxyacetone phosphate (M3 species; Supplementary Fig. S1B),and glucose-6-phosphate (Supplementary Fig. S1C) weredecreased in FBP1-overexpressing cells, supporting the view thatFBP1 suppressed glucose uptake via inhibition of aerobic glycol-ysis. Similar to ccRCC cells (6), ectopic FBP1 also tended to inhibitM2 enrichment of TCA cycle intermediates such as malate andcitrate (Fig. 2G; Supplementary Fig. S1D and S1E). Furthermore,isotopomer distribution analysis showed that M5 enrichment of

adenosine triphosphate (ATP) and adenosine diphosphate weredecreased in FBP1-overexpressing cells (Supplementary Fig. S1Fand S1G), suggesting that FBP1 suppressed de novo nucleic syn-thesis through the pentose phosphate pathway (PPP) inHCC cells(five 13C atoms were preserved through the PPP). Total ATPsynthesis was also decreased when FBP1 was overexpressed (Sup-plementary Fig. S1H), consistent with the results of reducedaerobic glycolysis and TCA cycle in FBP1-overexpressing cells.Overall, the results of functional analysis suggested that FBP1 actedas a tumor suppressor through inhibition of glucose uptake andaerobic glycolysis, which encouraged us to further explore thesignificance of FBP1 in HCC.

Lower FBP1 expression promoted tumor progression in HCCcases

Next, to clarify the clinical significance of FBP1 expression inHCC, we analyzed FBP1 expression in tumor tissues andnormal liver tissues from three independent cohorts of HCCcases (Fig. 3A). For the analysis of patients who underwentresection of primary HCC at our institution (Kyushu HCC set),FBP1 expression in tumors was significantly lower than that innormal liver tissues (P < 0.001). Given the observation thatFBP1 was expressed at low levels in both tumor tissues and livercancer cell lines, these data implied that downregulation ofFBP1 expression was essential for HCC tumorigenesis. Further-more, decreased FBP1 expression was significantly associatedwith advanced tumor stage (stage I vs. stage II, P¼ 0.017; stage Ivs. stage III, P ¼ 0.005). The median expression levels of FBP1in each tumor stage (stage I, stage II, and stage III) relative tothose in normal liver tissues were 0.427, 0.179, and 0.059,respectively. To confirm the results of our series, we analyzedthe public datasets of HCC cases from the GSE14520 and theTCGA datasets. In accordance with our cases, FBP1 expressionin tumors was significantly lower than that in normal livertissues (P < 0.001 and P < 0.001, respectively). Moreover, thesignificant association between lower FBP1 expression andadvanced tumor stage was validated in the GSE14520 (stageI vs. stage III, P < 0.001; stage II vs. stage III, P < 0.009) and theTCGA (stage I vs. stage II, P < 0.001; stage I vs. stage III, P <0.001) datasets. The median expression levels of FBP1 in eachtumor stage (stage I, stage II, and stage III) relative to those innormal liver tissues were 0.262, 0.202, and 0.090, respectively,in the GSE14520 dataset and 0.280, 0.143, and 0.153, respec-tively, in the TCGA dataset. These results suggested that the lossof FBP1 expression was associated with tumor progression inHCC cases.

Promoter methylation and copy-number loss inhibited FBP1expression in HCC cases

To investigate the mechanisms suppressing FBP1 expression inHCC cases, promoter methylation and copy-number profiles ofFBP1were analyzed in the TCGA dataset (Fig. 3B). We found thatthe FBP1 promoter exhibited significantly higher methylation intumors than in normal liver tissues (P < 0.001; Fig. 3C). Inaddition, FBP1 expressionwas inversely correlatedwith thedegreeof FBP1methylation in tumors (Pearson correlation coefficient¼�0.437; P < 0.001; Fig. 3D), indicating that FBP1 promotermethylation repressed the transcriptional activity of FBP1 inHCC.Similarly, copy-number loss of FBP1 was also significantly asso-ciatedwith lower FBP1 expression (themedian expression level inHCCs harboring copy-number loss relative to that in HCCs

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harboring copy-number neutral/gain was 0.519; Fig. 3E). Foran integrated assessment of the influence of both promotermethylation and copy-number alterations on FBP1 expressionlevels, multiple linear regression analysis was performed. Thestandardized effect sizes (t values) for promoter methylationand copy-number alterations were �7.55 (P < 0.001) and 3.48

(P < 0.001; Fig. 3B), respectively. These findings suggested thatpromotermethylation and copy-number losswere independentlyassociated with downregulation of FBP1 expression. We did notfind any correlations between FBP1 mutations and FBP1 expres-sion because only two nonsynonymousmutations were observedin the TCGA dataset (Supplementary Fig. S2).

Figure 1.FBP1 was expressed at low levels andrepressed tumor progression in HCCcells. A, FBP1 expression profiles across967 cell lines from theCCLE. Labeled celllines used for overexpressionexperiments in the present and previousstudies are highlighted in black. Labeledcell lines used for FBP1-knockdownexperiments in a previous study arehighlighted in gray. The horizontaldotted-line represents the z-scorethreshold. Note that there were no livercancer cell lines expressing high levels ofFBP1. The numbers in parentheses nextto the labeled cell lines represent thereference numbers. B, box plots of FBP1expression based on FBP1 copy-numberalterations in liver cancer cell lines fromCCLE. Data on copy-number profileswere available for 23 of 27 liver cancercell lines. C, overexpression of FBP1 wasverified by RT-PCR and Westernblotting analysis. D, the number ofspheres was significantly decreasedwhen FBP1 was overexpressed. E,xenograft tumor growth of HuH7cells with or without ectopic FBP1expression. Values represent the means� SDs of triplicate experiments. RCC,renal cell carcinoma; EV, empty vectorcontrol cells.

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Figure 2.FBP1 improved altered glucose metabolism in HCC cells. A, intracellular glucose uptake was evaluated by 2-NBDG, a fluorescently tagged glucose derivative,in HCC cells with or without ectopic FBP1 expression. B, lactate secretion was significantly decreased when FBP1 was overexpressed. C, the expression ofglucose-induced TXNIP was used to sense intracellular glucose uptake. Cells were incubated in glucose-free medium for 12 hours, followed by glucose stimulationfor an additional 3 hours. D, the expression levels of genes encoding enzymes involved in aerobic glycolysis, that is, HK2, PFK1, and PKM2, were assessed byRT-PCR after glucose stimulation, as described in C. E–G, isotopomer distribution analysis in vector control or FBP1-overexpressing HuH7 cells labeled with[U-13C]glucose for 4 hours. M3 enrichment of lactate (E), M6 enrichment of 1,6-diphosphate (F-1,6-BP; F), and M2 enrichment of malate and citrate (G) werequantified. a, in the analysis of F-1,6-BP, the metabolite concentrations of one experiment in the vector control and all three experiments in FBP1-expressingcells were below the detection limit. P values were calculated using Welch's t test. Values represent the means � SDs of triplicate experiments. EV, emptyvector control cells; ND, not detectable.






Figure 3.Downregulation of FBP1 expression via promoter methylation and copy-number loss was associated with tumor progression in HCC cases. A, box plots ofFBP1 expression in normal liver and tumor tissues grouped into stages I to III in three independent cohorts of HCC cases. Information on tumor stage wasavailable for 114 of 118 patients from the Kyushu HCC set (left), 225 of 242 cases from GSE14520 (middle), and 214 of 234 cases from TCGA (right). Five patients withstage IV cancer were excluded from the analysis of TCGA data. B, an integrative view of expression, promoter methylation, and copy-number profiles ofFBP1 across 234 HCC cases in TCGA. The samples are sorted according to FBP1 expression levels. C, box plots of FBP1methylation status in normal liver and tumortissues from TCGA (data were available for 46/48 normal tissues), D FBP1 expression levels in tumor samples were inversely correlated with the degreeof FBP1methylation in TCGA. E, box plots of FBP1 expression based on FBP1 copy-number alterations in TCGA (data were available for 229/234 cases). F, box plotsof FBP1 expression based on tumor size in GSE14520 (data were available for 241/242 cases). Information on tumor size was not available in TCGA. G, boxplots of FBP1 expression based on tumor differentiation in TCGA (data were available for 230/234 cases, and one anaplastic HCC was excluded). Informationon tumor differentiation was not available in GSE14520. NS, not significant.

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FBP1 expression levels predicted survival in patients with HCCTo further investigate the clinical significance of FBP1 expres-

sion, we assessed survival rates in three independent datasets ofHCC. On the basis of the FBP1 expression level, cases weredivided into two groups using the minimum P value approach,which is a comprehensive method to identify the optimal riskseparation cutoff point in continuous gene-expression mea-surements for survival analysis in multiple datasets (24). Thecutoff values for high and low FBP1 expression groups were 0.6(FBP1/GAPDH expression) in the Kyushu HCC set, 6.8 (log2expression) in the GSE14520 dataset, and 13.0 (log2 expres-sion) in the TCGA dataset. For the analysis of 118 patients inthe Kyushu HCC set, clinicopathologic factors were comparedbetween the groups (Table 1). Notably, the low FBP1 expres-sion group exhibited larger tumor size (P ¼ 0.010) and poorerdifferentiation grade (P < 0.001) than the high FBP1 expressiongroup. These findings were validated with the available publicdata. Lower FBP1 expression was significantly associated with

larger tumor size (GSE15420: � 5 cm vs. > 5 cm; P < 0.001; Fig.3F) and poorly differentiated HCC (TCGA: poorly vs. well; P ¼0.003; Fig. 3G), suggesting that highly malignant HCCexpressed low levels of FBP1. For the analysis of OS (Fig.4A), the low FBP1 expression group had significantly poorerOS than the high FBP1 expression group (P ¼ 0.005). Consis-tent with our cohort, lower FBP1 expression was significantlyassociated with poorer OS in the GSE14520 (P < 0.001) andTCGA (P ¼ 0.010) datasets. In the multivariate analysis thatexcluded tumor stage as a confounding variable, the associationbetween lower FBP1 expression and poorer OS remained sig-nificant in the Kyushu HCC [HR, 2.49; 95% confidence interval(CI), 1.22–5.06; P ¼ 0.012) and TCGA (HR, 2.84; 95% CI,1.22–6.64; P ¼ 0.016) datasets (Table 2).

To validate the effects of FBP1 expression on survival in patientswithHCC,weapplied gene set enrichment analysis (GSEA; ref. 25)on the GSE14520 and TCGA datasets using two gene sets accord-ing to OS following resection of HCC (26). GSEA revealed thatupregulation of FBP1 was significantly correlated with bettersurvival in the GSEA14520 (P < 0.001) and TCGA (P < 0.001)datasets (Fig. 4B). Conversely, downregulation of FBP1 wassignificantly correlated with worse survival in the GSEA14520(P < 0.001) and TCGA (P < 0.001) dataset (Fig. 4C). Collectively,survival analysis with Kaplan–Meier curves and GSEA demon-strated that lower FBP1 expression was significantly associatedwith poor survival in patients with HCC.

Higher FBP1 expression was associated with a lower risk ofrecurrence in HCC

Subsequently, we assessed the effects of FBP1 expression onrecurrence following resection of primary HCC (Fig. 4D). In ouranalysis of the Kyushu HCC set, RFS in the high FBP1 expressiongroupwas significantly better than that in the low FBP1 expressiongroup (P¼ 0.016). Consistent with the results of our series, higherFBP1 expression was also significantly associated with better RFSin the available public data (GSE14520; P < 0.001). The associ-ation between lower FBP1 expression and poorer RFS remainedsignificant after excluding tumor stage as a confounding variablein the GSE14520 dataset (HR, 1.49; 95% CI, 1.01–2.20; P ¼0.045; Table 2).

We then performed GSEA to determine whether the gene setsassociated with HCC recurrence were differentially regulateddependingon the expression levels ofFBP1 inHCC.GSEA showedthat higher FBP1 expression was significantly correlated with alower risk of early recurrence (27) in the GSE14520 and TCGAdatasets (P < 0.001 and P < 0.001, respectively; Fig. 4E), andwith that of late recurrence (28) in the GSE14520 and TCGAdatasets (P < 0.001 and P < 0.001, respectively; Fig. 4F). Theseresults demonstrated that higher FBP1 expression was associatedwith a lower risk of recurrence.

FBP1 expression levels reflected altered glucosemetabolism in HCC

To further investigate the role of FBP1 in HCC progression, wefocused on glucose metabolism. First, FBP1 expression and glu-coneogenic activity were compared using genome-wide gene-expression profiles of the GSE14520 and TCGA datasets. Tocomprehensively analyze gluconeogenic activity, we defined thegluconeogenic module composed of genes specific to gluconeo-genesis (4–6). This module included genes encoding enzymesinvolved in irreversible steps of gluconeogenesis, for example,

Table 1. FBP1 expression and clinicopathologic factors in patients in the KyushuHCC set (n ¼ 118)

High expression(n ¼ 88)

Low expression(n ¼ 30)

Factors Number (%) Number (%) P

Age (y; mean � SD) 66.3 � 7.9 66.4 � 9.4 0.956Gender 0.955Male 65 (73.9) 22 (73.3)Female 23 (26.1) 8 (26.7)

HBV 0.661(þ) 20 (22.7) 8 (26.7)(�) 68 (77.3) 22 (73.3)

HCV 0.711(þ) 59 (67.0) 19 (63.3)(�) 29 (33.0) 11 (36.7)

Child-Pugh grade 0.684A 79 (89.8) 26 (86.7)B 8 (9.1) 4 (13.3)C 1 (1.1) 0 (0.0)

Tumor size (cm, mean � SD) 3.72 � 2.91 5.42 � 3.50 0.010b

Number of tumors 0.527Single 64 (72.7) 20 (66.7)Multiple 24 (27.3) 10 (33.3)

Tumor differentiationa <0.001b

Well/moderately 79 (89.8) 19 (63.3)Poorly/undifferentiated 6 (6.8) 10 (33.3)NA 3 (3.4) 1 (3.3)

Capsular formation 0.517(þ) 58 (65.9) 21 (70.0)(�) 30 (34.1) 8 (26.7)NA 0 (0.0) 1 (3.3)

Portal vein invasion 0.061(þ) 37 (42.0) 18 (60.0)(�) 51 (58.0) 11 (36.7)NA 0 (0.0) 1 (3.3)

Hepatic vein invasion 0.869(þ) 25 (28.4) 9 (30.0)(�) 60 (68.2) 20 (66.7)NA 3 (3.4) 1 (3.3)

Bile duct invasion 0.063(þ) 3 (3.4) 4 (13.3)(�) 85 (96.6) 25 (83.3)NA 0 (0.0) 1 (3.3)

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; NA, not available.aWHO grades 1/2 or Edmondson-Steiner grades 1/2 were classified as well/moderately; WHO grades 3/4 or Edmondson-Steiner grades 3/4 were classifiedas poorly/undifferentiated.bP < 0.05.






Figure 4.FBP1 expression levels predicted survival and recurrence in patients with HCC. A, Kaplan–Meier OS curve based on FBP1 expression in three independent datasetsof HCC. Left, Kyushu HCC set; middle, GSE14520; right, TCGA. B and C, gene set enrichment analysis of the GSE14520 and TCGA datasets showed thathigher FBP1 expression was significantly correlated with better survival in HCC (B). In contrast, lower FBP1 expression was significantly correlated with poorersurvival in HCC (C). D, Kaplan–Meier recurrence-free survival in patients with HCC based on FBP1 expression. Left, Kyushu HCC set; middle, GSE14520; right,information on recurrence statuswas not available in TCGA. E and F, gene set enrichment analysis of the GSE14520 and TCGA datasets revealed that upregulation ofFBP1 expression was significantly correlated with a lower risk of early (E) and late (F) recurrence. ES, enrichment score.

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glucose-6-phosphatase, catalytic subunit (G6PC); phosphoenol-pyruvate carboxykinase 1; phosphoenolpyruvate carboxykinase2; and pyruvate carboxylase (Fig. 5A). Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1a), a tran-scription factor regulating the expression of gluconeogenic genes(29), was also contained in this module. Consistent with aprevious report (4), all of these genes were expressed at lowerlevels in tumors than normal liver tissues supporting the obser-vation that hepatic gluconeogenesis was reduced in HCC (Sup-plementary Fig. S3A–S3E). Moreover, we found a significantpositive correlation between FBP1 expression levels and gluco-neogenic module activity in the GSE14520 (Pearson correlationcoefficient ¼ 0.719; P < 0.001) and TCGA (Pearson correlationcoefficient ¼ 0.716; P < 0.001) datasets (Fig. 5B). Our findingssuggested that hepatic gluconeogenesis was impaired in HCCsexpressing low levels of FBP1.

Subsequently, we evaluated aerobic glycolysis in HCC cases.Three enzymes are involved in catalyzing the irreversible steps ofglycolysis: hexokinase, phosphofructokinase, and pyruvate kinase(Fig. 5A). Among these three enzymes, we focused on the HK2(20, 21),PFK1 (22, 23), and pyruvate kinaseM2 (PKM2; refs. 30, 31)genes,whichhavebeen reported toplay a pivotal role inpromotingaerobic glycolysis in cancer cells; therefore, an aerobic glycolyticmodule containing these three genes was defined. Contrary withgluconeogenesis, significant inverse correlations were observedbetween FBP1 expression and aerobic glycolytic module activityin the GSE14520 (Pearson correlation coefficient ¼ �0.458;P < 0.001) and TCGA (Pearson correlation coefficient ¼ �0.536;P < 0.001) datasets (Fig. 5C), indicating that lower FBP1 expressionwas associated with high aerobic glycolysis in HCC cases.

To confirm whether FBP1 expression levels were associatedwith altered glucose metabolism in HCC, GSEA was performed.Predefined gene sets involved in normal glucosemetabolismweresignificantly enriched in HCCs expressing high levels of FBP1, as

follows: KEGG_GLYCOLYSIS_GLUCONEOGENESIS in theGES14520 (P < 0.001) and TCGA (P < 0.001) datasets (Fig.5D); and REACTOME_GLUCOSE_METABOLISM in theGES14520 (P < 0.001) and TCGA (P ¼ 0.023) datasets (Fig.5E). The results of GSEA indicated that normal glucose metabo-lism was significantly impaired in HCCs expressing low levels ofFBP1 compared with that in HCCs expressing high levels of FBP1.

Finally, we assessed the effects of the other genes involved ingluconeogenesis/aerobic glycolysis on prognosis in patientswith HCC. Lower G6PC and PGC-1a expression and higherPKM2 expression were significantly associated with poor OS inboth the GSEA14520 and TCGA datasets (Supplementary TableS3). Consistent with these observations, higher expressionlevels of genes specific to gluconeogenesis, particularly thoseinvolved in maintenance of normal glucose metabolism (ele-vated gluconeogenesis and decreased aerobic glycolysis), weresignificantly associated with better OS (Supplementary Fig.S4A–S4C), suggesting that the glucose metabolic shift affectedprognosis. Together with the results of functional analysis andsurvival analysis, our results showed that FBP1 was not onlyone of the key regulators of HCC progression but a promisingbiomarker-predicting prognosis in patients with HCC. Amonggenes involved in gluconeogenesis, downregulation of FBP1may contribute to a vicious cycle of altered glucose metabolismin HCC considering the observation that loss of FBP1 directlyenhanced tumor progression and aerobic glycolysis. Overall,we found that lower FBP1 expression was significantly associ-ated with altered glucose metabolism, which contributed totumor progression in HCC.

DiscussionWedemonstrated the function and clinical significance of FBP1

expression in HCC. Promoter methylation and copy-number loss

Table 2. Effect of FBP1 expression on OS and recurrence-free survival in the multivariate analysis that excluded tumor stage as a confounding variable

Univariate analysis Multivariate analysisFactor HR 95% CI P HR 95% CI P

OSKyushu HCC setFBP1 expression (low/high) 2.47 (1.28–4.77) 0.007b 2.49 (1.22–5.06) 0.012b

TNM stage (II/I) 0.97 (0.45–2.10) 0.939 0.96 (0.44–2.07) 0.910TNM stage (III/I) 2.12 (0.75–5.95) 0.156 1.37 (0.45–4.07) 0.577

GSE14520FBP1 expression (low/high) 2.08 (1.37–3.14) <0.001b 1.45 (0.91–2.29) 0.116TNM stage (II/I) 2.09 (1.20–3.64) 0.009b 1.99 (1.14–3.47) 0.016a

TNM stage (III/I) 5.30 (3.05–9.20) <0.001b 4.64 (2.60–8.29) <0.001b

TCGAFBP1 expression (low/high) 2.68 (1.23–5.85) 0.013b 2.84 (1.22–6.64) 0.016b

TNM stage (II/I) 0.64 (0.31–1.29) 0.210 0.55 (0.27–1.13) 0.102TNM stage (III/I) 1.44 (0.83–2.50) 0.196 1.35 (0.77–2.34) 0.292

Recurrence-free survivala

Kyushu HCC setFBP1 expression (low/high) 1.79 (1.11–2.88) 0.017b 1.48 (0.87–2.51) 0.150TNM stage (II/I) 1.67 (0.94–2.96) 0.081 1.657 (0.93–2.94) 0.084TNM stage (III/I) 3.27 (1.49–7.18) 0.003b 2.74 (1.20–6.25) 0.017b

GSE14520FBP1 expression (low/high) 1.90 (1.34–2.70) <0.001b 1.49 (1.01–2.20) 0.045b

TNM stage (II/I) 1.96 (1.29–2.99) <0.001b 1.86 (1.22–2.84) 0.004b

TNM stage (III/I) 3.08 (1.95–4.86) <0.001b 2.62 (1.61–4.26) <0.001b

Abbreviation: CI, confidential interval.aInformation on recurrence status was not available in the TCGA dataset.bP < 0.05.






were responsible for the decreased expression of FBP1, whichcontributed to tumor progression, poor prognosis, and alteredglucose metabolism. Our data indicated that FBP1 acted as atumor suppressor by restoring altered glucose metabolism, andimplied that FBP1 was a more central regulator of the glucosemetabolic pathway in HCC.

In the present study, we validated the prognostic impact ofFBP1 expression using three independent large cohorts, and theresults were reproducible. For eitherOS or RFS in each cohort, thisprognostic impact persisted even after adjusting for tumor stage.Moreover, our data indicated that decreased expression of FBP1promoted tumor progression, thereby affecting the clinical

Figure 5.FBP1 expression levels reflected altered glucose metabolism in HCC cases. A, illustration of the glucose metabolic pathway. Irreversible gluconeogenicenzyme genes, that is, G6PC; phosphoenolpyruvate carboxykinase 1 (PEPCK1); phosphoenolpyruvate carboxykinase 2 (PEPCK2); pyruvate carboxylase (PC);and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1a), a transcription factor regulating G6PC and PEPCK1, are highlightedin red. Enzymes catalyzing three irreversible steps of glycolysis and isoforms involved in aerobic glycolysis, that is, HK2, PFK1, and PKM2, are highlightedin blue. B, correlations between FBP1 expression level and gluconeogenic activity in the GSE14520 and TCGA datasets are shown as heat maps. Themodule activity of gluconeogenesis was calculated for each case using the average of the normalized expression of genes specific to gluconeogenesis.C, correlations between FBP1 expression levels and the activity of aerobic glycolysis in the GSE14520 and TCGA datasets are shown as heat maps. Themodule activity of aerobic glycolysis was calculated for each case using the average of the normalized expression of HK2, PFK1, and PKM2. D and E,gene set enrichment analysis demonstrated that two predefined gene sets involved in normal glucose metabolism were significantly enriched in HCC casesexhibiting high FBP1 expression, suggesting that normal glucose metabolism was preserved in these cases. ES, enrichment score.


Hirata et al.




outcomes and clinicopathologic characteristics of patients withHCC. Therefore, FBP1 expression could be a dependable bio-marker for predicting prognosis and provide additional prognos-tic information beyond tumor stage in patients with HCC.

The proposed mechanism underlying the tumor-suppressiveproperties of FBP1 involves the inhibition of aerobic glycolysis(6, 7). Our results were consistent with this proposal and sug-gested that the reduced transcriptional activity of HK2 and PFK1induced by ectopic FBP1 played a role in the decreased glucose-6-phosphate and F-1,6-BP levels. One of the limitations of thisstudy was that glucose metabolism was mainly examined bytranscriptional profiling of genes specific to gluconeogenesis orglycolysis in clinical cases. Enzyme activity can also be regulatedby protein structure and expression. Indeed, FBP1 is involved inthe posttranslational modification of PKM2 in the glycolyticpathway (30, 31). The loss of FBP1 directly increases the level ofF-1,6-BP, which enhances PKM2 activity through dynamic struc-tural switching from the less active dimer to the active tetramer.Furthermore, the effect of FBP1 knockdown on altered glucosemetabolismwas not evaluated because FBP1was expressed at lowlevels in all available liver cancer cell lines from the CCLE. Despitethese limitations, our findings provide a reasonable explanationfor the effects of FBP1 expression on altered glucose metabolismin HCC. In addition to the enzymatic activity of FBP1 in glucosemetabolism, Li and colleagues (6) reported that the direct bindingof FBP1 to hypoxia-inducible factor (HIF) inhibits ccRCC pro-gression by reducingHIF activity. Our data showed that the effectsof FBP1 on malate, citrate, and other metabolites were not asrobust as that seen in ccRCC cells, likely due to the fact that ccRCCcells have constitutive HIF activity and HCC cells do not innormoxic conditions. Considering these diverse functions ofFBP1, further studies are needed to clarify the detailed mechan-isms through which FBP1 suppresses cancer progression in HCC.

In HCC, FBP1 promoter methylation has been reported only incell lines and a few clinical cases (8). However, in our largeranalysis in this study, we found that copy-number loss andpromoter methylation could explain the decreased expression ofFBP1. FBP1 promoter methylation downregulated FBP1 expres-sion in patients with basal-like breast cancer (7), gastric cancer(32), and small intestinal neuroendocrine tumor (33). Copy-number loss of FBP1was also observed in ccRCC cases (34). Thus,our data indicated that genomic alterations as well as epigeneticmodification were responsible for inhibiting FBP1 expression inHCC.

In summary, we demonstrated the clinical significance of FBP1expression in HCC using three independent clinical datasets.Downregulation of FBP1 via promoter methylation and copy-number loss promoted tumor progression by altering glucosemetabolism. FBP1 could be a useful biomarker for predicting theprognosis of patients with HCC and may represent a noveltherapeutic target for the treatment of HCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Hirata, K. Sugimachi, K. MimoriDevelopment of methodology: H. Hirata, H. Komatsu, M. Ueda, T. Masuda,R. UchiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H.Hirata, K. Sugimachi, H. Komatsu, H. Eguchi, S. ItoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Hirata, K. Sugimachi, H. Komatsu, M. Ueda,T. Masuda, R. Uchi, S. Sakimura, S. Nambara, T. Saito, M. HirakawaWriting, review, and/or revision of the manuscript: H. Hirata, K. Sugimachi,T. Iguchi, K. MimoriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Terashima, K. SakamotoStudy supervision: Y. Shinden, H. Honda, K. Mimori

AcknowledgmentsThis research used the supercomputing resource provided by the Human

Genome Center, Institute ofMedical Science, the University of Tokyo (http://sc.hgc.jp/shirokane.html). The authors thank Dr. A. Niida for providing bioin-formatics tools, K. Oda, M. Kasagi, T. Kawano, and J. Takano for technicalassistance, Hiroshima Red Cross Hospital, Iizuka Hospital, and Oita Red CrossHospital for providing clinical samples. The authors also appreciate the tech-nical assistance from The Research Support Center, Research Center for HumanDisease Modeling, Kyushu University.

Grant SupportThis work was supported in part by the Japan Society for the Promotion of

Science Grant-in-Aid for Scientific Research (grant numbers 24592005,26861003, 15K10168, and 15H04921).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 23, 2015; revised March 28, 2016; accepted March 31,2016; published OnlineFirst April 6, 2016.

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2016;76:3265-3276. Published OnlineFirst April 6, 2016.Cancer Res Hidenari Hirata, Keishi Sugimachi, Hisateru Komatsu, et al. Carcinomawith Glucose Metabolism and Tumor Progression in Hepatocellular Decreased Expression of Fructose-1,6-bisphosphatase Associates

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