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Tumor and Stem Cell Biology

Lysine Demethylase LSD1 Coordinates Glycolyticand Mitochondrial Metabolism in HepatocellularCarcinoma CellsAkihisa Sakamoto1,2, Shinjiro Hino1, Katsuya Nagaoka1, Kotaro Anan1, Ryuta Takase1,Haruka Matsumori1, Hidenori Ojima3,Yae Kanai3, Kazunori Arita2, and Mitsuyoshi Nakao1,4

Abstract

The hallmark of most cancer cells is the metabolic shift frommitochondrial to glycolytic metabolism for adapting to thesurrounding environment. Although epigenetic modification isintimately linked to cancer, the molecular mechanism, by whichepigenetic factors regulate cancer metabolism, is poorly under-stood. Here, we show that lysine-specific demethylase-1 (LSD1,KDM1A) has an essential role in maintaining the metabolic shiftin human hepatocellular carcinoma cells. Inhibition of LSD1reduced glucose uptake and glycolytic activity, with a concurrentactivation of mitochondrial respiration. These metabolic changes

coexisted with the inactivation of the hypoxia-inducible factorHIF1a, resulting in a decreased expression of GLUT1 and glyco-lytic enzymes. In contrast, during LSD1 inhibition, a set ofmitochondrial metabolism genes was activated with the concom-itant increase ofmethylated histoneH3at lysine 4 in the promoterregions. Consistently, both LSD1 and GLUT1 were significantlyoverexpressed in carcinoma tissues. These findings demonstratethe epigenetic plasticity of cancer cellmetabolism, which involvesan LSD1-mediated mechanism. Cancer Res; 75(7); 1445–56. �2015AACR.

IntroductionPatterns of gene expression are maintained and often repro-

grammed by the combination of transcription factors and epige-netic factors involved in modifications of DNAs and histones,leading to conversion of cellular phenotypes. In particular, altera-tions of these epigenetic marks are hallmarks of many types ofcancer cells (1–3). As another notable feature, proliferative cancercells are thought to rely on energy production from the glycolyticpathway, even under high oxygen conditions: the so-called aer-obic glycolysis (4–6). This metabolic remodeling may be inter-preted as an adaptation to the hypoxic microenvironment wherethe cancer cells originally reside (7). Recent reports havehighlight-ed that metabolic enzymes involved in such process are manip-ulated in cancer cells (8).Mitochondrial function is alsomodifiedsuch that it supports the production of biomacromolecules and

reactive oxygen species rather than ATP (9). These lines of evi-dence support the notion that metabolic alteration in cancer cellsis not merely a consequence of impaired cellular functions bytransformation, but rather an ordered reprogramming of energyflow that fuels the accelerated cell growth. Epigenetic plasticity hasbeen discussed as an underlying mechanism for metabolic repro-gramming (10). In addition, recent studies on isocitrate dehy-drogenase mutations in various cancers defined that misguidedmetabolic flow leads to the impaired activities of epigeneticfactors (11). Thus metabolism–epigenome crosstalk may pro-foundly contribute to the dysregulated gene expression in cancer(12, 13). However, little is known about how specific epigeneticfactors control cancer cell metabolism.

Lysine-specific demethylase-1 (LSD1, also known as KDM1A)is aflavin-dependent amine oxidase, which, in general, suppressesgene expression by removing the methyl group from mono- anddimethylated histone H3 at lysine 4 (H3K4; ref. 14). LSD1knockout mice die early in development (15), and LSD1-nullembryonic stem cells showed impaired viability (16), suggestingthat LSD1 plays a crucial role in cell functions. Several studiesshowed that overexpression of LSD1 drives cell proliferation invarious cancers (17–20). We have recently found that LSD1suppresses mitochondrial respiration and maintains energy stor-age in murine adipocytes under the obese condition (21). There-fore, it is fascinating to test whether LSD1 facilitates themetabolicreprogramming in cancer cells.

Materials and MethodsCell culture

HepG2 and Huh-7 cells from human hepatocellular carcino-mas (HCC) were grown in DMEM (Sigma), supplemented with10% (v/v) heat-inactivated FBS. Human telomerase-immortal-ized hepatic NeHepLxHT cells were cultured in DMEM/Nutrient

1Department of Medical Cell Biology, Institute of Molecular Embryo-logy and Genetics, Kumamoto University, Kumamoto, Japan 2Depart-ment of Neurosurgery, Faculty of Medicine, Graduate School of Med-ical and Dental Sciences, Kagoshima University, Kagoshima, Japan3Division of Molecular Pathology, National Cancer Center ResearchInstitute, Tokyo, Japan 4Core Research for Evolutional Science andTechnology (CREST), Japan Science and Technology Agency, Tokyo,Japan.

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

CorrespondingAuthors:Mitsuyoshi Nakao, Department ofMedical Cell Biology,Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1Honjo, Chuo-ku, Kumamoto, 860-0811, Japan. Phone: 819-6373-6800; Fax: 819-6373-6804; E-mail: [email protected]; and Shinjiro Hino, E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1560

�2015 American Association for Cancer Research.

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Mixture F-12 Ham (Sigma), supplemented with 10% (v/v) heat-inactivated FBS, dexamethasone, insulin, and G-418. MDA-MB-231 cells from breast cancer were cultured in RPMI1640 (Sigma),supplemented with 10% (v/v) heat-inactivated FBS. All cell lineswere purchased from ATCC or JCRB Cell Bank, and were authen-ticated by the providers by short-tandem repeat analysis orisoenzyme analysis. All cell lines were thawed within a short termafter receipt, and were used at early passages. We did not observeany morphologic changes or altered growth rates during ourmaintenance culture. For the knockdown experiments, specificsiRNAs were introduced into the cells using RNAiMAX reagent(Invitrogen) when they were approximately 50% confluent. Mostexperiments were done at 96 hours after siRNA introduction, andwere carried out at 72 hours after tranylcypromine treatment,unless otherwise described. For hypoxic conditions, cells wereincubated with 200 mmol/L cobalt chloride (CoCl2) or 1 mmol/LDMOGfor 12hours. Informationon the siRNAsused in this studyis listed in Supplementary Table S1. For cell-cycle analyses, cellswere fixedwith ethanol, stained with propidium iodide, andweresubjected to FACS analysis.

Tumor xenograft experimentsAnimal experiments were conducted in accordance with the

guidelines of the Animal Care and Use Committee of KumamotoUniversity (Kumamoto, Japan). Control siRNAor LSD1 siRNA-1–treated HepG2 cells were dissociated, and resuspended in Matri-gel/HBSS (BD Biosciences) mixture. Twenty-four hours aftersiRNA introduction, 5 � 106 control or LSD1-KD cells wereinjected into right and left flanks, respectively, of 6-week-oldSCID mice (FOX CHASE SCID C.B-17/lcr-scid/scidJcl). Threeweeks after transplantation, tumors were harvested, and collectedtumors were weighed and dissected for RNA analysis.

Patients and histologic assessmentThirty-eight patients with HCC, who had undergone tumor

resection at the National Cancer Center Hospital (Tokyo, Japan),between May 2003 and December 2005, were enrolled in thisstudy. As described previously (22), histologic classification wasassessed according to theWorldHealthOrganizationHistologicalClassification of Tumors. The Ethics Committee of the NationalCancerCenter approved this study,withwritten informed consentfrom all patients.

ImmunohistochemistryImmunohistochemistry (IHC) for GLUT1 and LSD1 was per-

formed using a polymer-basedmethodwith EnvisionþDual LinkSystem-horseradish peroxidase (DK-2600 Glostrup; Dako).Sources and dilutions of primary antibodies were as follows:anti-GLUT1 (rabbit polyclonal; ab15309, Abcam; 1:200) andanti-LSD1 (rabbit polyclonal; ab17721, Abcam; 1:200). Forma-lin-fixed, paraffin-embedded serial tissue sections (4 mm) wereplaced on silane-coated slides for IHC. Antigen retrieval wascarried out by heating in a target retrieval solution (Tris/EDTAbuffer, pH 9; Dako Cytomation) for GLUT1 and in a 0.01 mol/Lcitrate buffer (pH 6.0) for LSD1 at 121�C for 10 minutes in apressure cooker. The other procedures used in IHCwere similar toour previous report (22).

Evaluation of immunohistochemistry and correlationsImmunoreactivities of GLUT1 were defined as follows: 0þ, no

membrane staining; 1þ, membrane staining, lower than the

intensity of the membrane in red blood cells; 2þ, membranestaining, equivalent or higher than the intensity of red blood cellsmembrane within the same section. Immunoreactivities of LSD1were defined as follows: 0þ, no nuclear staining; 1þ, nuclearstaining, equivalent to the intensity of the normal hepatocyteepithelium (NHE); 2þ, nuclear staining, higher than the intensityof the NHE within the same section. The IHC score of 1þ and 2þof GLUT1 and 2þ of LSD1were defined as positive for expressionof eachprotein. Thepercentage of positive areaofGLUT1was thendefined as the percentage of positively membrane stained area(IHC scores of 1þ and 2þ) divided by the total membrane area ofthe tumor cells. When there was a positive area, it was defined aspositive case of GLUT1. The positive index of LSD1 was assessedby counting the positive cells in randomly selected three 40�high-power fields (more than 300 tumor cells). Finally, immu-noreactivities of GLUT1 were positive in 13 (34.2%) of the 38cases. Correlations between positive case of GLUT1 and positiveindex of LSD1 were evaluated by the Student t test.

Statistical analysesData are presented as means � SD. Statistical analyses were

performed by the two-tailed Student t test.

ResultsLSD1 depletion compromises glycolysis-shifted metabolism incancer cells

To determine the involvement of LSD1 inmetabolic pathways,livemonitoringusing an extracellularflux analyzerwas performedin humanHepG2 cells, which are well-established hepatocellularcarcinoma (HCC) cells. In this study, LSD1 knockdown(KD) wasachieved using two different siRNAs, which conferred limitedinfluence on the cell cycle and growth under the current exper-imental setting (Supplementary Fig. S1A–S1C). Compared withcontrol cells, the oxygen consumption rate (OCR) increased inLSD1-KD cells (Fig. 1A, left), while the loss of LSD1 decreased theextracellular acidification rate (ECAR), an index of glycolyticactivity (Fig. 1A, middle). The increased OCR/ECAR ratio sug-gested that LSD1 depletion converted energy production fromglycolysis tomitochondrial respiration in the cells (Fig. 1A, right).Real-time monitoring of glycolytic activities was then carried outunder glucose starvation and subsequent readdition (Fig. 1B). Theglucose-dependent increase in the ECARwas significantly blockedby LSD1 depletion (Fig. 1B, left). Glucose exposure induced arapid shift frommitochondrial respiration to glycolysis in controlcells, whereas this metabolic shift was attenuated in LSD1-KDcells (Fig. 1B, right and Supplementary Fig. S1D).

To further examine glucose utilization under the LSD1-depleted conditions, glucose uptake was measured using afluorescent glucose analog, 2-[N-(7-Nitrobenz-2-Oxa-1,3-Dia-zol-4-yl)Amino]-2-Deoxy-D-Glucose (2-NBDG). Flow cyto-metric analysis revealed a significant decrease of 2-NBDGuptake in the LSD1-KD cells (Fig. 1C and Supplementary Fig.S1E). On the other hand, fluorescent staining of mitochondriawith JC-1 dye revealed that LSD1 inhibition, either by siRNAsor by the enzymatic inhibitor tranylcypromine, significantlyincreased both mitochondrial mass (FL1) and membranepotential (FL2; Fig. 1D and Supplementary Fig. S1F), whichcorresponded with the active oxygen consumption in the LSD1-KD cells. Similar results were obtained in another human HCCcell line, Huh-7 (Supplementary Fig. S4A and S4B).

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The metabolic landscapes in HepG2 cells were then character-ized, using a capillary electrophoresis time-of-flight mass spec-trometry (CE-TOFMS)-based metabolomics analysis (Fig. 1E andSupplementary Fig. S2; Supplementary Table S2). The loss of LSD1significantly reduced the products fromtheearly stepsof glycolysis,such as glucose-6-phosphate and fructose-6-phosphate, togetherwith a relative decrease in lactate levels. Products of pentosephosphate pathway branching from glycolysis were also dramat-ically reduced, which is consistent with the limited glucose utili-zation under LSD1-KD. On the other hand, acetyl-CoA was ele-vated to a detectable level, which might have contributed to theenhanced mitochondrial respiration by LSD1-KD. Collectively,LSD1 inhibition activates mitochondrial respiration and repressesglycolysis, suggesting that LSD1 controls energy flow inHCC cells.

LSD1 depletion selectively reverses the gene expressioncharacteristic of glycolytic shift

Weperformed expressionmicroarray analyses to clarify the roleof LSD1 in metabolic gene regulation. Gene set enrichmentanalysis revealed that the gene set of "glycolysis and gluconeo-genesis" was ranked as significantly affected by LSD1 depletion(Fig. 2A and Supplementary Fig. S3). The data were validatedusing quantitative RT-PCR in HepG2 cells (Fig. 2B and C) andHuh-7 cells (Supplementary Fig. S4C and S4D). Importantly,among this gene set, glycolytic genes were downregulated byLSD1-KD (Fig. 2B), whereas genes encoding the rate-limitingenzymes of gluconeogenesis were upregulated (Fig. 2C). In addi-tion, genes involved in mitochondrial fatty acid b-oxidationwere also induced in the LSD1-KD cells (Fig. 2A and D and

Figure 1.LSD1 depletion converts glycolytic metabolism to mitochondrial metabolism in cancer cells. A, effect of LSD1-KD on energy metabolism in HepG2 cells. Using anextracellular flux analyzer, mitochondrial respiration and glycolytic activities were determined by measuring OCR and ECAR, respectively. Values are means � SDof four assays. �� , P < 0.01 versus control siRNA. B, real-time monitoring of glycolytic activity under glucose starvation and readdition (25 mmol/L). ECARandOCR/ECARweremeasuredup to approximately 44minutes after glucose readdition. Values are normalized to the values just before glucose addition. C, effect ofLSD1-KD on glucose uptake in HepG2 cells. 2-NBDG incorporation was determined by flow cytometry. The representative histograms of triplicate samples(left) and mean fluorescence intensities (right) are shown. D, effect of LSD1-KD onmitochondrial mass and membrane potential. JC-1 incorporation was determinedby flow cytometry. Green histograms indicate the unstained control. Means � SD of triplicate samples (bottom panels). E, summary of metabolome andtranscriptome data in the glycolytic pathway and mitochondrial fatty acid b-oxidation in HepG2 cells. Histograms indicate cellular metabolite concentrationsdeterminedbyCE-TOFMS in control and LSD1-KD cells (black and redbars, respectively, in box). Genes upregulated anddownregulated by LSD1-KD are shown in redand blue, respectively. Full descriptions of the gene symbols are provided in Supplementary Table S3. The values are means � SD of three samples. � , P < 0.05;�� , P < 0.01 versus control siRNA. ND, not detected.

LSD1 Directs Metabolic Shift in HCC

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Supplementary Fig. S4C). In Fig. 1E, these gene expressionprofiles are shown with the metabolomics data [downregulated(blue) and upregulated genes (red) by LSD1-KD]. To furthervalidate the importance of LSD1 in glycolytic regulation, weproduced HepG2 cells that overexpress LSD1, and observed asignificant increase of glycolytic gene expression (Supplemen-tary Fig. S6). Thus, LSD1 regulates metabolic genes that linkglucose uptake to the glycolytic pathway in HCC cells. We alsotested a breast cancer cell line, MDA-MB-231, and observed thatthe important glycolytic genes were downregulated by LSD1-KD (Supplementary Fig. S7).

LSD1 supports the expression of GLUT1 gene and is associatedwith the increased expression of GLUT1 in human HCC

As glucose uptake was significantly reduced by LSD1-KD inHCC cells (Fig. 1C), we postulated that glucose transporterproteins had been decreased. To verify this, we examined themRNA expression of well-characterized glucose transporter genesGLUT1-4 (also known as SLC2A1–4) under LSD1-KD. We foundthat both the mRNA and protein levels of GLUT1 were decreasedby LSD1-KD (Fig. 3A and B and Supplementary Fig. S4D). GLUT1has been reported to be highly expressed and rate limiting forglucose transport in HCC cells (23). Consistent with this report,

glucose uptakewas strikingly impaired inGLUT1-KDHepG2 cells(Fig. 3C and Supplementary Fig. S5A and S5B).

Recent reports have shown that LSD1 is overexpressed invarious cancers (18, 20, 24–26). To test the importance of LSD1in glucose metabolism in human HCC, we conducted immuno-histochemical studies of HCC tissues from 38 patients. Represen-tative images are shown in Fig. 3D: LSD1 and GLUT1 were notdetected in case 1, while both were positively stained in case 2. Assummarized in Fig. 3E, the index of the LSD1 immunoreactivitieswas significantly high in the GLUT1 positively stained tissues(34.2% of the examined cases), indicating a close correlationbetween LSD1 and GLUT1 expression in clinical specimens. Totest whether the metabolic function of LSD1 correlates with thecancerous state, similar experiments were repeated in telomerase-immortalized NeHepLxHT cells of human hepatic origin (27).Expression of LSD1mRNAs was remarkably higher in HCC cells,compared with normal liver and NeHepLxHT cells (Supplemen-tary Fig. S8A). Interestingly, the loss of LSD1 significantlyincreased glucose uptake in NeHepLxHT cells (SupplementaryFig. S8B and S8C), with the concomitant upregulation of GLUT1and glycolytic genes (Supplementary Fig. S8D). These resultssuggest that LSD1 is uniquely involved in the glucosemetabolismof cancer cells.

Figure 2.LSD1 uniquely regulates glucose metabolism genes in cancer cells. A, gene set enrichment analysis of LSD1-regulated genes in HepG2 cells. In each panel, nominalP values and false discovery rates (FDR) are indicated. B–D, expression changes of representative glycolytic genes (B), gluconeogenic genes (C), and fattyacid b-oxidation genes (D) in LSD1-depleted cells. Two different siRNAs against LSD1 were introduced into HepG2 cells. Full descriptions of the gene symbolsare provided in Supplementary Table S3. Quantitative RT-PCR values, which were normalized to the expression levels of the 36B4 gene, are shown as thefold difference against control siRNA-treated samples.

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LSD1 directly suppresses mitochondrial metabolism genes viathe H3K4 demethylation

LSD1 was originally identified as an H3K4 demethylaseinvolved in gene repression (14), and some reports have shownits alternative role in H3K9 demethylation (17). To test theepigenetic regulation of metabolic genes by LSD1, chromatinimmunoprecipitation (ChIP) analyses were performed in HepG2cells. LSD1 inhibition induced PGC-1a, which is involved intranscriptional control of mitochondrial oxidative metabolism,with a significant enrichment of transcriptionally active marks,such as methylated H3K4 (Fig. 4A–C). In addition, the geneencoding long chain specific acyl-CoA dehydrogenase (LCAD,also known as ACADL), which is involved in fatty acid oxidation,was enriched with methylated H3K4 in the LSD1-KD cells

(Fig. 4C, right). ChIP analyses further showed that both PGC-1a and LCAD gene promoters were directly bound by LSD1(Fig. 4D). Thus, the results show that LSD1 epigeneticallyrepresses genes for mitochondrial metabolism and fatty acidoxidation by H3K4 demethylation. On the other hand, modifiedhistone H3, including methylated H3K4 and H3K9 on GLUT1and hexokinase-2 (HK2) gene promoters, did not markedlychange in LSD1-depleted cells (Fig. 5A).

LSD1 depletion impairs HIF1a-mediated expression ofglycolytic genes

As glucose metabolism genes did not exhibit notable changesin histone modifications after LSD1-KD, we reasoned thattranscriptional control of these genes had been affected.

Figure 3.LSD1 supportsGLUT1 gene expression and is associatedwith the increased expression of GLUT1 in humanHCC. A, expression changes of glucose transporter genes inLSD1-KD HepG2 cells. qRT-PCR was performed similarly as Figure 2 B–D, and values are shown relative to GLUT1 expression in control cells. Values are means� SDof three samples. ��, P < 0.01 versus control siRNA. B, decrease of GLUT1 protein in LSD1-depleted HepG2 cells. Two different siRNAs against LSD1 were usedto deplete LSD1 expression. Proteins expressed in the LSD1-depleted cells were subjected to Western blot analysis, followed by densitometric quantification(bottom). Values are means � SD of three samples. �� , P < 0.01 versus control siRNA. C, reduced glucose uptake induced by GLUT1 knockdown in HepG2 cells.The representative histograms of triplicate samples (left) and the mean fluorescent intensities (right) are shown. D, immunohistochemical studies of GLUT1and LSD1 in 38 HCC tissues. Two representative cases are exemplified: score 0 for both GLUT1 and LSD1 in case 1 (left), and score 2þ for both GLUT1 and LSD1 in case 2(right). Score 2þ for GLUT1 is equivalent or higher than the intensity of the red blood cell membrane in the same section (red arrows in top panels). The nuclearLSD1 staining in noncancerous hepatocytes was classified as 1þ, as shown in the inset of the right panel. Bar, 50 mm. E, significant correlation between LSD1-positiveand GLUT1-positive in HCC tissues.






Hypoxia-inducible facter-1a (HIF1a) plays a central role inglycolytic gene regulation both in normal and cancer cells (28).As the majority of LSD1-regulated glucose metabolism geneswere also the targets of HIF1a, we hypothesized that LSD1might functionally interact with HIF1a (24, (25). To addressthis, we first examined the HIF1a enrichment in the regulatoryregions of glucose metabolism genes. Under aerobic condition,HIF1a is hydroxylated by HIF prolyl hydroxylases (PHD), andthe hydroxylated HIF1a is ubiquitinated by von Hippel-Lindau(VHL) protein followed by the proteasomal degradation. Uponhypoxic stress, O2-dependent activities of PHDs are attenuatedthereby HIF1a protein accumulates (29). Under CoCl2 treat-ment, which inhibits O2-dependent PHD activity (30, 31), weobserved HIF1a binding at the vicinity of the putative hypoxia-responsive element (HRE at site 3) of the GLUT1 gene (Fig. 5B,gray bars in the top panel). Interestingly, HIF1a occupancy inthe predicted HRE decreased to approximately 40% in LSD1-depleted cells (orange bars). A similar reduction in HIF1abinding by LSD1-KD was found at the transcription start site(exon 1) ofHK2 (Supplementary Fig. S9A). LSD1 also appearedto be present on the promoter region of GLUT1 (Fig. 5B,bottom panel) and HK2, further suggesting the coregulationof these genes by LSD1 and HIF1a (Supplementary Fig. S9A).The expression of metabolic genes under the combination ofLSD1 depletion and CoCl2 treatment was then assayed. Inaccordance with the reduced HIF1a binding, LSD1 depletionabolished the induction of GLUT1 and most of the glycolyticgenes by hypoxic stress (Fig. 5C, genes shown in red). In

addition, hypoxic stress-induced suppression of metabolicgenes (e.g., PGC-1a) was also ablated by LSD1-KD (Fig. 5C,genes shown in blue). Moreover, depletion of HIF1a and LSD1exhibited analogous effects on the expression of glucose metab-olism genes under normoxia, suggesting that these factorscooperate under both normoxia and hypoxia (SupplementaryFig. S9B and S9C). Thus, LSD1 regulates glycolytic genesthrough the HIF1a-mediated pathway in HCC cells.

LSD1 is involved in the maintenance of HIF1a proteinlevel

To further explore the regulation of HIF1a function by LSD1,we examined the protein expression of HIF1a. Notably, we foundthat LSD1-KD significantly decreased HIF1a protein, as well asGLUT1, in the presence of CoCl2 in HepG2 and Huh-7 cells (Fig.6A and Supplementary Fig. S9D, shown quantitatively in thegraphs). We also obtained similar data under hypoxia (1% O2)(Fig. 6B). The LSD1-HIF1a-GLUT1 connection was further veri-fied by immunostaining analyses of a mixed culture that con-tained control and LSD1-KD cells. Under CoCl2 treatment, bothnuclear LSD1 and HIF1a were enriched in the control cells,whereas HIF1a expression clearly decreased in the LSD1-depletedcells (Fig. 6C and D). Consistent with this, GLUT1 protein wasreduced in the LSD1-KD cells, compared with the control cells(Fig. 6E and F), indicating the coexistence of the LSD1-HIF1a-GLUT1 at the single-cell level.

We then examined whether LSD1 transcriptionally or post-transcriptionally controls HIF1a protein in cultured HCC cells.

Figure 4.LSD1 directly represses PGC-1a via H3K4 demethylation. A, increased expression of PGC-1a mRNAs in LSD1-KD and tranylcypromine (TC)-treated HepG2 cells.Values are means � SD of three samples. �� , P < 0.01 versus control siRNA or vehicle. B, effect of tranylcypromine on PGC-1a promoter activity. The reporterplasmid PGC-1a/Luc and internal control pRL-TK were transfected into HepG2 cells. Cells were treated with TC for 24 hours before the measurement ofluciferase activities. Values are means � SD of three samples. � , P < 0.05; ��P < 0.01 versus vehicle. C, effect of LSD1-KD on histone modifications in thePGC-1a and LCAD promoters. In ChIP analyses, enrichment values were normalized to total histone H3. Values are means � SD of three samples. D, localizationof LSD1 in the PGC-1a and LCAD promoters (left and right, respectively). Values are normalized to input and are the mean � SD of three samples. � , P < 0.05;�� , P < 0.01 versus the region 1 in the PGC-1a gene.

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The HIF1a mRNA level was not affected by either LSD1 deple-tion or by CoCl2 addition (Supplementary Fig. S9E). It has beenreported that activated PI3K–AKT–mTOR signaling is implicat-ed in the enhanced HIF1a translation in cancer cells (32, 33),and that the phosphorylation of S6K and 4E-BP1 is the majordownstream effector of the mTOR signaling, which leads toprotein synthesis (34). However, we did not find any reductionin their phosphorylation level by LSD1-KD (SupplementaryFig. S9F). Thus, transcriptional and translational pathwaysdid not seem to be responsible for the decrease of HIF1a byLSD1-KD.

To clarifywhetherHIF1aprotein is reduced by LSD1-KDvia theubiquitin proteasome degradation, we treated the cells with aproteasome inhibitor MG-132 or a PHD inhibitor dimethylox-alylglycine (DMOG; Supplementary Fig. S9G). The treatmentwithCoCl2, or withDMOG to a lesser extent, stabilized theHIF1aprotein with absence of the accumulated hydroxyl-form. In con-

trast, proteasome inhibition clearly augmented the hydroxyl-HIF1a protein with detectable smeared bands of high molecularweight (>120 kDa; Supplementary Fig. S9G), indicating thatHIF1a was degraded via the PHD-mediated proteasomal path-way in LSD1-KD cells as well as control cells. Furthermore, wechecked the ubiquitination of HIF1a protein in FLAG-taggedHIF1a introduced HepG2 cells (Supplementary Fig. S9H).Polyubiquitinated HIF1a proteins accumulated under protea-some inhibition in LSD1-KD cells as well as in control cells.Our repeated experiments showed that (i) HIF1a was destabi-lized via the ubiquitin proteasome pathway in LSD1-KD cellsand (ii) DMOG treatment did not reverse the effect of LSD1-KDon HIF1a.

Recently, Qin and colleagues demonstrated that LSD1 contri-butes to the stabilization of HIF1a protein through facilitating itsdeacetylation by HDAC2 in pancreatic cancer cells (35). Wetherefore tested whether this model could also be applied to

Figure 5.LSD1 maintains glycolytic metabolism via the HIF1a-mediated pathway in cancer cells. A, distinct effects of LSD1-KD on histone modifications in GLUT1 andHK2 promoters. H3K4me1, me2, me3, and acetylated (ac) histone H3 were tested by ChIP. By ChIP–qPCR, enrichments were normalized to total histone H3.Values aremeans� SDof triplicate results. B, LSD1-dependent enrichment of hypoxia-inducible factor, HIF1a, at theGLUT1 gene promoter. ChIP-qPCR analyseswereperformed in control and LSD1-depleted cells under CoCl2 treatment. Enrichment values at the indicated sites (1–7) were normalized to input DNAs. Valuesare means� SD of triplicate results. C, distinct effects of LSD1-KD on metabolic genes under hypoxic conditions. The gene sets upregulated and downregulated byCoCl2 treatment are indicated by red and blue, respectively, while the unaffected genes are shown in green. Values are means � SD of triplicate results, asshown relative to control siRNA-treated samples (black bars). � , P < 0.05; �� , P < 0.01.






HCC cells in our study. However, we did not observe a physicalinteraction of HIF1a protein with either LSD1 or HDAC1/2 inHepG2 cells (Supplementary Fig. S10A). Accordingly, LSD1depletion did not affect the acetylation status of HIF1a (Supple-mentary Fig. S10B). In contrast, interaction between LSD1 andHDAC1/2 could be clearly observed (Supplementary Fig. S10A),consistent with previous studies (36, 37). These findings suggestthat HIF1a is not the target of LSD1/HDAC-mediated deacetyla-tion in HCC cells.

Collectively, these results suggest that LSD1 protects HIF1afrom proteasomal degradation at least in part through a hydrox-ylation-independent mechanism.

LSD1 depletion suppresses engraftment and growth of HCCxenograft tumor

To gain insight into the in vivo role of LSD1 in HCC develop-ment, we transplanted siRNA-introduced HepG2 cells subcuta-neously into SCID mice. Twenty-four hours after siRNA transfec-tion, control and LSD1-KDHepG2 cells were injected into left andright flanks of each mouse, respectively, and then the mice werekept for 3weeks until sacrifice. Interestingly, in 6of 9mice, tumorsderived from LSD1-KD cells were markedly smaller than thecontrols, with approximately 36% reduction in the cumulativetumor weight (Fig. 7A and B). In 3 mice, we observed dramaticreduction in tumor size by LSD1-KD. In these tumors,many of theglycolytic genes under regulation by LSD1 in vitro showed

decreased expression, compared with the control tumors fromthe samemice (Fig. 7C, left).On the other hand, in themicewhereLSD1-KD did not reduce the tumor size, there was no obviousdifference in the glycolytic gene expression between the controland LSD1-KD (Fig. 7C, right). These results suggest that LSD1supports the HCC tumor engraftment and/or growth. Next, weadministrated tranylcypromine, a chemical inhibitor of LSD1,in mice after the xenograft tumors had been expanded (Sup-plementary Fig. S11B). We observed that tranylcypromine-treated tumors tended to show smaller size compared with thecontrols but without statistical significance (SupplementaryFig. S11C).

Taken together, these results show that LSD1 coordinatelymaintains the increased glucose uptake, followed by glycolysisand the suppression of mitochondrial respiration in cancer cells,thereby leading to the typical cancer metabolism known as theWarburg effect (shown schematically in Fig. 7D).

DiscussionIn the current study, we demonstrated that LSD1 coordinates

energy production through activating glycolytic pathway andsuppressing the mitochondrial respiration in HCC cells. Of greatinterest, LSD1 repressed mitochondrial metabolism genes viathe H3K4 demethylating activity, while glycolytic genes wereinduced, at least in part, via the LSD1-mediated HIF1a

Figure 6.HIF1a protein is reduced by LSD1depletion. A, reduction of HIF1a andGLUT1 proteins by LSD1-KD underCoCl2 treatment. The data obtainedby Western blot analyses arequantitated by densitometry.� , P < 0.05; �� , P < 0.01. B, reduction ofHIF1a and GLUT1 proteins by LSD1-KDunder hypoxia. The data obtained byWestern blot analyses werequantitated by densitometry. C and E,immunostaining analyses of HIF1a (C)and GLUT1 (E) in HepG2 cells. Bar,10 mm. The control and LSD1-KD cellswere mixed and grown in the sameculture dish. D and F, single-cellquantification of the coexistence ofLSD1 with either HIF1a (D) or GLUT1(F). The data are displayed as a box-whisker plot. The control and LSD1-KDcells were grown separately.

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transactivation. Orchestration of the glycolytic shift by LSD1 iswell reflected on the metabolomic profile of LSD1-KD cells, inwhich the early glycolytic products and pentose phosphate path-way intermediates were clearly decreased compared with thecontrol. An increase of acetyl-CoA level in LSD1-KD cells was ingood agreement with the upregulation of fatty acid oxidation

genes. Acetyl-CoA is metabolized by citrate synthase in the mito-chondria into citrate that inhibits PFK activity thusmay contributeto the reduced glycolysis. However, we did not find a changein the citrate level by LSD1-KD (Supplementary Fig. S2), suggest-ing that elevated acetyl-CoA was utilized to fuel respiratorychain activity as demonstrated by the extracellular flux analysis.

Figure 7.In vivo role of LSD1 in the tumorigenesis of HepG2 cells in mouse xenograft model. A–C, the effect of LSD1-KD on the transplanted HepG2-derived tumor in mice.A, representative images of isolated tumors. Each image shows a pair of tumors derived from the same mouse. B, cumulative weight of tumors collectedfrom nine mice. Connected lines indicate the pair of tumors derived from the same mouse. C, mRNA expression of LSD1, glycolytic genes, and VEGFA in control andLSD1-KD tumors. LSD1-KD tumors with distinctively smaller size compared to the control showed decreased expression of glycolytic genes (left), while these geneswere not affected in LSD1-KD tumors that did not show growth reduction (right). D, schematic model of the LSD1-mediated metabolic shift in cancer cells.Overexpressed LSD1 suppressesmitochondrialmetabolismgenes viaH3K4demethylation and inducesglycolytic genesvia theHIF1a-mediatedpathway, resulting inthe active glucose utilization (left). LSD1 inhibition reactivates mitochondrial metabolism and downregulates glycolysis and HIF1a function, leading to ametabolic correction (right). Pyr, pyruvate; Lac, lactate.






Our metabolomic data together with extracellular flux, glucoseuptake, and mitochondrial potential data clearly define the con-tribution of LSD1 in the maintenance of glycolytic activation andmitochondrial suppression in HCC cells.

Coordinated expression of metabolic genes is crucial for theadaptive metabolic remodeling in response to severe environ-mental conditions. Recent work by Duteil and colleaguesdemonstrated that LSD1 promotes the oxidative respiration inwhite adipocytes in response to strong catabolic stimuli such asb-adrenergic activation (38), whereas we have previouslyshown that LSD1 suppresses the mitochondrial respirationunder adipogenic and lipogenic conditions (21), suggestingthat LSD1 translates environmental fluctuations to diversemetabolic outcomes. In the current study, even though LSD1depletion resulted in the impaired glucose utilization in HCCcells in vitro, we did not observe a clear effect on cell growth andcell-cycle progression. As sufficient amount of nutrients andoxygen are present under normal culture condition, LSD1-depleted cancer cells might have used alternative pathways tosupply energy sources. Interestingly, we demonstrated thatLSD1 depletion led to the size reduction of xenograft tumors.As we employed transient siRNA introduction before the trans-plant, it is likely that LSD1 expression had been reduced onlyfor the first 3 to 4 days. This raises an intriguing possibility thatLSD1 was required for the cells to adapt to hypoxic andnutrient-poor condition. Of note, both LSD1-depleted xeno-graft tumors and HepG2 cells in vitro exhibited a reducedexpression of VEGFA, a HIF1a-responsive gene that is a keyfactor for the angiogenesis and tumor growth (Fig. 7C andSupplementary Fig. S11A; ref. 39). Together, these lines ofevidence suggest that LSD1 plays an essential role in themetabolic reprogramming of cancer cells under fluctuatingenvironment.

Our data indicate that LSD1 maintains the HIF1a-mediatedtranscriptome in two HCC lines, HepG2 and Huh-7. At thesame time, we observed that a subset of glycolytic genes wasdifferentially regulated by LSD1 (Supplementary Fig. S4).Previous report has identified, using integrative genomicapproach, that HIF1a responsive genes varied across cell types,and some but not all glycolytic genes were the universal targets(40). As most of the LSD1-target glycolytic genes were core-gulated by HIF1a (Supplementary Fig. S9C), it is possible thatthe target gene preference could be attributed to the respon-siveness to HIF1a. In addition, the maintenance of glycolyticpathway by LSD1 was not found in nontransformed cells(Supplementary Fig. S8B and S8D). The difference in LSD1expression level may contribute to this selectivity, as HCC cellsshowed markedly higher expression compared with nontrans-formed cells and the normal liver (Supplementary Fig. S8A).This possibility is backed up by our data that the forcedexpression of LSD1 resulted in the increase of glycolytic geneexpression (Supplementary Fig. S6). The difference betweencancer and nontransformed cells also suggests that activationof glucose utilization by LSD1 may require additional onco-genic signals. Phosphorylation signaling cascades mediated byPI3K and MAPK regulate the levels of HIF1a (41). Consistent-ly, enhanced HIF1a activity has been observed in HCC in bothhuman and mice (33, 42). In addition, LSD1 interacts withMyc, which is also an important regulator of glucose flux, andactivates the Myc target genes (43), implying that PI3K/MAPKand LSD1/Myc pathways may contribute to the oncogenic

energy utilization. Moreover, p53 is a negative regulator ofglycolytic enzyme phosphoglycerate mutase (PGAM; ref. 44)and glucose transporters (GLUT1 and GLUT4; ref. 45), and wasreported to be inactivated through the LSD1-mediateddemethylation (46). Considering the dual role of LSD1 inglycolysis and mitochondrial respiration, these evidences raisean intriguing possibility that LSD1 plays a pivotal role inregulating cancer cell metabolism in response to oncogenicand antioncogenic signals.

We found in the current study that LSD1 is required formaintaining the HIF1a protein level, thus contributing to theglycolytic gene expression. Our data indicate that LSD1 pro-tects HIF1a from proteasomal degradation. The functionalassociation of LSD1 with HIF1a was enhanced under hypoxicstress, suggesting that LSD1 facilitates the canonical HIF1astabilization pathway. However, we did not find a change inthe hydroxylation level of HIF1a nor physical interactions ofLSD1 with PHDs (data not shown). Direct interpretation ofthis is that LSD1 affects the ubiquitination and/or proteasomaldegradation of HIF1a at least in part through a hydroxylation-independent mechanism. Intriguingly, we observed that theratio of conjugated to unconjugated forms of HIF1a washigher in LSD1-KD cells, compared with control cells, owingin part to the decrease of unconjugated HIF1a in the LSD1-KD(Supplementary Fig. S9H). Thus LSD1 may protect HIF1afrom ubiquitination and the subsequent proteasomal degra-dation. LSD1 has been reported to remove the methyl groupfrom some nonhistone proteins either to augment or to represstheir functions (47). Known or unknown proteins involved inHIF1a stabilization might be modulated by LSD1 throughsuch mechanisms. In summary, the current study demonstrat-ed that LSD1 maintains the glycolytic gene expression incancer cells without affecting the major chromatin marks, butthrough the modulation of a transcriptional activator.

Our current study provides evidence that LSD1 functions as apivotal regulator of cancer cell metabolism, and highlights theepigenetic plasticity of the cellular metabolic state under LSD1inhibition. Moreover, recent studies demonstrated that LSD1has an essential role in the maintenance of pluripotency inembryonic stem cells and cancer stem cells (48, 49), and thatsuch highly proliferative cells obtain energy preferentially byglycolysis (50). Therefore, the LSD1-mediated metabolic switchmay be generally crucial for cancer metabolism, as well as forproliferative capacity.

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

Authors' ContributionsConception and design: A. Sakamoto, S. Hino, M. NakaoDevelopment of methodology: A. Sakamoto, S. Hino, M. NakaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Sakamoto, S. Hino, K. Nagaoka, K. Anan, R. Takase,H. Matsumori, Y. KanaiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Sakamoto, S. Hino, K. Anan, H. Ojima, K. Arita,M. NakaoWriting, review, and/or revision of the manuscript: A. Sakamoto, S. Hino,Y. Kanai, M. NakaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. NakaoStudy supervision: K. Arita, M. Nakao

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Grant SupportThis work was supported by a Grant-in-Aid for Scientific Research on Priority

Areas and on Innovative Areas (3307) from the Ministry of Education, Culture,Sports, Science and Technology of Japan, by the Japan Science and TechnologyAgency (CREST), and by a grant from The Uehara Memorial Foundation(M. Nakao). This work was also supported by a Grant-in-Aid for ScientificResearch from Japan Society for the Promotion of Science and by the NakatomiFoundation (S. Hino).

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 May 27, 2014; revised December 4, 2014; accepted December 22,2014; published OnlineFirst February 3, 2015.

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2015;75:1445-1456. Published OnlineFirst February 3, 2015.Cancer Res Akihisa Sakamoto, Shinjiro Hino, Katsuya Nagaoka, et al. Mitochondrial Metabolism in Hepatocellular Carcinoma CellsLysine Demethylase LSD1 Coordinates Glycolytic and

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