Long Noncoding RNA MALAT1 RegulatesCancer Glucose Metabolism by EnhancingmTOR-Mediated Translation of TCF7L2Pushkar Malakar1, Ilan Stein2,3, Amijai Saragovi2,3, Roni Winkler4, Noam Stern-Ginossar4,Michael Berger2,3, Eli Pikarsky2,3, and Rotem Karni1
Abstract
Reprogrammed glucose metabolism of enhanced aerobicglycolysis (or the Warburg effect) is known as a hallmark ofcancer. The roles of long noncoding RNAs (lncRNA) in regu-lating cancer metabolism at the level of both glycolysis andgluconeogenesis are mostly unknown. We previously showedthat lncRNAmetastasis-associated lung adenocarcinoma tran-script 1 (MALAT1) acts as a proto-oncogene in hepatocellularcarcinoma (HCC). Here, we investigated the role of MALAT1in regulating cancer glucose metabolism. MALAT1 upregu-lated the expression of glycolytic genes and downregulatedgluconeogenic enzymes by enhancing the translation of themetabolic transcription factor TCF7L2. MALAT1-enhancedTCF7L2 translation was mediated by upregulation of SRSF1and activation of the mTORC1–4EBP1 axis. Pharmacologicalor genetic inhibition of mTOR and Raptor or expression of ahypophosphorylatedmutant versionof eIF4E-binding protein(4EBP1) resulted in decreased expression of TCF7L2. MALAT1
expression regulated TCF7L2 mRNA association with heavypolysomes, probably through the TCF7L2 50-untranslatedregion (UTR), as determined by polysome fractionationand 50UTR-reporter assays. Knockdown of TCF7L2 inMALAT1-overexpressing cells and HCC cell lines affectedtheir metabolism and abolished their tumorigenic poten-tial, suggesting that the effects of MALAT1 on glucosemetabolism are essential for its oncogenic activity. Takentogether, our findings suggest that MALAT1 contributes toHCC development and tumor progression by reprogram-ming tumor glucose metabolism.
Significance: These findings show that lncRNA MALAT1contributes to HCC development by regulating cancer glucosemetabolism, enhancing glycolysis, and inhibiting gluconeo-genesis via elevated translation of the transcription factorTCF7L2.
IntroductionLong noncoding RNAs (lncRNA) constitute a large class of
mRNA-like transcripts, greater than 200 nucleotides with noprotein coding capability (1). In the past few years, severallncRNAs have been shown to play a role in cancer by promotingproliferation, invasion andmetastasis (2–4). LncRNAs have beenshown to regulate almost every step of gene expression (5).MALAT1 was one of the first lncRNAs to have a designated rolein cancer (4, 6). MALAT1 is highly conserved among mammals,approximately 7Kb in length and highly abundant (7). Previous-ly, we showed that MALAT1 acts as a proto-oncogene in hepato-cellular carcinoma throughWnt pathway activation, induction of
the splicing factor SRSF1 and mTORC1 activation (8). Further-more, we showed that mTORC1 activation is required forMALAT1-mediated tumorigenesis (8).
Both the Wnt and mTOR signaling pathways have been shownto play an important role in altering the glucose metabolicprogram in cancers (9, 10). Altered glucose metabolism is oneof the first identified hallmarks of cancer (11), discovered by OttoWarburg in the late 1920s (12). Cancer cells predominantly carryout glycolysis in the cytosol rather thanoxidativephosphorylationthrough the TCA cycle in the mitochondria (13). It is generallybelieved that in most cancers, oncogenic lesions are largely thecause of enhanced glycolysis and the "Warburg effect" (14).c-MYC, a downstream target of Wnt signaling, was shown to playan important role in the regulation of glycolysis in cancercells (15). Glucose metabolism genes were shown to be directlyregulated by c-MYC. The key modulator of the canonical Wntsignaling pathway is the bipartite transcription factor b-Cat(b-catenin)/TCF, formed by b-catenin and a member of the TCFfamily (TCF-1, LEF-1, TCF-3 and TCF-4/TCF7L2; ref. 16). TCF7L2was shown to be an effector of the Wnt signaling pathway andbinds directly to multiple genes that are important in regulatingglucose metabolism. Moreover, genome-wide association studies(GWAS) have identified SNPs in the TCF7L2 gene associated withobesity and diabetes (17). mTOR activation regulates glucosemetabolism through activation ofHIF1a. HIF1a is a transcriptionfactor that is known to induce the expressionof at least 9 glycolyticenzymes, thereby regulating glucose metabolism in many can-cers (18). Several lncRNAs have been shown to regulate, or to be
1Department of Biochemistry and Molecular Biology, Institute for MedicalResearch Israel Canada (IMRIC), Hebrew University-Hadassah Medical School,Jerusalem, Israel. 2TheLautenbergCenter for Immunology andCancerResearch,Institute for Medical Research Israel Canada (IMRIC), Jerusalem, Israel. 3Depart-ment of Pathology, Hebrew University—Hadassah Medical School, Jerusalem,Israel. 4Department of Molecular Genetics, Weizmann Institute of Science,Rehovot, Israel.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
regulated by, the Wnt and mTOR signaling pathways (19, 20).Even though lncRNAs have been shown to affect cancer initiationand progression, only a handful of studies have focused on theinvolvement of lncRNAs in cancer glucosemetabolism at the levelof glycolysis (21, 22). Gluconeogenesis is essentially the reverse ofthe glycolysis pathway and usually occurs in the liver when bloodglucose levels drop and the liver regenerates glucose, sending it toother tissues (23). There is a single report describing the role oflncRNAs in cancer development through regulating gluconeogen-esis. lncRNA Nur77 was shown to suppress HCC through upre-gulating gluconeogenesis (24). There are no reports describing therole of MALAT1 in cancer glucose metabolism at the level ofglycolysis or gluconeogenesis. Several studies have shown gluco-neogenesis to be downregulated in HCC (25, 26). However, theregulation of both glycolysis and gluconeogenesis by lncRNAs inHCC development or progression has not been reported.
In this study, we investigated the roles of MALAT1 in regulatingglucose metabolism of HCC cancer cells and found that MALAT1enhanced aerobic glycolysis and repressed gluconeogenesis. Wefurther discovered that MALAT1 regulates glycolytic gene expres-sion through increased translation of transcription factor TCF7L2.We demonstrate, both pharmacologically and genetically, thatTCF7L2 upregulation is mediated by mTORC1 activation of cap-dependent translation. MALAT1-mediated tumorigenesis isdependent on TCF7L2. In addition, using Mdr2�/� mice livertumor samples, we show elevated levels of MALAT1, nuclearTCF7L2 and glycolytic gene expression, and decreased expressionof gluconeogenic gene expression, suggesting a positive correla-tion with glycolysis and a negative correlation with gluconeogen-esis. Thus, we present here a novel function for MALAT1 intumorigenesis and provide a previously unappreciated mecha-nism by which cancer cells switch to aerobic glycolysis, repressinggluconeogenesis, during cancer progression. This is the first reportshowing the regulation of TCF7L2 by mTORC1-mediated cap-dependent translation and suggests that the mTORC1 pathwaycan regulate Wnt signaling through TCF7L2 translation.
Materials and MethodsCell culture
PHM-1 cells are mouse liver progenitor cells derived fromembryonic day 18 fetal livers from TP53�/� mice and immor-talized with MSCV-based retroviruses expressing MYC-IRES-GFP (27). PHM-1, FLC4, and HepG2 cells were grown inDMEM supplemented with 10% FCS, 0.1 mg/mL penicillin,and 0.1 mg/mL streptomycin. All cell lines have been tested andauthenticated using STR loci plus Amelogenin for gender iden-tification for human cell line authentication by the BiosynthesisDNA Identity Testing Centre.
Stable cell linespCD513B1 empty (System Biosciences) and pCD513B1-
hMALAT1 lentiviruses were prepared using the manufacturer'sinstructions. These viruses were used to infect PHM-1 cells. Cellswere selected by the addition of puromycin (2 mg/mL) for 72 to96 hours. In the case of infection withMLP-puro-shRNA viruses,cells were selected with puromycin (2 mg/mL) for 96 hours.
siRNA treatmentDouble-stranded siRNAs (Sigma) were used at specified con-
centrations to deplete MALAT1 or TCF7L2 from cells. siRNAs
against Luciferase (Dharmacon Thermo Scientific) or siRNA Uni-versal Negative Control (Sigma) was used as a control at specifiedconcentrations. Lipofectamine 2000 reagent (Invitrogen) wasused for transfection as per the manufacturer's instructions.
qRT-PCRTotal RNA was extracted with TRI Reagent (Sigma), and 1 mg of
total RNA was reverse transcribed using M-MLV reverse transcrip-tase (Promega) after DNase treatment (Promega). qPCR wasperformed on the cDNA using SYBR Green Mix (Roche) andCFX96 (Bio-Rad) real-time PCR machine. Primer list is suppliedin Supplementary Table S1.
ImmunoblottingCells were lysed in Laemmli buffer and analyzed for total
protein concentration. Twenty micrograms of total proteinfrom each cell lysate were separated by SDS-PAGE and transferredto a polyvinylidene difluoride (PVDF) membrane. Primaryantibodies used were TCF7L2 EP20334 (1:10,000; Abcam),GAPDH (1:5,000; Sigma), b-catenin (1:2,000; Sigma), b-actin(1:2,000; Sigma). a-Tubulin (1:1,000; Santa Cruz Biotechnol-ogy), b-Tubulin (1:2000;Sigma). SRSF1 (AK96 culture super-natant 1:300), T7 Tag (1:5,000; BD Transduction laboratories),mTOR (1:1,000; Cell Signaling Technology), Raptor (1:1,000;Cell Signaling Technology), p4EBP1 (1:1,000; Cell SignalingTechnology), Total 4EBP1 (1:1,000; Cell Signaling Technolo-gy). Secondary antibodies used were HRP-conjugated goat anti-mouse, goat anti-rabbit, donkey anti-goat IgG (HþL; 1:10,000;The Jackson Laboratory).
Colony formation assayCells were seeded in 6-well plates (1,000 cells/well) and grown
for 10 days. After fixation with 2.5% glutaraldehyde, the plateswere washed three times. Fixed cells were then stained withmethylene blue solution (1%methylene blue in 0.1mol/L boratebuffer, pH 8.5) for 60 minutes at room temperature. Plates werephotographed after extensive washing and air drying (28).
Anchorage-independent growthColony formation in soft agar was assayed as described previ-
ously [41]. After 14 to 21 days, colonies from 10 different fields ineach of twowells were counted for each treatment and the averagenumber of colonies per well was calculated. The colonies werestained and photographed under a light microscope at �10magnification (28).
Lactate assayCells (2 � 105) were seeded in 6-well culture plates. The cells
were trypsinized 48 hours after culture or siRNA treatment. Cellswere homogenized in the presence of lactate assay buffer andcentrifuged at 13,000 � g for 10 minutes. Lactate quantificationwas performed using commercially available lactate assay kit(Abcam, ab65330) in a 96-well plate as per the manufacturer'sinstructions. Lactate levels were measured using a plate reader atan optical density of 570 nm (29). Lactate levels were normalizedto total cellular protein concentration.
Glucose secretion assayHepG2 and FLC4 cells were cultured and treated with siRNAs.
After 48 hours of siRNA treatment, themediumwas replacedwithDMEM containing 0.1% serum for 16 hours. Cells were washed
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twice with PBS to remove glucose and then incubated for 6 hoursin glucose production assaymedium(glucose andphenol red-freeDMEM containing 2 mmol/L sodium pyruvate, 20 mmol/Lsodium lactate, 2 mmol/L L-glutamine and 15 mmol/L HEPES).Medium (200 mL) was sampled for measurement of glucoseconcentration. Glucose level quantification was performed usinga commercially available glucose assay kit (Amplex Red GlucoseAssay Kit, ThermoFisher Scientific) in a 96-well plate. Glucoselevels were normalized to total cellular protein concentration.
MiceAll animal experiments were performed in accordance with the
institutional animal care and use committee. Mdr2�/� mice (30)were bred and maintained in specific pathogen-free conditions.
ImmunohistochemistryImmunohistochemistry for TCF7L2 was performed on 5-mm
formalin-fixed paraffin-embedded sections. After citrate-basedantigen retrieval (Vector Labs # H-3300), endogenous perox-idase was blocked by 3% H2O2. Slides were incubated withprimary antibody (a-TCF7L2, Abcam # ab76151), washed, andincubated with anti-Rabbit-HRP ImmPRESS Reagent (VectorLabs # MP-7401). Slides were developed with the HRP substratediaminobenzidine (Thermo Scientific) and counterstained withhematoxylin.
Polysome profilingPolysome profile analysis was carried out as described previ-
ously (31). Briefly, cells were cultured in 10cm dishes. Beforeharvesting cells were treated with cyclohexamide (20 mL CHXfrom 50 mg/mL stock) for 3 minutes. Then cells were washedtwice with cold PBS containing 50 mg/mL cycloheximide, col-lected, and lysed in a 250 mL of lysis buffer [Lysis Buffer (5 mL):250 mL 20% Triton (RNAse free) þ 4.75 mL Polysome buffer þ60 mL DNAse (120 U)]. Lysates were loaded onto 10% to 50%sucrose density gradients prepared in polysomebuffer. [PolysomeBuffer (20mL): 250mLof 1mol/L Tris pH7, 150mLof 1mol/L TrispH 8, 600 mL of 5mol/L NaCl, 100 mL of 1mol/LMgCl2, 40 mL ofCHX (50mg/mL in EtOH), 20 mL of DTT (1mol/L) and 18.84mLof DEPC Water]. Extracts were fractionated for 3 hours at35,000 rpm at 4�C in a Beckman rotor, and the gradients wererecovered in 12 fractions using gradient fractionators. RNA wasextracted from each fraction. Translational status of TCF7L2mRNA on polysome fractions was determined by qRT-PCR.
Luciferase reporter assay507 bp of human TCF7L2, 50-untranslated region (UTR)
upstream of the start codon, were amplified from FLC4 cellscDNA by RT-PCR using a forward primer with a KpnI restrictionsite and a reverse primerwith a XhoI restriction site and subclonedinto the KpnI and XhoI restriction sites of the pSG5 Luc plasmid.The insert was verified by sequencing. pSG5 Luc Plasmid was akind gift from Prof. Fatima Gebauer, Center for Genomic Regu-lation (CRG), Barcelona. PHM-1, HepG2 and FLC4 cells wereseeded in 6-well plates (2 � 105 cells/well) under standardconditions. After 24 hours, cells were transfected with MALAT1siRNAs using Lipofectamine 2000. After another 48 hours thesecells were further transfected, using polyethylenimine, with 2 mgof TCF7L2-50UTR-Firefly construct and 0.5mg pRenilla constructper well. Forty-eight hours later, the cells were harvested andluciferase activity was analyzed using Dual-Glo Luciferase Assay
System according to the protocol provided by Promega andInfinite M200 PRO. Renilla activity was used to normalize fortransfection efficiency. Firefly-luciferase mRNA expression wasalso measured as an additional control for luciferase activity.
Glucose uptake in FLC4 cellsFLC4 cells were treated with siRNAs targeted against luciferase
or MALAT1 for 48 hours. Subsequently, they were treated withmedium without glucose for 16 hours and then exposed to2NBDG (a fluorescent derivative of glucose) for 30 minutes.2NBDG fluorescence was recorded using flow cytometry.
Statistical analysisError bars for all data represent SDs from the mean. P values
were calculated using two-tailed type 2 Student t tests except fora few cases where tail one type 2 Student t test was used.Statistical significance is displayed as �, P < 0.05; ��, P < 0.01;and ���, P <0.001.
One of the first identified hallmarks of cancer is altered glucosemetabolism (32). Tumor cells enhance glycolysis even in thepresence of oxygen and in many cases reduce oxidative phos-phorylation (13). In addition, many oncogenes enhance glycol-ysis by alternativemechanisms (18).Wepreviously found that thelncRNA MALAT1 acts as a proto-oncogene in HCC developmentand activates the mTORC1 pathway (8). The mTORC1 pathwayaffects tumor metabolism by several mechanisms and specificallyglucosemetabolism (33). Thus,we sought to examine the effect ofMALAT1 on glucose metabolism in HCC cancer cells. One of thecharacteristics of hepatocytes is their ability to produce glucose bygluconeogenesis, to supply glucose to the body when bloodglucose levels drop (23). This process acts in a reverse pathwayto glycolysis. We measured lactate production as a measure forglycolysis in PHM-1 cells either overexpressing or knocked-downforMALAT1. Lactate productionwasmeasuredby the intracellularlactate content. Overexpression of MALAT1 led to enhancedlactate production (Fig. 1A and B). Conversely, transient knock-down of MALAT1 by siRNAs resulted in reduced lactate produc-tion (Fig. 1C and D). These data suggest that MALAT1 expressionregulates glucose metabolism in PHM-1 cells by enhancing gly-colysis. To eliminate possible effects of cell proliferation orcellular density on glucose metabolism, we examined glucoseuptake at a single cell level. Cells were labeled with fluorescentglucose (2NBDG) and glucose uptake was measured by flowcytometry. We found that glucose uptake was lower followingMALAT1 knockdown in HCC FLC4 cells (SupplementaryFig. S1A–S1B). In previously performed RNA-seq analysis onPHM-1 cells overexpressing MALAT1, we detected increasedexpression of several glycolytic genes (8). To confirm the tran-scriptional regulation of the glucose metabolism program inhepatocytes by MALAT1 we validated several of the upregulatedgenes. In agreement with enhanced glycolysis, the expression ofseveral glycolytic enzymeswas upregulated in cells overexpressingMALAT1 (Fig. 1E–G) and reduced by knockdown of MALAT1(Supplementary Fig. S1C–S1E). These data suggest that MALAT1expression promotes glycolytic metabolism in cancer cells.
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MALAT1 affects cancer glucose metabolism. A, qRT-PCR of PHM-1 cells stably expressing hMALAT1 or an empty vector. B, Extracellular lactate productionwasmeasured in cells described in A using a lactate assay kit (n¼ 3). C, PHM-1 cells overexpressing MALAT1 knocked down for MALAT1 by siRNAs (siMALAT#1, #2)were analyzed by qRT-PCR.D, Extracellular lactate productionwas measured in cells described in D using a lactate assay kit (n¼ 3). E, Schematic representationof the glycolytic and gluconeogenetic pathways of glucose metabolism. The enzymes marked in red were selected for gene expression analysis. F, A gel imageof semiquantitative RT-PCR of glycolytic gene expression in PHM-1 cells described in A.G, Expression of genes in the glucose metabolic pathway in cellsdescribed inAmeasured by qRT-PCR. All samples were normalized to GAPDHmRNA levels. Error bars, SD (n¼ 3). Student t test was used. � , P < 0.05;�� , P < 0.01; ��� , P < 0.001.
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MALAT1 negatively affects gluconeogenesisGluconeogenesis is a major component of glucose metabolism
in normal liver cells, regulating whole-body glucose homeosta-sis (34). In HCC, gluconeogenesis plays a tumor-suppressive roleopposing aerobic glycolysis and preventing the "Warburgeffect" (35). To examine the effect ofMALAT1on gluconeogenesisin HCC, we knocked-down MALAT1 in HCC cell lines andexamined gluconeogenic gene expression. Expression of gluco-neogenic genes is downregulated in HCC compared with normalhepatocytes (25, 26). We found that transient knockdown ofMALAT1 by siRNAs in HepG2 cells (Fig. 2A) and FLC4 cells(Supplementary Fig. S2A) resulted in increased glucose secretion(Fig. 2B; Supplementary Fig. S2B) and reduced lactate production(Fig. 2C; Supplementary Fig. S2C). Transient knockdown ofMALAT1 in these cells also resulted in increased expression ofgluconeogenic genes,G6PCandPCK1 (Fig. 2D–F; SupplementaryFig. S2D and S2E).
MALAT1 controls TCF7L2 expression at the protein levelThe transcription factor TCF7L2 was shown to modulate glu-
cose homeostasis in the liver (36, 37). Moreover, it has beenshown that TCF7L2 negatively regulates gluconeogenesis (38).Although the role of TCF7L2 in theWnt signaling pathway is wellstudied, its role in modulating glucose metabolism is less wellcharacterized, and in some cases conflicting (39). Thus, we exam-ined the regulation of TCF7L2 byMALAT1. To probe the potentialmechanism by which MALAT1 regulates TCF7L2, we examinedthe effects ofMALAT1manipulation on the expression of TCF7L2.We detected no significant change in the mRNA levels of TCF7L2in response to MALAT1 overexpression or knockdown (Fig. 3A).In contrast with these results, western blot analysis showedthat MALAT1 overexpression resulted in enhanced TCF7L2 pro-tein expression (Fig. 3B). Transient knockdown of MALAT1 didnot change TCF7L2 mRNA level (Fig. 3C), but resulted indecreased protein expression of TCF7L2 (Fig. 3D; Supplementary
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Figure 2.
MALAT1 negatively regulates gluconeogenesis in HCC cells. A, HepG2 cells were transfected with either MALAT1 siRNA (siMALAT#1, #2) or control siRNA(siLuciferase). MALAT1 RNA levels were analyzed by qRT-PCR. B. Cellular glucose secretion was measured in cells described in A using a glucose assay kit(n¼ 3). C, Extracellular lactate production was measured in cells described in A using a lactate assay kit (n¼ 2).D, Schematic representation of the glycolyticand gluconeogenetic pathways. The enzymes marked in blue are involved in gluconeogenesis. E and F,mRNA expression of the G6PC (E) and PCK1 (F) genesin the gluconeogenesis pathway in cells described in A. All samples were normalized to actin or GAPDHmRNA levels. Error bars, SD (n¼ 3). Student t test wasused. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.
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MALAT1 upregulates TCF7L2 translation. A, TCF7L2 mRNA expression in PHM-1 cells stably expressing hMALAT1 or an empty vector analyzed by qRT-PCR. B,Left, TCF7L2 protein levels in cells described in A analyzed byWestern blot. Right, quantification of TCF7L2 protein levels (n¼ 4). C, TCF7L2 mRNA levels inPHM-1 cells overexpressing MALAT1 treated with the indicated siRNAs, as determined by qRT-PCR. D, Left, TCF7L2 protein levels in cells described in Cweredetermined byWestern blot. Right, quantification of TCF7L2 protein levels (n¼ 3). E, Relative distribution of TCF7L2 mRNA across the polysome fractions inPHM-1 cells stably expressing hMALAT1 or an empty vector. F, Relative distribution of TCF7L2 mRNA across the polysome fractions in cells transfected witheither MALAT1 siRNA or control siRNA (siLuciferase). G, PHM-1 cells were transfected with MALAT1 siRNAs (siMALAT1#1, #2) or siControl. Luciferase activity (foldchange compared with siControl) produced after transfection with luciferase construct containing theWT TCF7L2 50UTR was measured. Luciferase activity wasnormalized to Renilla expression. H, The graph shows the luciferase transcript expression measured by qRT-PCR. All bars show the average of two to threeexperiments. Error bars, SD. Student t test was used. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.
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Fig. S3A–S3B). These results suggest that the regulation of TCF7L2byMALAT1 is post-transcriptional. To investigate the mechanismof TCF7L2 translational regulation by MALAT1, we characterizedpolysome-associated TCF7L2 mRNA in MALAT1-manipulatedcells. Overexpression of MALAT1 resulted in enhanced associa-tion of TCF7L2 mRNA with heavy polysomal fractions andreduced association with light and free polysomal fractions(Fig. 3E). Furthermore, transient knockdown ofMALAT1 resultedin increased association of TCF7L2 to free and light ribosomefractions and decreased association of TCF7L2 mRNA to heavyribosome fractions (Fig. 3F). This result suggests that MALAT1regulates TCF7L2 translation. Translation initiation ismediated inmany cases through the 50UTR, which can contain secondary RNAstructures or upstream open reading frames (ORF) that inhibittranslation (40). To examine whether MALAT1 regulates TCF7L2translation through the TCF7L2 50UTR, we subcloned TCF7L250UTR upstream of a luciferase reporter construct and measuredluciferase protein and mRNA levels following transient MALAT1knockdown. Knockdown of MALAT1 lead to a significant reduc-tion in luciferase activity from the TCF7L2 50UTR luciferasereporter whereas the mRNA levels of luciferase were not affected(Fig. 3G and H; Supplementary Fig. S3C and S3D). This resultsuggests thatMALAT1 regulates TCF7L2 translation, at least partly,through TCF7L2 50UTR.
TCF7L2 translation is regulated by mTORC1Wehave previously shown thatMALAT1 upregulation activates
the mTORC1 pathway. This was evident from the increasedphosphorylation of eIF4E binding protein (4EBP1) in PHM-1cells overexpressing MALAT1, whereas knockdown of MALAT1 inthese cells resulted in decreased phosphorylation of 4EBP1 (8).mTORC1 regulates numerous components involved in proteinsynthesis, ranging from initiation and elongation factors to thebiogenesis of ribosomes themselves (41). mTORC1 promotesprotein synthesis largely through the phosphorylation of two keyeffectors, p70 S6 Kinase 1(S6K1) and 4EBP1 (41). We took threedifferent approaches to assess the importance of mTORC1 acti-vation inMALAT1-mediated regulation of TCF7L2protein expres-sion. First,weused themTOR inhibitor rapamycin toblockmTORcatalytic activity as part of mTORC1. Increased expression ofTCF7L2 in MALAT1-overexpressing PHM-1 cells was reduced bytreatment of cells with rapamycin (Fig. 4A). Second, we usedshRNAs to knockdown either mTOR itself or Raptor, distinctivecomponents of mTORC1. Knockdown of either of these factorsreduced protein expression of TCF7L2 in PHM-1 cells (Fig. 4B andC). Thirdly, we overexpressed a mutant 4EBP1, 4EBP1-5A, inwhich the five known phosphorylation sites were replaced withalanine (42).Hyperphosphorylation of 4EBP1 is known to lead toactivation of cap-dependent translation. This mutant cannot bephosphorylated and binds constitutively to eIF4E, thus inhibitingits ability to enhance cap-dependent translation (43). Expressionof the dominant negative 4EBP1-5Amutant profoundly repressedexpression of TCF7L2, as compared with vector control (Fig. 4D).To further confirm the importance of cap-dependent translationin the TCF7L2 protein expression and MALAT1-mediated glyco-lytic effect, we ectopically expressed 4EBP1 wild type (WT) andmutant 4EBP1-4A (in which the four known phosphorylationsites were replaced with alanine; ref. 44) in PHM-1MALAT1 cells.Expression of the dominant negative 4EBP1-4A mutant pro-foundly repressed expression of TCF7L2 compared with 4EBP1WT (Supplementary Fig. S4A) and repressed the expression of
glycolytic genes (Supplementary Fig. S4B–S4E). These data sug-gest that the phosphorylation status of 4EBP1 is important forregulation of TCF7L2 protein expression and consequently itsdownstream targets.
MALAT1 regulates TCF7L2 translation through SRSF1MALAT1 was shown to activate the mTOR pathway by enhanc-
ing the expression and function of the splicing oncoproteinSRSF1 (8). It was shown previously that SRSF1 can activatemTORand protein translation. To investigate the molecular mechanismby which MALAT1 (which is a nuclear lncRNA), regulates thetranslation of TCF7L2 in the cytoplasm, we examined the regu-lationof TCF7L2by SRSF1. Stable knockdownof SRSF1 inHepG2cells resulted in reduced protein expression of TCF7L2 (Fig. 5A).Furthermore, knockdown of SRSF1 in PHM-1 cells resulted inreduced protein expression of TCF7L2 (Supplementary Fig. S5A).We detected no significant change in the mRNA levels of TCF7L2in response to SRSF1 knockdown in HepG2 and PHM-1 cells(Fig. 5B; Supplementary Fig. S5B). Ribosome fractionationshowed reducedbinding of TCF7L2mRNA to the heavy polysomefraction and elevated binding to the light polysome fraction uponSRSF1 knockdown (Fig. 5C). These results suggest that SRSF1regulates the expression of TCF7L2 post-transcriptionally byregulating its translation in hepatocellular carcinoma cells, aphenomenon seen for other proteins (45, 46).
TCF7L2mediates the effects of MALAT1 on glucosemetabolismSilencing of TCF7L2 protein levels in hepatocytes leads to an
increase in glucose output associated with elevated expression ofmultiple gluconeogenic genes (36, 47). TCF7L2 was shown to beoverexpressed and contribute to the malignant phenotype inHCC (48). TCF7L2 is an important mediator of theWnt signalingpathway, a signal transduction pathway that directly contributesto the regulationof cellularmetabolism. Becauseweobserved thatknockdown of MALAT1 resulted in increased expression of glu-coneogenic genes (Fig. 2E and F), we decided to examine whetherthe effect of MALAT1 on glucose metabolism is mediated viaTCF7L2. We introduced siRNAs targeting TCF7L2 into PHM-1MALAT1 cells (Fig. 6A and B). Knockdown of TCF7L2 reducedlactate production by MALAT1-overexpressing cells (Fig. 6C).Similarly, transient knockdown of TCF7L2 in HepG2 cellsincreased glucose secretion and reduced lactate production (Sup-plementary Fig. S6A–S6C). This suggests that TCF7L2 is involvedin MALAT1-mediated glucose metabolism. Next, we examinedglycolytic gene expression following transduction of PHM-1 cellsoverexpressing MALAT1 with lentiviruses encoding shRNAsagainst TCF7L2 (Fig. 6D). TCF7L2 knockdown reduced glycolyticgene expression in these cells (Fig. 6E–H). InHepG2 cells, TCF7L2knockdown increased the expression of gluconeogenic genesG6PC and PCK1 (Supplementary Fig. S6D and S6E). Collectively,these findings suggest that TCF7L2 is involved in MALAT1-regulated glucose metabolism in cancer cells.
MALAT1 and TCF7L2 regulate gluconeogenesis through thesame pathway
To examine whether MALAT1 and TCF7L2 are regulating glu-coneogenesis through the same pathway, we knocked down bothMALAT1 and TCF7L2, either individually or together, in FLC4cells (Supplementary Fig. S7A and S7B). We examined gluconeo-genesis gene expression in these cells. We found that transientknockdown of both MALAT1 and TCF7L2, either individually or
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TCF7L2 translation is regulated bymTORC1-4EBP1. A, Left, Western blot analysis of PHM-1 cells transduced with lentivirus encoding either MALAT1 or an emptyvector in the presence or absence (DMSO) of rapamycin. Right, quantification of TCF7L2 protein levels upon rapamycin treatment (n¼ 4). B, Left, Western blotanalysis of PHM-1 cells transduced with retroviruses encoding mTOR shRNAs. Right, quantification of mTOR and TCF7L2 protein levels upon shRNA knockdown(n¼ 3). C, Left, Western blot analysis of PHM-1 cells transduced with retroviruses encoding Raptor shRNAs. Right, quantification of Raptor and TCF7L2 proteinlevels upon shRNA knockdown (n¼ 4). D, Left, Western blot analysis of PHM-1 cells transduced with retroviruses encoding for empty vector pWZL-Hygro(empty) or PWZL-4EBP phosphorylation defective mutant (4EBP1-5A). Right, quantification of phosphorylated 4E-BP1 (n¼ 2) and TCF7L2 protein levels uponoverexpression of PWZL-4EBP1-5A (n¼ 2). Error bars, SD. Student t test was used. � , P < 0.05; ��, P < 0.01; ��� , P < 0.001.
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together, resulted in increased gluconeogenic gene expressionwithout a significant additive effect (Supplementary Fig. S7C andS7D) without a significant additive effect. Similar results wereobtained in HepG2 cells (Supplementary Fig. S7E–S2H). Theseresults suggest that MALAT1 and TCF7L2modulate gluconeogen-esis through the same pathway.
TCF7L2 acts downstream of MALAT1To examine the potential of TCF7L2 as a downstream mod-
ulator of MALAT1, we transduced PHM-1 cells with lentivirusesencoding either TCF7L2 or an empty vector (Fig. 6I and J).Overexpression of TCF7L2 lead to enhanced lactate production(Fig. 6K). In contrast, transient knockdown ofMALAT1 by siRNAsin TCF7L2 overexpressing PHM-1 cells (Fig. 6L) did not showsignificant changes in lactate production (Fig. 6M), suggestingthat TCF7L2 acts as a downstream effector of MALAT1.
TCF7L2 is required for MALAT1-mediated transformationTo examine whether TCF7L2 upregulation mediates MALAT1
induced transformation, we knocked down TCF7L2 in PHM-1cells overexpressing humanMALAT1 (Fig. 7A). Stable knockdownof TCF7L2 in these cells resulted in decreased survival in aclonogenic assay (Fig. 7B) and reduced formation of colonies insoft agar (Fig. 7C), demonstrating that cells overexpressingMALAT1 require TCF7L2 overexpression for their oncogenicproperties. Knockdown of TCF7L2 in PHM-1 cells overexpressinghuman MALAT1 did not show a strong effect on the proliferativecapacity of these cells (Fig. 7D and E). To validate the importanceof TCF7L2 upregulation in HCC cells, we stably or transientlyknocked down TCF7L2 inHepG2 (Supplementary Fig. S8A) cells.TCF7L2 knockdown in these cells resulted in reduced formationof colonies in soft agar (Supplementary Fig. S8B). TCF7L2 knock-
down in FLC4 cells showed similar results (Supplementary Fig.S8C and S8D). These results suggest that TCF7L2 is required forthe maintenance of the oncogenic properties of HCC cells.
Elevated nuclear expression of TCF7L2 in tumors from amousemodel of HCC
Wenext sought todeterminewhether in an in vivomousemodelof HCC (Mdr2�/� mice), which is known to upregulateMALAT1 (8), TCF7L2 is upregulated and if its expression corre-lates with expression of genes controlling glycolysis and gluco-neogenesis. We examined TCF7L2 mRNA and protein expressionin tumor and non-tumor inflamed liver samples from Mdr2�/�
mice. Both Western blot analysis and immunohistochemistryshow that the protein expression of TCF7L2 was elevated in thetumor samples compared with the non-tumor liver samples, withnuclear localization in HCC tumors compared with adjacentparenchyma (Fig. 7F; Supplementary Fig. S9A). Similar to whatwas detected in cell lines, TCF7L2 mRNA levels were not signif-icantly different in HCC tumors compared with non-tumor livers(Supplementary Fig. S9B). Increased TCF7L2 protein levels wasstatistically significant (Supplementary Fig. S9C). Normalizationof protein to mRNA levels in these tumors suggests that there isincreased protein to mRNA ratios of TCF7L2 in most of thetumor samples compared with the normal samples (Supple-mentary Fig. S9D). Furthermore, mRNA analysis by qRT-PCRshowed upregulation of multiple glycolytic genes (Supplemen-tary Fig. S9E), and downregulation of gluconeogenic genes(Supplementary Fig. S9F), in most of the tumor samplescompared with normal samples. Taken together, our resultssuggest a potential role for MALAT1 in the regulation ofglycolytic and gluconeogenic gene expression in HCC throughTCF7L2. This reveals yet another oncogenic function of
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SRSF1 regulates TCF7L2 translation.A, Left, Western blot analysis ofHepG2 cells transduced withlentiviruses containing shRNAsagainst SRSF1 (shSF2#1, #2, #3) oran empty vector (shCon). b-Actinwas used as loading control. Right,quantification of SRSF1 and TCF7L2protein levels upon shRNAknockdown (n¼ 3). B, qRT-PCR ofTCF7L2 mRNA levels in cellsdescribed inA. C. Relativedistribution of TCF7L2 mRNAacross the polysome fractions inHepG2 cells transfected with eithercontrol shRNA or shRNAs againstSRSF1. Error bars, SD. Student t testwas used. � , P < 0.05; �� , P < 0.01;���, P < 0.001.
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MALAT1, promoting the "Warburg effect" and repressing glu-coneogenesis during the development of human HCC.
DiscussionIncreased aerobic glycolysis, or the "Warburg effect," is one of
the first identified hallmarks of cancer (32). However, the under-
lyingmolecularmechanisms leading to this phenomenon remainunclear in many tumors. The present study reveals an unexpectedfunction of lncRNAMALAT1 in promoting aerobic glycolysis andrepressing gluconeogenesis in HCC, adding to its previouslyknown oncogenic activities (8, 49, 50). Our initial observationthat MALAT1 overexpression in PHM-1 cells changes the color ofthe cell medium, led us to explore the role of this oncogenic
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Figure 6.
TCF7L2 mediates MALAT1 effectson glucose metabolism. A, Left,Western blot of PHM-1 cellsoverexpressing MALAT1transfected with siTCF7L2. Right,quantification of TCF7L2 proteinlevels upon TCF7L2 siRNAtreatment (n¼ 2). B, qRT-PCR ofcells described in A. C, Extracellularlactate production was measured incells described in A using a lactateassay kit (n¼ 3). D,Western blotanalysis of PHM-1 cellsoverexpressing MALAT1 aftertransduction with lentivirusesexpressing TCF7L2 shRNAs.E–H, qRT-PCR of the indicatedgenes in the glucose metabolicpathway in cells described in D. Allsamples were normalized toGAPDHmRNA levels. I,Westernblot analysis of PHM-1 cells stablyexpressing TCF7L2 or an emptyvector. J, qRT-PCR of cellsdescribed in I. K, Extracellularlactate production was measured incells described in I using a lactateassay kit (n¼ 2). L, qRT-PCR ofPHM-1 cells overexpressing TCF7L2and knocked down for MALAT1 bysiRNA.M, Extracellular lactateproductionwas measured in cellsdescribed in L using a lactate assaykit (n¼ 2). Error bars, SD. Studentt test was used. � , P < 0.05;�� , P < 0.01; ��� , P < 0.001.
LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism
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TCF7L2 is required for MALAT1-induced transformation. A, Left, Western blot of PHM-1 cells overexpressing MALAT1 transduced with lentiviruses encodingTCF7L2 shRNAs. Right, quantification of TCF7L2 protein levels upon TCF7L2 knockdown by shRNA (n¼ 2). B, Clonogenic assay of control PHM-1 cells stablyexpressing empty vector and cells described in A. C,Growth in soft agar assay of control PHM-1 cells stably expressing empty vector and cells describedinA. Colonies were counted 28 days after seeding. Error bars, SD (n¼ 3). Two tailed Student t test was used. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. D,Proliferation assay of PHM-1 cells stably expressing hMALAT1 or an empty vector. E, Proliferation assay of cells described in A. F, Immunohistochemistry stainingfor TCF7L2 in liver tumor (T) specimens, including surrounding parenchymal (P) tissue from two 12-months-old Mdr2�/�mice (scale bar, 50 mm). Note theenhanced nuclear TCF7L2 staining in malignant hepatocytes. G, Scheme summarizing the role of MALAT1 in regulating glucose metabolism in HCC. Red linesrepresent pathways by which MALAT1 regulates glucose metabolism in HCC as described here. Blue lines represent pathways described by others.
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lncRNA in facilitating aerobic glycolysis. Indeed, we found afunctional correlation between MALAT1 expression, glucosesecretion and lactate production in PHM-1 cells overexpressingMALAT1 and HCC cells (Figs. 1 and 2). Using flow cytometry, wedemonstrate glucose uptake at a single cell level, eliminating thepossibility that it is due to cell density or cell proliferation.Furthermore, MALAT1 overexpression in HCC cells increasedthe expression of glycolytic genes and its knockdown resultedin increased expression of gluconeogenic genes (Figs. 1 and 2;Supplementary Fig. S2).
Gluconeogenesis is a process that consumes energy to regen-erate glucose in the liver, secreting it to the blood when bloodglucose levels drop (34). In this pathway three specific steps,catalyzed by gluconeogenesis enzymes, are used to bypass theirreversible reactions of glycolysis. In this regard, simultaneousactivation of both pathways may result in a futile cycling ofglucose that is detrimental to cell survival (51). To avoid suchfutility, activationof either pathway should bemutually exclusive.Results of our experiments, with knockdown of MALAT1 in HCCcell lines, demonstrate increased expression of gluconeogenicgenes. Considering the essential requirement of energy and build-ing blocks for cell doubling, it is evident that upregulation ofgluconeogenesis, an energy consuming process, would result inthe suppression of HCC proliferation. This is indeed the case asknockdown of MALAT1 in HCC cell lines resulted in decreasedproliferation (8). As gluconeogenesis is dramatically impaired inmalignant hepatocytes, similar to what we observed in Mdr2�/�
liver tumor samples compared with adjacent normal liver paren-chyma, it is possible that gluconeogenesis represents a metabolicbarrier to HCC development. This is the first report showing theregulation of gluconeogenesis by MALAT1 in HCC. Importantly,we also establish regulation of various glycolytic genes (GLUT1,HK2, ENO1, and PKM2) byMALAT1 in PHM-1 cells. Tomaintainthe survival and rapid proliferation, cancer cells normally elevateexpression of glycolytic genes. Several oncoproteins and tumorsuppressors were found to regulate enzymes that facilitate glyco-lytic tumor glucose metabolism. In this study, we report for thefirst time that lncRNA MALAT1 regulates an array of glycolyticgenes in HCC.
Gluconeogenesis has been shown to be negatively regulatedby TCF7L2 in various studies (36, 47). Even though there are nostudies showing the role of TCF7L2 in glycolysis or the "War-burg effect," TCF7L2 has been implicated in HCC in variousstudies (48) and has been shown to be an important mediatorof the Wnt signaling pathway. Wnt signaling has been shown tomodulate the "Warburg effect" (15). This prompted us to lookat the regulation of TCF7L2 by MALAT1. The elevation inTCF7L2 protein levels upon MALAT1 overexpression, as wellas the decrease in TCF7L2 protein levels upon MALAT1 knock-down, results from translational regulation and is not a resultof changes in transcription or stability (Fig. 3). Control ofmRNA translation constitutes a critical step in the regulationof gene expression and in cancer (52, 53). Polysome profilingof TCF7L2 mRNA showed increased translation of TCF7L2 inMALAT1-overexpressing cells while reduced translation ofTCF7L2 in MALAT1 knockdown cells (Fig. 3). Translationinitiation efficiency can be regulated by the 50UTR. SecondaryRNA structures at the 50UTR of many mRNAs inhibit translationinitiation and this inhibition can be alleviated by RNA heli-cases, which are recruited by the eIF4G–eIF4E complex (40).Knockdown of MALAT1 resulted in reduced 50UTR activity of
TCF7L2 as measured by luciferase activity without an effect onluciferase mRNA levels (Fig. 3).
Signaling by the PI3K/AKT/mTOR pathway profoundly affectsmRNA translation through phosphorylation of downstream tar-gets, such as 4EBP1 and S6K1 (54). The cap-dependent proteinsynthesis pathway serves as a pleotropic integrator and amplifierof many essential oncogenic signals (53, 55). Our data show thatTCF7L2 is specifically regulated by the mTORC1–4EBP1 axis(Fig. 4). Because TCF7L2 is a major transcriptional regulator ofthe Wnt pathway, it is possible that the mTORC1 pathway,through its regulation of TCF7L2 translation, can modulate theWnt pathway. The crosstalk between these two signaling pathwayshas not been demonstrated to our knowledge. Next, to furthersubstantiate the regulation of cytoplasmic TCF7L2 translation bynuclearMALAT1,we lookedat the regulationof TCF7L2by SRSF1.SRSF1 is a nuclear splicing factor and MALAT1 was shown toregulate the expression and function of SRSF1. SRSF1 was shownto activatemTOR and protein translation (45, 46, 56). Indeed, wefound that SRSF1 knockdown reduced TCF7L2 translation inHepG2 cells (Fig. 5). This result suggests that increased proteinexpression of TCF7L2 by MALAT1 could be, in part, attributed toincreased expression of SRSF1 by MALAT1. This result also pro-vides an explanation for the reduced expression of TCF7L2 in thepresence of Rapamycin, as knockdown of SRSF1, which is knownto activate mTORC1, in PHM-1 and HepG2 cells resulted inreduced protein expression of TCF7L2 (Figs. 4 and 5).
The result of increased expression of gluconeogenesis genesupon TCF7L2 knockdown is in agreement with previous studieswhere it was shown that TCF7L2 is a negative regulator ofgluconeogenesis (36, 47). Regarding glycolytic gene expressionregulation by TCF7L2, this is the first description of the regulationof glycolytic gene expression by TCF7L2 in HCC.
As expected from the alteration in glycolytic and gluconeogenicenzyme expression, overexpression of TCF7L2 resulted inincreased lactate production while knockdown of TCF7L2resulted in decreased lactate production (Fig. 6). These resultssuggest direct regulation of cancer glucose metabolism byTCF7L2. Furthermore, knockdown of MALAT1 in TCF7L2-over-expressing cells did not change lactate production, suggesting thatTCF7L2 acts downstream to MALAT1.
Because TCF7L2 regulates glucose metabolism and actsdownstream of MALAT1, we sought to examine the importanceof TCF7L2 in MALAT1-mediated oncogenic activity. To this endwe knocked-down TCF7L2 in PHM-1 cells overexpressingMALAT1. Stable knockdown of TCF7L2 in these cells resultedin decreased survival in a clonogenic assay and reduced for-mation of colonies in soft agar (Fig. 7). Furthermore, knock-down of TCF7L2 in HCC cell lines (HepG2 and FLC4) resultedin reduced oncogenic properties as seen by reduced formationof colonies in soft agar (Supplementary Fig. S8). These resultssuggested that TCF7L2 expression is essential for MALAT1-mediated transformation. TCF7L2 overexpression in HCC hasbeen reported in several studies (48). Our western blot andgene expression analysis on Mdr2�/� mice liver tumor samplesrevealed strong upregulation of MALAT1 at the RNA level andTCF7L2 at the protein level (Fig. 7; Supplementary Fig. S9).Furthermore, Mdr2�/� liver tumor samples showed elevatedglycolytic and repressed gluconeogenic gene expression (Sup-plementary Fig. S9), suggesting a significant relevance ofMALAT1-mediated tumor glucose metabolism in the develop-ment of tumors in vivo.
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It is important to note that the true clinical implications of theseresults needs to be examined further in human normal liver andHCC clinical samples.
ConclusionTaken together, our data suggest thatMALAT1acts as a regulator
of glucose metabolism in HCC. Our results add insight to themechanisms of cancer glucose metabolism and cancer progres-sion. The novel findings from the present study, together with thesignificant discoveries fromprevious studies, placeMALAT1 at thecrossroad of cellular metabolism and carcinogenesis (Fig. 7G).MALAT1 regulates the expression of TCF7L2 at the translationallevel. TCF7L2 regulation by MALAT1 is through a mTORC1-dependent pathway via cap-dependent translation. TCF7L2 playsan important role in MALAT1-induced tumorigenesis and alteredglucose metabolism in HCC development. These results pointtoward the fact that knockdown of MALAT1 or reduction ofTCF7L2 levels might serve as new strategies based on tumorglucose metabolism for the treatment of HCC.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: P. Malakar, R. KarniDevelopment of methodology: P. MalakarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Malakar, I. Stein, A. Saragovi, R. Winkler,N. Stern-Ginossar, M. Berger, E. PikarskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.Malakar, I. Stein, A. Saragovi, R.Winkler,M. Berger,R. KarniWriting, review, and/or revision of the manuscript: P. Malakar, R. KarniStudy supervision: R. Karni
AcknowledgmentsThe authors wish to thank Dr. Zahava Siegfried for comments on the article
and Fatima Gebauer (CRG, Barcelona) for the pSG5 Luc Plasmid. This studywas supported in part by Israel Science Foundation (ISF; ISF Grant no. 1290/12to R. Karni).
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 10, 2018; revised January 10, 2019; accepted March 20, 2019;published first March 26, 2019.
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www.aacrjournals.org Cancer Res; 79(10) May 15, 2019 2493
LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism
2019;79:2480-2493. Published OnlineFirst March 26, 2019.Cancer Res Pushkar Malakar, Ilan Stein, Amijai Saragovi, et al. Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2Long Noncoding RNA MALAT1 Regulates Cancer Glucose
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