Long Noncoding RNA MALAT1 regulates cancer glucose ... · 3/26/2019 · 1 Long Noncoding RNA...

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Long Noncoding RNA MALAT1 regulates cancer glucose metabolism by enhancing

mTOR-mediated translation of TCF7L2

Pushkar Malakar1, Ilan Stein

2, Amijai Saragovi

2, Roni Winkler

3, Noam Stern-Ginossar

3, Michael

Berger2, Eli Pikarsky

2 and Rotem Karni

1*

1. Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel

Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem, 9112001 Israel,

2. The Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research

Israel Canada (IMRIC) and Department of Pathology, Hebrew University–Hadassah Medical

School, Jerusalem, 9112001 Israel. 3. Department of Molecular Genetics, Weizmann Institute of

Science, 76100, Rehovot, Israel.

* Correspondence should be addressed to Rotem Karni, Department of Biochemistry and

Molecular Biology, Hebrew University-Hadassah Medical School, 9112001, Jerusalem, Israel.

e-mail: [email protected]; Phone: +972-2-6758289.

Running title: Long Noncoding RNA MALAT1 regulates cancer glucose metabolism.

Conflict of interest statement: The authors declare that there are no conflicts of interest to

disclose.

Keywords: Long Noncoding RNAs, MALAT1, Glycolysis, Gluconeogenesis, Hepatocellular

Carcinoma, TCF7L2, mTOR

Research. on December 8, 2020. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 26, 2019; DOI: 10.1158/0008-5472.CAN-18-1432

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http://cancerres.aacrjournals.org/

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Abstract

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is

known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating

cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We

previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1

(MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here we investigated

the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the

expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the

translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2

translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis.

Pharmacological or genetic inhibition of mTOR and Raptor or expression of a

hypophosphorylated mutant version of eIF4E binding protein (4EBP1) resulted in decreased

expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy

polysomes, probably through the TCF7L2 5'UTR as determined by polysome fractionation and

5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell

lines affected their metabolism and abolished their tumorigenic potential, suggesting that the

effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken

together, our findings suggest that MALAT1 contributes to HCC development and tumor

progression by reprogramming tumor glucose metabolism.

Significance

Findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer

glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated

translation of the transcription factor TCF7L2.




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Introduction

Long noncoding RNAs (lncRNAs) constitute a large class of mRNA-like transcripts, greater than

200 nucleotides with no protein coding capability (1). In the past few years, several lncRNAs

have been shown to play a role in cancer by promoting proliferation, invasion and metastasis (2-

4). LncRNAs have been shown to regulate almost every step of gene expression (5). MALAT1

was one of the first lncRNAs to have a designated role in cancer (6) (4). MALAT1 is highly

conserved among mammals, approximately 7Kb in length and highly abundant (7). Previously,

we showed that MALAT1 acts as a proto-oncogene in hepatocellular carcinoma through Wnt

pathway activation, induction of the splicing factor SRSF1 and mTORC1 activation (8).

Furthermore, we showed that mTORC1 activation is required for MALAT1 mediated

tumorigenesis (8).

Both the Wnt and mTOR signaling pathways have been shown to play an important role in

altering the glucose metabolic program in cancers (9,10). Altered glucose metabolism is one of

the first identified hallmarks of cancer (11), discovered by Otto Warburg in the late 1920s (12).

Cancer cells predominantly carry out glycolysis in the cytosol rather than oxidative

phosphorylation through the TCA cycle in the mitochondria (13). It is generally believed that in

most cancers, oncogenic lesions are largely the cause of enhanced glycolysis and the “Warburg

effect” (14). c-Myc, a downstream target of Wnt signaling, was shown to play an important role

in the regulation of glycolysis in cancer cells (15). Glucose metabolism genes were shown to be

directly regulated by c-Myc . The key modulator of the canonical Wnt signaling pathway is the

bipartite transcription factor β-Cat (β-catenin)/TCF, formed by β-Catenin and a member of the

TCF family (TCF-1, LEF-1, TCF-3 and TCF-4/TCF7L2) (16). TCF7L2 was shown to be an

effector of the Wnt signaling pathway and binds directly to multiple genes that are important in

regulating glucose metabolism . Moreover, genome-wide association studies (GWAS) have

identified SNPs in the TCF7L2 gene associated with obesity and diabetes (17). mTOR activation

regulates glucose metabolism through activation of HIF-1α. HIF-1α is a transcription factor that

is known to induce the expression of at least 9 glycolytic enzymes, thereby regulating glucose

metabolism in many cancers (18). Several lncRNAs have been shown to regulate, or to be

regulated by, the Wnt and mTOR signaling pathways (19,20). Even though lncRNAs have been




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shown to affect cancer initiation and progression, only a handful of studies have focused on the

involvement of lncRNAs in cancer glucose metabolism at the level of glycolysis (21,22).

Gluconeogenesis is essentially the reverse of the glycolysis pathway and usually occurs in the

liver when blood glucose levels drop and the liver regenerates glucose, sending it to other tissues

(23). There is a single report describing the role of lncRNAs in cancer development through

regulating gluconeogenesis. lncRNA Nur77 was shown to suppress HCC through upregulating

gluconeogenesis (24). There are no reports describing the role of MALAT1 in cancer glucose

metabolism at the level of glycolysis or gluconeogenesis. Several studies have shown

gluconeogenesis to be downregulated in HCC (25,26). However, the regulation of both

glycolysis and gluconeogenesis by lncRNAs in HCC development or progression has not been

reported.

In this study, we investigated the roles of MALAT1 in regulating glucose metabolism of HCC

cancer cells and found that MALAT1 enhanced aerobic glycolysis and repressed

gluconeogenesis. We further discovered that MALAT1 regulates glycolytic gene expression

through increased translation of transcription factor TCF7L2. We demonstrate, both

pharmacologically and genetically, that TCF7L2 upregulation is mediated by mTORC1

activation of cap-dependent translation. MALAT1-mediated tumorigenesis is dependent on

TCF7L2. In addition, using Mdr2-/- mice liver tumor samples we show elevated levels of

MALAT1, nuclear TCF7L2 and glycolytic gene expression and decreased expression of

gluconeogenic gene expression suggesting a positive correlation with glycolysis and a negative

correlation with gluconeogenesis. Thus, we present here a novel function for MALAT1 in

tumorigenesis and provide a previously unappreciated mechanism by which cancer cells switch

to aerobic glycolysis, repressing gluconeogenesis, during cancer progression. This is the first

report showing the regulation of TCF7L2 by mTORC1-mediated cap-dependent translation and

suggests that the mTORC1 pathway can regulate Wnt signaling through TCF7L2 translation.

Materials and Methods

Cell culture

PHM-1 cells are mouse liver progenitor cells derived from embryonic day 18 fetal livers from

TP53−/−

mice and immortalized with MSCV-based retroviruses expressing MYC-IRES-GFP.




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(27). PHM-1, FLC4 and HepG2 cells were grown in DMEM supplemented with 10% FCS, 0.1

mg/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines have been tested and authenticated

using STR loci plus Amelogenin for gender identification for human cell line authentication by

the Biosynthesis DNA Identity Testing Centre.

Stable cell lines

pCD513B1 empty (System Biosciences) and pCD513B1-hMALAT1 lentiviruses were prepared

using the manufacturer's instructions. These viruses were used to infect PHM-1 cells. Cells were

selected by the addition of puromycin (2 μg/mL) for 72–96 hours. In the case of infection with

MLP-puro-shRNA viruses, cells were selected with puromycin (2 μg/mL) for 96 hours.

siRNA treatment

Double-stranded siRNAs (Sigma) were used at specified concentrations to deplete MALAT1 or

TCF7L2 from cells. siRNAs against Luciferase (Dharmacon Thermo Scientific) or siRNA

Universal Negative Control (Sigma) was used as a control at specified concentrations.

Lipofectamine 2000 reagent (Invitrogen) was used for transfection as per the manufacturer's

instructions.

qRT-PCR

Total RNA was extracted with TRI Reagent (Sigma), and 1 μg of total RNA was reverse

transcribed using M-MLV reverse transcriptase (Promega) after DNase treatment (Promega).

qPCR was performed on the cDNA using SYBR Green Mix (Roche) and CFX96 (Bio-Rad) real-

time PCR machine. Primer list is supplied in Supplementary Table S1.

Immunoblotting

Cells were lysed in Laemmli buffer and analyzed for total protein concentration. Twenty

micrograms of total protein from each cell lysate was separated by SDS-PAGE and transferred to

a polyvinylidene difluoride (PVDF) membrane. Primary antibodies used were TCF7L2 EP20334

(1:10,000; Abcam), GAPDH (1:5000; Sigma), β-catenin (1:2,000; Sigma), β-actin (1:2,000;

Sigma). α-tubulin (1:1,000; Santa Cruz Biotechnology) ,β-Tubulin (1:2000;Sigma). SRSF1

(AK96 culture supernatant 1:300), T7 Tag (1:5000; BD Transduction laboratories), mTOR




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(1:1000; Cell Signaling Technology), Raptor (1:1000; Cell Signaling Technology), p4EBP1

(1:1000; Cell Signaling Technology), Total 4EBP1 (1:1000; Cell Signaling Technology).

Secondary antibodies used were HRP-conjugated goat anti-mouse, goat anti-rabbit, donkey anti-

goat IgG (H+L; 1:10,000; Jackson Laboratories).

Colony formation assay

Cells were seeded in six-well plates (1,000 cells/well) and grown for 10 days. After fixation with

2.5% glutaraldehyde, the plates were washed three times. Fixed cells were then stained with

methylene blue solution (1% methylene blue in 0.1 mol/L borate buffer, pH 8.5) for 60 minutes

at room temperature. Plates were photographed after extensive washing and air drying (28).

Anchorage-independent growth

Colony formation in soft agar was assayed as described previously [41]. After 14 to 21 days,

colonies from 10 different fields in each of two wells were counted for each treatment and the

average number of colonies per well was calculated. The colonies were stained and

photographed under a light microscope at ×10 magnification (28).

Lactate assay

Cells (2x105) were seeded in six-well culture plates. The cells were trypsinized 48 hours after

culture or siRNA treatment. Cells were homogenized in the presence of lactate assay buffer and

centrifuged at 13,000g for 10 minutes. Lactate quantification was performed using commercially

available lactate assay kit (Abcam, ab65330) in a 96-well plate as per the manufacturer’s

instructions. Lactate levels were measured using a plate reader at an optical density of 570 nm

(29). Lactate levels were normalized to total cellular protein concentration.

Glucose secretion assay

HepG2 and FLC4 cells were cultured and treated with siRNAs. After 48 hours of siRNA

treatment, the medium was replaced with DMEM containing 0.1% serum for 16 hours. Cells

were washed twice with PBS to remove glucose and then incubated for 6 hours in glucose

production assay medium (glucose and phenol red-free DMEM containing 2 mM sodium

pyruvate, 20 mM sodium lactate, 2 mM L-glutamine and 15 mM HEPES). Medium (200µl) was




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sampled for measurement of glucose concentration. Glucose level quantification was performed

using a commercially available glucose assay kit (Amplex Red Glucose Assay Kit,

ThermoFisher Scientific) in a 96-well plate. Glucose levels were normalized to total cellular

protein concentration.

Mice

All 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.

Immunohistochemistry

Immunohistochemistry for TCF7L2 was performed on 5-μm formalin-fixed paraffin-embedded

sections. After citrate-based antigen retrieval (Vector Labs # H-3300), endogenous peroxidase

was blocked by 3% H2O2. Slides were incubated with primary antibody (-TCF7L2, abcam #

ab76151), washed, and incubated with anti-Rabbit-HRP ImmPRESS™ Reagent (Vector Labs #

MP-7401). Slides were developed with the HRP substrate diaminobenzidine (Thermo Scientific)

and counterstained with hematoxylin.

Polysome Profiling

Polysome profile analysis was carried out as described previously (31). Briefly, cells were

cultured in 10cm dishes. Before harvesting cells were treated with cyclohexamide (20 µl CHX

from 50mg/ml stock) for 3 minutes. Then cells were washed twice with cold PBS containing 50

mg/mL cycloheximide, collected, and lysed in a 250 µl of lysis buffer [Lysis Buffer (5 ml):

l 20% Triton (RNAse free) + 4.75ml Polysome buffer + 60 µl DNAse (120 units) ].

Lysates were loaded onto 10% to 50% sucrose density gradients prepared in polysome

buffer. [Polysome Buffer (20ml) : 250 µl of 1M Tris pH 7, 150 µl of 1M Tris pH 8, 600 µl of

5M NaCl, 100 µl of 1M MgCl2, 40 µl of CHX (50mg/mL in EtOH), 20 µl l of DTT (1M) and

18.84 ml of DEPC Water]. Extracts were fractionated for 3 hrs at 35,000 rpm at 4°C in a

Beckman rotor, and the gradients were recovered in 12 fractions using gradient fractionators.

RNA was extracted from each fraction. Translational status of TCF7L2 mRNA on polysome

fractions was determined by qRT-PCR.




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Luciferase reporter assay

507 bp of human TCF7L2, 5’ untranslated region (UTR) upstream of the start codon, were

amplified from FLC4 cells cDNA by RT-PCR using a forward primer with a KpnI restriction

site and a reverse primer with a XhoI restriction site and subcloned into the KpnI and XhoI

restriction sites of the pSG5 Luc plasmid. The insert was verified by sequencing. pSG5 Luc

Plasmid was a kind gift from Prof. Fatima Gebauer, Centre for Genomic Regulation (CRG),

Barcelona. PHM-1, HepG2 and FLC4 cells were seeded in six-well plates (2x105 cells/ well)

under standard conditions. After 24 hrs, cells were transfected with MALAT1 siRNAs using

Lipofectamine 2000. After another 48 hrs these cells were further transfected, using

polyethylenimine, with 2µg of TCF7L2-5’UTR-Firefly construct and 0.5µg pRenilla construct

per well. 48 hours later, the cells were harvested and luciferase activity was analyzed using Dual-

Glo Luciferase Assay System according to the protocol provided by Promega and Infinite M200

PRO. Renilla activity was used to normalize for transfection efficiency. Firefly-luciferase mRNA

expression was also measured as an additional control for luciferase activity.

Glucose uptake in FLC4 cells

FLC4 cells were treated with siRNAs targeted against luciferase or MALAT1 for 48 hours.

Subsequently, they were treated with medium without glucose for 16 hours and then exposed to

2NBDG (a fluorescent derivative of glucose) for 30 minutes. 2NBDG fluorescence was recorded

using flow cytometry.

Statistical analysis.

Error bars for all data represent SDs from the mean. P values were calculated using two tailed

type 2 Student t-tests except for a few cases where tail one type 2 student t test was used.

Statistical significance is displayed as *<0.05, **<0.01 and ***<0.001.

Results

MALAT1 affects glucose metabolism in immortalized and cancerous liver cells, promoting

aerobic glycolysis.




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One of the first identified hallmarks of cancer is altered glucose metabolism (32). Tumor cells

enhance glycolysis even in the presence of oxygen and in many cases reduce oxidative

phosphorylation (13). In addition, many oncogenes enhance glycolysis by alternative

mechanisms (18). We previously found that the lncRNA MALAT1 acts as a proto-oncogene in

HCC development and activates the mTORC1 pathway (8). The mTORC1 pathway affects

tumor metabolism by several mechanisms and specifically glucose metabolism (33). Thus, we

sought to examine the effect of MALAT1 on glucose metabolism in HCC cancer cells. One of

the characteristics of hepatocytes is their ability to produce glucose by gluconeogenesis, in order

to supply glucose to the body when blood glucose levels drop (23). This process acts in a reverse

pathway to glycolysis. We measured lactate production as a measure for glycolysis in PHM-1

cells either overexpressing or knocked-down for MALAT1. Lactate production was measured

by the intracellular lactate content. Overexpression of MALAT1 led to enhanced lactate

production (Fig. 1A and B). Conversely, transient knockdown of MALAT1 by siRNAs resulted

in reduced lactate production (Fig. 1C and D). These data suggest that MALAT1 expression

regulates glucose metabolism in PHM-1 cells by enhancing glycolysis. To eliminate possible

effects of cell proliferation or cellular density on glucose metabolism, we examined glucose

uptake at a single cell level. Cells were labelled with fluorescent glucose (2NBDG) and glucose

uptake was measured by flow cytometry. We found that glucose uptake was lower following

MALAT1 knockdown in HCC FLC4 cells (Fig. S1A-B). In previously performed RNA-seq

analysis on PHM-1 cells overexpressing MALAT1 we detected increased expression of several

glycolytic genes in (8). To confirm the transcriptional regulation of the glucose metabolism

program in hepatocytes by MALAT1 we validated several of the upregulated genes. In

agreement with enhanced glycolysis, the expression of several glycolytic enzymes was

upregulated in cells overexpressing MALAT1 (Fig. 1E- G) and reduced by knockdown of

MALAT1 (Fig. S1C-E). These data suggest that MALAT1 expression promotes glycolytic

metabolism in cancer cells.

MALAT1 negatively affects gluconeogenesis.

Gluconeogenesis is a major component of glucose metabolism in normal liver cells, regulating

whole body glucose homeostasis (34). In HCC, gluconeogenesis plays a tumor suppressive role

opposing aerobic glycolysis and preventing the “Warburg effect” (35). To examine the effect of




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MALAT1 on gluconeogenesis in HCC, we knocked-down MALAT1 in HCC cell lines and

examined gluconeogenic gene expression. Expression of gluconeogenic genes is downregulated

in HCC compared to normal hepatocytes (25,26). We found that transient knockdown of

MALAT1 by siRNAs in HepG2 cells (Fig. 2A) and FLC4 cells (Fig. S2A) resulted in increased

glucose secretion (Fig. 2B; Fig. S2B) and reduced lactate production (Fig. 2C; Fig. S2C).

Transient knockdown of MALAT1 in these cells also resulted in increased expression of

gluconeogenic genes, G6PC and PCK1 (Fig. 2D; Fig. 2E; Fig. 2F; Fig. S2D and E).

MALAT1 controls TCF7L2 expression at the protein level.

The transcription factor TCF7L2 was shown to modulate glucose homeostasis in the liver

(36,37). Moreover, it has been shown that TCF7L2 negatively regulates gluconeogenesis (38).

Although the role of TCF7L2 in the Wnt signalling pathway is well studied, its role in

modulating glucose metabolism is less well characterized and in some cases conflicting (39).

Thus, we examined the regulation of TCF7L2 by MALAT1. To probe the potential mechanism

by which MALAT1 regulates TCF7L2, we examined the effects of MALAT1 manipulation on

the expression of TCF7L2. We detected no significant change in the mRNA levels of TCF7L2

in response to MALAT1 overexpression or knockdown (Fig. 3A). In contrast to these results,

western blot analysis showed that MALAT1 overexpression resulted in enhanced TCF7L2

protein expression (Fig. 3B). Transient knockdown of MALAT1 did not change TCF7L2 mRNA

level (Fig. 3C), but resulted in decreased protein expression of TCF7L2 (Fig. 3D; Fig. S3A-B),

without a These results suggest that the regulation of TCF7L2 by MALAT1 is post-

transcriptional. To investigate the mechanism of TCF7L2 translational regulation by MALAT1,

we characterized polysome-associated TCF7L2 mRNA in MALAT1 manipulated cells.

Overexpression of MALAT1 resulted in enhanced association of TCF7L2 mRNA with heavy

polysomal fractions and reduced association with light and free polysomal fractions (Fig. 3E).

Furthermore, transient knockdown of MALAT1 resulted in increased association of TCF7L2 to

free and light ribosome fractions and decreased association of TCF7L2 mRNA to heavy

ribosome fractions (Fig. 3F). This result suggests that MALAT1 regulates TCF7L2 translation.

Translation initiation is mediated in many cases through the 5’ UTR, which can contain

secondary RNA structures or upstream open reading frames (ORFs) that inhibit translation (40).

To examine if MALAT1 regulates TCF7L2 translation through the TCF7L2 5’UTR, we




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subcloned TCF7L2 5’UTR upstream of a luciferase reporter construct and measured luciferase

protein and mRNA levels following transient MALAT1 knockdown. Knockdown of MALAT1

lead to a significant reduction in luciferase activity from the TCF7L2 5’UTR luciferase reporter

while the mRNA levels of luciferase were not affected (Fig. 3G and H; Fig. S3C and D). This

result suggests that MALAT1 regulates TCF7L2 translation, at least partly, through TCF7L2

5’UTR.

TCF7L2 translation is regulated by mTORC1.

We have previously shown that MALAT1 upregulation activates the mTORC1 pathway. This

was evident from the increased phosphorylation of eIF4E binding protein (4EBP1) in PHM-1

cells overexpressing MALAT1, while knockdown of MALAT1 in these cells resulted in

decreased phosphorylation of 4EBP1 (8). mTORC1 regulates numerous components involved

in protein synthesis, ranging from initiation and elongation factors to the biogenesis of

ribosomes themselves (41). mTORC1 promotes protein synthesis largely through the

phosphorylation of two key effectors, p70 S6 Kinase 1(S6K1) and 4EBP1 (41). We took three

different approaches to assess the importance of mTORC1 activation in MALAT1 mediated

regulation of TCF7L2 protein expression. Firstly, we used the mTOR inhibitor rapamycin to

block mTOR catalytic activity as part of mTORC1. Increased expression of TCF7L2 in

MALAT1 overexpressing PHM-1 cells was reduced by treatment of cells with rapamycin (Fig.

4A). Secondly, we used shRNAs to knockdown either mTOR itself or Raptor, distinctive

components of mTORC1. Knockdown of either of these factors reduced protein expression of

TCF7L2 in PHM-1 cells (Fig. 4B and C). Thirdly, we overexpressed a mutant 4EBP1, 4EBP1-

5A, in which the five known phosphorylation sites were replaced with alanine (42).

Hyperphosphorylation of 4EBP1 is known to lead to activation of cap-dependent translation.

This mutant cannot be phosphorylated and binds constitutively to eIF4E, thus inhibiting its

ability to enhance cap-dependent translation (43). Expression of the dominant negative

4EBP1-5A mutant profoundly repressed expression of TCF7L2, as compared to vector control

(Fig. 4D). To further confirm the importance of cap-dependent translation in the TCF7L2

protein expression and MALAT1-mediated glycolytic effect, we ectopically expressed 4EBP1

wild type (WT) and mutant 4EBP1-4A (44) (in which the four known phosphorylation sites

were replaced with alanine) in PHM-1 MALAT1 cells. Expression of the dominant negative




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4EBP1-4A mutant profoundly repressed expression of TCF7L2 compared to 4EBP1 (WT)

(Fig. S4A) and repressed the expression of glycolytic genes (Fig. S4B-E). These data suggest

that the phosphorylation status of 4EBP1 is important for regulation of TCF7L2 protein

expression and consequently its downstream targets.

MALAT1 regulates TCF7L2 translation through SRSF1.

MALAT1 was shown to activate the mTOR pathway by enhancing the expression and function

of the splicing oncoprotein SRSF1(8). It was shown previously that SRSF1 can activate mTOR

and protein translation. To investigate the molecular mechanism by which MALAT1 (which is a

nuclear lncRNA), regulates the translation of TCF7L2 in the cytoplasm, we examined the

regulation of TCF7L2 by SRSF1. Stable knockdown of SRSF1 in HepG2 cells resulted in

reduced protein expression of TCF7L2 (Fig. 5A). Furthermore, knockdown of SRSF1 in PHM-1

cells resulted in reduced protein expression of TCF7L2 (Fig. S5A). We detected no significant

change in the mRNA levels of TCF7L2 in response to SRSF1 knockdown in HepG2 and PHM-1

cells (Figs. 5B and S5B). Ribosome fractionation showed reduced binding of TCF7L2 mRNA to

the heavy polysome fraction and elevated binding to the light polysome fraction upon SRSF1

knockdown (Fig. 5C). These results suggest that SRSF1 regulates the expression of TCF7L2

post-transcriptionally by regulating its translation in hepatocellular carcinoma cells, a

phenomenon seen for other proteins (45,46).

TCF7L2 mediates the effects of MALAT1 on glucose metabolism.

Silencing of TCF7L2 protein levels in hepatocytes leads to an increase in glucose output

associated with elevated expression of multiple gluconeogenic genes (36,47). TCF7L2 was

shown to be overexpressed and contribute to the malignant phenotype in HCC (48). TCF7L2 is

an important mediator of the Wnt signaling pathway, a signal transduction pathway that directly

contributes to the regulation of cellular metabolism. Since we observed that knockdown of

MALAT1 resulted in increased expression of gluconeogenic genes (Fig. 2E and F), we decided

to examine whether the effect of MALAT1 on glucose metabolism is mediated via TCF7L2. We

introduced siRNAs targeting TCF7L2 into PHM-1 MALAT1 cells (Fig. 6A-B). Knockdown of

TCF7L2 reduced lactate production by MALAT1 overexpressing cells (Fig. 6C). Similarly,

transient knockdown of TCF7L2 in HepG2 cells increased glucose secretion and reduced lactate




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production (Fig. S6A, B and C). This suggests that TCF7L2 is involved in MALAT1 mediated

glucose metabolism. Next, we examined glycolytic gene expression following transduction of

PHM-1 cells overexpressing MALAT1 with lentiviruses encoding shRNAs against TCF7L2

(Fig. 6D). TCF7L2 knockdown reduced glycolytic gene expression in these cells (Fig. 6E-H). In

HepG2 cells, TCF7L2 knockdown increased the expression of gluconeogenic genes G6PC and

PCK1 (Fig. S6D and E). Collectively, these findings suggest that TCF7L2 is involved in

MALAT1-regulated glucose metabolism in cancer cells.

MALAT1 and TCF7L2 regulate gluconeogenesis through the same pathway.

In order to examine whether MALAT1 and TCF7L2 are regulating gluconeogenesis through the

same pathway, we knocked down both MALAT1 and TCF7L2, either individually or together, in

FLC4 cells (Fig. S7A and B). We examined gluconeogenesis gene expression in these cells. We

found that transient knockdown of both MALAT1 and TCF7L2, either individually or together,

resulted in increased gluconeogenic gene expression (Fig. S7C and D) without a significant

additive effect. Similar results were obtained in HepG2 cells (Fig. S7E and F; Fig. S7G and H).

These results suggest that MALAT1 and TCF7L2 modulate gluconeogenesis through the same

pathway.

TCF7L2 acts downstream of MALAT1.

To examine the potential of TCF7L2 as a downstream modulator of MALAT1, we transduced

PHM-1 cells with lentiviruses encoding either TCF7L2 or an empty vector (Fig. 6I and J).

Overexpression of TCF7L2 lead to enhanced lactate production (Fig. 6K). In contrast, transient

knockdown of MALAT1 by siRNAs in TCF7L2 over-expressing PHM-1 cells (Fig. 6L) did not

show significant changes in lactate production (Fig. 6M), suggesting that TCF7L2 acts as a

downstream effector of MALAT1.

TCF7L2 is required for MALAT1 mediated transformation.

To examine whether TCF7L2 upregulation mediates MALAT1 induced transformation, we

knocked down TCF7L2 in PHM-1 cells overexpressing human MALAT1 (Fig. 7A). Stable

knockdown of TCF7L2 in these cells resulted in decreased survival in a clonogenic assay (Fig.

7B) and reduced formation of colonies in soft agar (Fig. 7C), demonstrating that cells




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overexpressing MALAT1 require TCF7L2 overexpression for their oncogenic properties.

Knockdown of TCF7L2 in PHM-1 cells overexpressing human MALAT1 did not show a

strong effect on the proliferative capacity of these cells (Fig. 7D and E). To validate the

importance of TCF7L2 upregulation in HCC cells, we stably or transiently knockdown

TCF7L2 in HepG2 (Fig. S8A) cells. TCF7L2 knockdown in these cells resulted in reduced

formation of colonies in soft agar (Fig. S8B). TCF7L2 knockdown in FLC4 cells showed

similar results (Fig. S8C and D). These results suggest that TCF7L2 is required to maintain

the oncogenic properties of HCC cells.

Elevated nuclear expression of TCF7L2 in tumors from a mouse model for HCC.

We next sought to determine if in an in vivo mouse model of HCC (Mdr2-/- mice), which is

known to upregulate MALAT1 (8), TCF7L2 is up-regulated and if its expression correlates

with expression of genes controlling glycolysis and gluconeogenesis. We examined TCF7L2

mRNA and protein expression in tumor and non-tumor inflamed liver samples from Mdr2-/-

mice. Both Western blot analysis and immunohistochemistry show that the protein expression

of TCF7L2 was elevated in the tumor samples compared to the non-tumor liver samples, with

nuclear localization in HCC tumors compared to adjacent parenchyma (Fig. 7F; Fig. S9A).

Similar to what was detected in cell lines, TCF7L2 mRNA levels were not significantly

different in HCC tumors compared to non-tumor livers (Fig. S9B). Increased TCF7L2 protein

levels was statistically significant (Fig. S9C). Normalization of protein to mRNA levels in

these tumors suggests that there is increased protein to mRNA ratios of TCF7L2 in most of the

tumor samples compared to the normal samples (Fig. S9D). Furthermore, mRNA analysis by

qRT-PCR showed upregulation of multiple glycolytic genes (Fig. S9E), and downregulation of

gluconeogenic genes (Fig. S9F), in most of the tumor samples compared to normal samples.

Taken together, our results suggest a potential role for MALAT1 in the regulation of glycolytic

and gluconeogenic gene expression in HCC through TCF7L2. This reveals yet another

oncogenic function of MALAT1, promoting the “Warburg effect” and repressing

gluconeogenesis during the development of human HCC.

Discussion




15

Increased aerobic glycolysis, or the "Warburg effect", is one of the first identified hallmarks of

cancer (32). However, the underlying molecular mechanisms leading to this phenomenon remain

unclear in many tumors. The present study, reveals an unexpected function of lncRNA MALAT1

in promoting aerobic glycolysis and repressing gluconeogenesis in HCC, adding to its previously

known oncogenic activities (8,49,50). Our initial observation that MALAT1 overexpression in

PHM-1 cells changes the color of the cell medium, led us to explore the role of this oncogenic

lncRNA in facilitating aerobic glycolysis. Indeed, we found a functional correlation between

MALAT1 expression, glucose secretion and lactate production in PHM-1 cells overexpressing

MALAT1 and HCC cells (Fig. 1 and Fig.2). Using flow cytometry we demonstrate glucose

uptake at a single cell level, eliminating the possibility that it is due to cell density or cell

proliferation. Furthermore, MALAT1 overexpression in HCC cells increased the expression of

glycolytic genes and its knockdown resulted in increased expression of gluconeogenic genes

(Figs. 1; Fig. 2; Fig. S2).

Gluconeogenesis is a process that consumes energy in order to regenerate glucose in the liver,

secreting it to the blood when blood glucose levels drop (34). In this pathway three specific

steps, catalysed by gluconeogenesis enzymes, are used to bypass the irreversible reactions of

glycolysis. In this regard, simultaneous activation of both pathways may result in a futile cycling

of glucose which is detrimental to cell survival (51). To avoid such futility, activation of either

pathway should be mutually exclusive. Results of our experiments, with knockdown of

MALAT1 in HCC cell lines, demonstrate increased expression of gluconeogenic genes.

Considering the essential requirement of energy and building blocks for cell doubling, it is

evident that upregulation of gluconeogenesis, an energy consuming process, would result in the

suppression of HCC proliferation. This is indeed the case as knockdown of MALAT1 in HCC

cell lines resulted in decreased proliferation (8). As gluconeogenesis is dramatically impaired in

malignant hepatocytes, similar to what we observed in Mdr2-/- liver tumor samples compared to

adjacent normal liver parenchyma, it is possible that gluconeogenesis represents a metabolic

barrier to HCC development. This is the first report showing the regulation of gluconeogenesis

by MALAT1 in HCC. Importantly, we also establish regulation of various glycolytic genes

(GLUT1, HK2, ENO1 and PKM2) by MALAT1 in PHM-1 cells. To maintain the survival and

rapid proliferation, cancer cells normally elevate expression of glycolytic genes. Several




16

oncoproteins and tumor suppressors were found to regulate enzymes that facilitate glycolytic

tumor glucose metabolism. In this study, we report for the first time that lncRNA MALAT1

regulates an array of glycolytic genes in HCC.

Gluconeogenesis has been shown to be negatively regulated by TCF7L2 in various studies

(36,47). Even though there are no studies showing the role of TCF7L2 in glycolysis or the

“Warburg effect”, TCF7L2 has been implicated in HCC in various studies (48) and has been

shown to be an important mediator of Wnt signaling pathway . Wnt signaling has been shown to

modulate the “Warburg effect” (15). This prompted us to look at the regulation of TCF7L2 by

MALAT1. The elevation in TCF7L2 protein levels upon MALAT1 overexpression, as well as

the decrease in TCF7L2 protein levels upon MALAT1 knockdown, results from translational

regulation and is not a result of changes in transcription or stability (Fig. 3). Control of mRNA

translation constitutes a critical step in the regulation of gene expression and in cancer (52,53).

Polysome profiling of TCF7L2 mRNA showed increased translation of TCF7L2 in MALAT1

overexpressing cells while reduced translation of TCF7L2 in MALAT1 knockdown cells (Fig.

3). Translation initiation efficiency can be regulated by the 5’UTR. Secondary RNA structures at

the 5'UTR of many mRNAs inhibit translation initiation and this inhibition can be alleviated by

RNA helicases which are recruited by the eIF4G-eIF4E complex (40). Knockdown of MALAT1

resulted in reduced 5’UTR activity of TCF7L2 as measured by luciferase activity without an

effect on luciferase mRNA levels (Fig. 3).

Signalling by the PI3K/AKT/mTOR pathway profoundly affects mRNA translation through

phosphorylation of downstream targets, such as 4EBP1 and S6K1 (54) The cap-dependent

protein synthesis pathway serves as a pleotropic integrator and amplifier of many essential

oncogenic signals (53,55). Our data shows that TCF7L2 is specifically regulated by the

mTORC1-4EBP1 axis (Fig. 4). Because TCF7L2 is a major transcriptional regulator of the Wnt

pathway, it is possible that the mTORC1 pathway, through its regulation of TCF7L2 translation,

can modulate the Wnt pathway. The crosstalk between these two signalling pathways has not

been demonstrated to our knowledge. Next, in order to further substantiate the regulation of

cytoplasmic TCF7L2 translation by nuclear MALAT1, we looked at the regulation of TCF7L2

by SRSF1. SRSF1 is a nuclear splicing factor and MALAT1 was shown to regulate the




17

expression and function of SRSF1. SRSF1 was shown to activate mTOR and protein translation

(45,46,56). Indeed, we found that SRSF1 knockdown reduced TCF7L2 translation in HepG2

cells (Fig. 5). This result suggests that increased protein expression of TCF7L2 by MALAT1

could be, in part, attributed to increased expression of SRSF1 by MALAT1. This result also

provides an explanation for the reduced expression of TCF7L2 in the presence of Rapamycin, as

knockdown of SRSF1, which is known to activate mTORC1, in PHM-1 and HepG2 cells

resulted in reduced protein expression of TCF7L2 (Figs. 4-5).

The result of increased expression of gluconeogenesis genes upon TCF7L2 knockdown is in

agreement with previous studies where it was shown that TCF7L2 is a negative regulator of

gluconeogenesis (36,47). Regarding glycolytic gene expression regulation by TCF7L2, this is the

first description of the regulation of glycolytic gene expression by TCF7L2 in HCC.

As expected from the alteration in glycolytic and gluconeogenic enzyme expression,

overexpression of TCF7L2 resulted in increased lactate production while knockdown of TCF7L2

resulted in decreased lactate production (Fig. 6). These results suggest direct regulation of cancer

glucose metabolism by TCF7L2. Furthermore, knockdown of MALAT1 in TCF7L2

overexpressing cells did not change lactate production suggesting that TCF7L2 acts downstream

to MALAT1.

Because TCF7L2 regulates glucose metabolism and acts downstream of MALAT1, we sought to

examine the importance of TCF7L2 in MALAT1-mediated oncogenic activity. To this end we

knocked-down TCF7L2 in PHM-1 cells overexpressing MALAT1. Stable knockdown of

TCF7L2 in these cells resulted in decreased survival in a clonogenic assay and reduced

formation of colonies in soft agar (Fig. 7). Furthermore, knockdown of TCF7L2 in HCC cell

lines (HepG2 and FLC4) resulted in reduced oncogenic properties as seen by reduced formation

of colonies in soft agar (Fig. S8). These results suggested that TCF7L2 expression is essential for

MALAT1-mediated transformation. TCF7L2 overexpression in HCC has been reported in

several studies (48). Our western blot and gene expression analysis on Mdr2-/- mice liver tumor

samples revealed strong up regulation of MALAT1 at the RNA level and TCF7L2 at the protein

level (Figs. 7 and S9). Furthermore, Mdr2-/- liver tumor samples showed elevated glycolytic and

repressed gluconeogenic gene expression (Fig. S9) suggesting a significant relevance of

MALAT1-mediated tumor glucose metabolism in the development of tumors in vivo.




18

It is important to note that the true clinical implications of these results needs to be examined

further in human normal liver and HCC clinical samples.

Conclusion

Taken together, our data suggest that MALAT1 acts as a regulator of glucose metabolism in

HCC. Our results add insight to the mechanisms of cancer glucose metabolism and cancer

progression. The novel findings from the present study, together with the significant discoveries

from previous studies, place MALAT1 at the crossroad of cellular metabolism and

carcinogenesis (Fig. 7G). MALAT1 regulates the expression of TCF7L2 at the translational

level. TCF7L2 regulation by MALAT1 is through a mTORC1-dependent pathway via cap-

dependent translation. TCF7L2 plays an important role in MALAT1-induced tumorigenesis and

altered glucose metabolism in HCC development. These results point towards the fact that

knockdown of MALAT1 or reduction of TCF7L2 levels might serve as new strategies based on

tumor glucose metabolism for the treatment of HCC.

Acknowledgements

The authors wish to thank Dr. Zahava Siegfried for comments on the manuscript and Prof.

Fatima Gebauer (CRG, Barcelona) for the pSG5 Luc Plasmid. This study was supported in part

by Israel Science Foundation (ISF) (ISF Grant no' 1290/12 to R.K.),

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Figure Legends

Figure 1: MALAT1 affects cancer glucose metabolism.

A. qRT-PCR of PHM-1 cells stably expressing hMALAT1 or an empty vector. B. Extracellular

lactate production was measured in cells described in (A) using a lactate assay kit (n=3). C.

PHM-1 cells over expressing MALAT1 knocked down for MALAT1 by siRNAs (siMALAT#1,

#2), were analyzed by qRT-PCR. D. Extracellular lactate production was measured in cells

described in (D) using a lactate assay kit (n=3). E. Schematic representation of the glycolytic and




22

gluconeogenetic pathways of glucose metabolism. The enzymes marked in red were selected for

gene expression analysis. F. A gel image of semi-quantitative RT-PCR of glycolytic gene

expression in PHM-1 cells described in (A). G. Expression of genes in the glucose metabolic

pathway in cells described in (A) measured by qRT-PCR. All samples were normalized to

GAPDH mRNA levels. The error bars indicate S.D. (n=3). Student T-Test was used. *, P<0.05,

**, P<0.01, *** P<0.001.

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 (n=3). C. Extracellular lactate

production was measured in cells described in (A) using a lactate assay kit (n=2). D. Schematic

representation of the glycolytic and gluconeogenetic pathways. The enzymes marked in blue are

involved in gluconeogenesis. E, F. mRNA expression of the G6PC (E) and PCK1 (F) genes in

the gluconeogenesis pathway in cells described in (A). All samples were normalized to actin or

GAPDH mRNA levels. The error bars indicate S.D. (n=3). Student T-Test was used. *, P<0.05,

**, P<0.01, *** P<0.001.

Figure 3: MALAT1 upregulates TCF7L2 translation.

A. TCF7L2 mRNA expression in PHM-1 cells stably expressing hMALAT1 or an empty vector

analyzed by qRT-PCR. B. TCF7L2 protein levels in cells described in (A) analyzed by Western

blot. Graph on the right shows the quantification of TCF7L2 protein levels (n=4). C. TCF7L2

mRNA levels in PHM-1 cells overexpressing MALAT1 treated with the indicated siRNAs, as

determined by qRT-PCR. D. TCF7L2 protein levels in cells described in (C) were determined by

Western blot. Quantification of TCF7L2 protein levels (n=3) (right panel). E. Relative

distribution of TCF7L2 mRNA across the polysome fractions in PHM-1 cells stably expressing

hMALAT1 or an empty vector. F. Relative distribution of TCF7L2 mRNA across the polysome

fractions in cells transfected with either MALAT1 siRNA or control siRNA (siLuciferase). G.

PHM-1 cells were transfected with MALAT1 siRNAs (siMALAT1#1, #2) or siControl.

Luciferase activity (fold change compared to siControl) produced after transfection with

luciferase construct containing the WT TCF7L2 5’UTR was measured. Luciferase activity was




23

normalized to Renilla expression. H. The graph shows the luciferase transcript expression

measured by qRT-PCR. All bars show the average of 2-3 experiments. Error bars indicate

standard deviation. Student T-Test was used. *, P<0.05, **, P<0.01, *** P<0.001.

Figure 4: TCF7L2 translation is regulated by mTORC1-4EBP1.

A. Western blot analysis of PHM-1 cells transduced with lentivirus encoding either MALAT1 or

an empty vector in the presence or absence (DMSO) of rapamycin (left panel). Quantification of

TCF7L2 protein levels upon rapamycin treatment (n=4) (right panel). B. Western blot analysis of

PHM-1 cells transduced with retroviruses encoding mTOR shRNAs (left panel). Quantification

of mTOR and TCF7L2 protein levels upon shRNA knockdown (n=3) (right panels). C. Western

blot analysis of PHM-1 cells transduced with retroviruses encoding Raptor shRNAs (left panel).

Quantification of Raptor and TCF7L2 protein levels upon shRNA knockdown (n=4) (right

panels). D. Western blot analysis of PHM-1 cells transduced with retroviruses encoding for

empty vector pWZL-Hygro (empty) or PWZL-4EBPphosphorylation defective mutant in which

all five phosphorylation sites were mutated to alanine (4EBP1-5A) (left panel). Quantification of

phosphorylated 4E-BP1 (n=2) and TCF7L2 protein levels upon overexpression of PWZL-4EBP1

phosphorylation defective mutant (n=2) (right panels). The error bars indicate S.D. Student T-

Test was used. *, P<0.05, **, P<0.01, *** P<0.001.

Figure 5: SRSF1 regulates TCF7L2 translation.

A. Western blot analysis of HepG2 cells transduced with lentiviruses containing shRNAs against

SRSF1 (shSF2#1,#2,#3) or an empty vector (shControl). β-Actin was used as loading control.

Quantification of SRSF1 and TCF7L2 protein levels upon shRNA knockdown (n=3) (right

panels). B. qRT-PCR of TCF7L2 mRNA levels in cells described in (A). Error bars indicate

standard deviation. Student T-Test was used. *, P<0.05, **, P<0.01, *** P<0.001. C. Relative

distribution of TCF7L2 mRNA across the polysome fractions in HepG2 cells transfected with

either control shRNA or shRNAs against SRSF1.

Figure 6: TCF7L2 mediates MALAT1 effects on glucose metabolism.

A. Western blot of PHM-1 cells over expressing MALAT1 transfected with siTCF7L2 (left

panel). Quantification of TCF7L2 protein levels upon TCF7L2 siRNA treatment (n=2) (right




24

panel). B. qRT-PCR of cells described in (A). C. Extracellular lactate production was measured

in cells described in (A) using a lactate assay kit (n=3). D. Western blot analysis of PHM-1 cells

overexpressing MALAT1 after transduction with lentiviruses expressing TCF7L2 shRNAs.

E,F,G and H. qRT-PCR of the indicated genes in the glucose metabolic pathway in cells

described in (D). All samples were normalized to GAPDH mRNA levels.

I. Western blot analysis of PHM-1 cells stably expressing TCF7L2 or an empty vector. J. qRT-

PCR of cells described in (I). K. Extracellular lactate production was measured in cells described

in (I) using a lactate assay kit (n=2). L. qRT-PCR of PHM-1 cells overexpressing TCF7L2 and

knocked down for MALAT1 by siRNA. M. Extracellular lactate production was measured in

cells described in (L) using a lactate assay kit (n=2). Error bars indicate standard deviation.

Student T-Test was used. *, P<0.05, **, P<0.01, *** P<0.001.

Figure 7: TCF7L2 is required for MALAT1-induced transformation.

A. Western blot of PHM-1 cells over expressing MALAT1 transduced with lentiviruses

encoding TCF7L2 shRNAs (left panel). Quantification of TCF7L2 protein levels upon TCF7L2

knockdown by shRNA (n=2) (right panel). B. Clonogenic assay of control PHM-1 cells stably

expressing empty vector and cells described in (A) . C. Growth in soft agar assay of control

PHM-1 cells stably expressing empty vector and cells described in (A). Colonies were counted

28 days after seeding. Error bars indicate standard deviations (n=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 staining for TCF7L2 in liver tumor (T) specimens including surrounding

parenchymal (P) tissue from two 12 months old Mdr2-/- mice (scale bar 50 μm). Note the

enhanced nuclear TCF7L2 staining in malignant hepatocytes. G. Scheme summarizing the role

of MALAT1 in regulating glucose metabolism in HCC. Red lines represent pathways by which

MALAT1 regulates glucose metabolism in HCC as described here. Blue lines represent

pathways described by others.




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1

1.5

2

2.5

3

Empty MALAT1

PHM-1

PKM2

***

0

1

2

3

4

5

Empty MALAT1

PHM-1

HK2 ***

GLUT1

HK2

ENO1

PKM2

GAPDH

PHM-1

0

0.2

0.4

0.6

0.8

1

1.2

siLuciferase siMALAT1#1 siMALAT1#2

Re

lati

ve

Ou

tpu

t

PHM-1 MALAT1

Lactate

***

***




0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

Ex

pre

ss

ion

HepG2

MALAT1

0

0.5

1

1.5

2

2.5

3

3.5

Re

lati

ve

Ex

pre

ss

ion

HepG2

PCK1

*** **

A C B

D E

Figure 2

F

0

0.4

0.8

1.2

Re

lati

ve

Ou

tpu

t

HepG2

Lactate

** ***

*** ***

0

1

2

3

4

5

6

7

Re

lati

ve

Ex

pre

ss

ion

HepG2

G6PC

**

*

0

0.5

1

1.5

2

2.5

3

3.5

4

Re

lati

ve

Ab

un

da

nc

e

HepG2

Glucose Secretion

**

*




0

0.4

0.8

1.2

1.6

2

Empty MALAT1

Re

lati

ve

Ex

pre

ss

ion

PHM-1

TCF7L2

TCF7L2

β-Catenin

PHM-1 0

0.5

1

1.5

2

2.5

3

3.5

Empty MALAT1

Re

lati

ve

In

ten

sit

y

PHM-1

***

0

0.4

0.8

1.2

1.6

2

Re

lati

ve

Ex

pre

ss

ion

PHM-1 MALAT1

TCF7L2

A B

C D

Figure 3

0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

In

ten

sit

y

PHM-1 MALAT1

*

TCF7L2

β-Actin

GAPDH

PHM-1 MALAT1

***

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Free Light Heavy

TC

F7

L2

mR

NA

F

rac

tio

n

PHM-1

Empty

MALAT1

*

*

*

0

0.2

0.4

0.6

0.8

1

1.2

siControl siMALAT1#1 siMALAT1#2

No

rmali

zed

Lu

cif

era

se

acti

vit

y

PHM-1

*** ***

0

0.5

1

1.5

2

2.5

siControl siMALAT1#1 siMALAT1#2

Rela

tive

Exp

ressio

n

PHM-1

Luciferase mRNA

E F

H G

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Free Light Heavy

TC

F7

L2

mR

NA

Fra

cti

on

PHM-1

siLuciferase

siMALAT1

**

** **

Tubulin




TCF7L2

β-Catenin

Tubulin

PHM-1 0

0.5

1

1.5

2

2.5

EmptyDMSO

MALAT1DMSO

MALAT1Rapamycin

Re

lati

ve

In

ten

sit

y

PHM-1

TCF7L2

**

**

mTOR

TCF7L2

β-Actin

Tubulin

PHM-1 0

0.2

0.4

0.6

0.8

1

1.2

Vector shmTOR#1 shmTOR#2

Re

lati

ve

In

ten

sit

y

PHM-1

mTOR

*** ***

0

0.2

0.4

0.6

0.8

1

1.2

Vector shmTOR#1 shmTOR#2

PHM-1

TCF7L2

*** ***

Raptor

TCF7L2

GAPDH

β-Tubulin

PHM-1

0

0.2

0.4

0.6

0.8

1

1.2

Vector shRaptor#1 shRaptor#2

Re

lati

ve

In

ten

sit

y

PHM-1

Raptor

** **

0

0.2

0.4

0.6

0.8

1

1.2

Vector shRaptor#1 shRaptor#2

PHM-1

TCF7L2

*** ***

TCF7L2

p4EBP1

Total 4EBP1

GAPDH

β-Actin

PHM-1

0

0.2

0.4

0.6

0.8

1

1.2

Empty 4EBP1(5A)

Re

lati

ve

In

ten

sit

y

PHM-1

p4EBP1

*

0

0.2

0.4

0.6

0.8

1

1.2

Empty 4EBP1(5A)

PHM-1

TCF7L2

***

A

B

C

D

Figure 4 Research.

on December 8, 2020. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from



SRSF1

TCF7L2

HepG2

β-Actin

A

Figure 5

0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

In

ten

sit

y

SRSF1 protein

**

***

***

0

0.2

0.4

0.6

0.8

1

1.2 TCF7L2 protein

***

*** ***

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Re

lati

ve

Ex

pre

ss

ion

TCF7L2 mRNA C

0

0.1

0.2

0.3

0.4

0.5

0.6

Free Light Heavy

TC

F7

L2

mR

NA

Fra

cti

on

ShControl

ShSRSF1#1

ShSRSF1#2

B

**

**

**

**

*

Polysome fractionation




TCF7L2

GAPDH

PHM-1 MALAT1 0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

In

ten

sit

y

TCF7L2

D

TCF7L2

β-Actin

PHM-1 MALAT1

α-Tubulin

E F

A

C

0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

Ex

pre

ss

ion

GLUT1

**

**

0

0.2

0.4

0.6

0.8

1

1.2

HK2

** ***

0

0.2

0.4

0.6

0.8

1

1.2

ENO1

*** ***

0

0.2

0.4

0.6

0.8

1

1.2

PKM2

**

***

Figure 6

G H

β-Actin 0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

Ex

pre

ss

ion

TCF7L2 qRT-PCR

***

***

*

*

0

0.2

0.4

0.6

0.8

1

1.2

Re

lati

ve

Ou

tpu

t Lactate

*** ***

B

T7

β-Actin

PHM-1 0

20

40

60

80

Empty TCF7L2

Rela

tive E

xp

ressio

n Human TCF7L2

**

0

0.5

1

1.5

2

2.5

Empty TCF7L2

Re

lati

ve

Ou

tpu

t

Lactate ** I J K

L M

0

0.2

0.4

0.6

0.8

1

1.2

SiLuc SiMAL#1 SiMAL#2

Re

lati

ve

Ex

pre

ss

ion

PHM-1 TCF7L2

MALAT1

***

***

0

0.2

0.4

0.6

0.8

1

1.2

1.4

SiLuc SiMAL#1 SiMAL#2

Re

lati

ve

Ou

tpu

t

PHM-1 TCF7L2

Lactate




PHM-1 MALAT1

TCF7L2

β-Actin

β-Tubulin

A

C D

0

0.2

0.4

0.6

0.8

1

1.2

shCon shTCF#1 shTCF#2

Re

lati

ve

In

ten

sit

y

PHM-1 MALAT1

*

**

E

B

F TCF7L2

T

P

P

T

TCF7L2

MALAT1

WNT Signaling

TCF7L2

Glycolysis

Gluconeogenesis

Hepatocellular Carcinoma

Gluconeogenesis

mTORC1

p-4EBP1

SRSF1G

Figure 7

0

10

20

30

40

50

60

70

80

0 24 48 72 96

Re

lati

ve

Ab

so

rba

nc

e

Time (Hrs)

shTCF#1

shTCF#2

shCon

MALAT1

shTCF#1

MALAT1

shTCF#2

Empty

MALAT1

PHM-1

0

0.5

1

1.5

2

2.5

Empty MALAT1 MALAT1shTCF#1

MALAT1shTCF#2

Re

lati

ve

No

. o

f A

ga

r C

olo

nie

s

PHM-1

***

*

**

0

5

10

15

20

25

30

0 24 48 72 96

Re

lati

ve

Ab

so

rba

nc

e

Time (Hrs)

Empty

MALAT1




Published OnlineFirst March 26, 2019.Cancer Res Pushkar Malakar, Ilan Stein, Amijai Saragovi, et al. TCF7L2metabolism by enhancing mTOR-mediated translation of Long Noncoding RNA MALAT1 regulates cancer glucose

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Long Noncoding RNA MALAT1 regulates cancer glucose ... · 3/26/2019 · 1 Long Noncoding RNA...

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