expression of long noncoding rna yiya promotes glycolysis ... · impaired glycolysis and tumor...

Molecular Cell Biology

Expression of Long Noncoding RNA YIYAPromotes Glycolysis in Breast CancerZhen Xing1, Yanyan Zhang1, Ke Liang1, Liang Yan1, Yu Xiang5, Chunlai Li1,Qingsong Hu1, Feng Jin2, Vasanta Putluri2, Nagireddy Putluri2, Cristian Coarfa2,Arun Sreekumar2, Peter K. Park1, Tina K. Nguyen1, Shouyu Wang1,3, Jianwei Zhou3,Yan Zhou4, Jeffrey R. Marks5, David H. Hawke6, Mien-Chie Hung1,7,8, Liuqing Yang1,7,9,Leng Han10, Haoqiang Ying1,7, and Chunru Lin1,7,9

Abstract

Long noncoding RNA (lncRNA) is yet to be linked tocancer metabolism. Here, we report that upregulation ofthe lncRNA LINC00538 (YIYA) promotes glycolysis, cellproliferation, and tumor growth in breast cancer. YIYA isassociated with the cytosolic cyclin-dependent kinaseCDK6 and regulated CDK6-dependent phosphorylationof the fructose bisphosphatase PFK2 (PFKFB3) in a cell-cycle–independent manner. In breast cancer cells,these events promoted catalysis of glucose 6-phosphateto fructose-2,6-bisphosphate/fructose-1,6-bisphosphate.CRISPR/Cas9-mediated deletion of YIYA or CDK6 silencing

impaired glycolysis and tumor growth in vivo. In clinicalspecimens of breast cancer, YIYA was expressed in approx-imately 40% of cases where it correlated with CDK6 expres-sion and unfavorable survival outcomes. Our results definea functional role for lncRNA in metabolic reprogrammingin cancer, with potential clinical implications for its thera-peutic targeting.

Significance: These findings offer a first glimpse into how along-codingRNA influences cancermetabolism todrive tumorgrowth. Cancer Res; 78(16); 4524–32. �2018 AACR.

IntroductionIt has been found that numerous long noncoding RNA

(lncRNA) transcripts are transcribed in the human genome (1)and play critical roles in diverse cellular processes and diseases,including neurodegenerative diseases, autoimmune diseases, and

cancer (2–4). An lncRNA, named YIYA, was originally demon-strated to be upregulated inmultiple cancer types. YIYA is locatedon 1q41, which is a cancer susceptibility locus (5). The YIYA geneis also amplified, and its expression is linked to cell-cycle regu-lation and cell proliferation (5).

One of the hallmarks of human cancer is the alteration ofglucose metabolism. Elevated glycolysis (also known as theWarburg effect) promotes cancer cell growth, angiogenesis,and metastasis (6). One of the key steps of glucose metabolismis the conversion of glucose 6-phosphate (G6P) to fructose-1,6-bisphosphate (F-1,6-BP) or fructose-2,6-bisphosphate (F-2,6-BP;refs. 7, 8), which ismediated by 6-phosphofructo-1-kinase (PFK1;ref. 9) or 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(PFKFB3), respectively (10). The enzymatic activity of PFK1 isallosterically activated by F-2,6-BP (11). Inhibition of PFKFB3reduces the enzymatic activity of PFK1 and glucose flux, leading todecreased cancer cell growth (12, 13).

In addition to cell-cycle regulation, cyclin-dependent kinase 6(CDK6) has been suggested to modulate other cellular activities(14). Mice with CDK6 knockout (KO) exhibited impairedhematopoietic development (15). In addition, recent studies havealso indicated that CDK6 regulates the pentose pathway (16).Inhibition of CDK6 by small-molecule inhibitors may impairthe lactate synthesis and pentose cycle activity (16). However,the regulation of CDK6 in cancer metabolic reprogrammingremains elusive.

Here, our data indicate that YIYA expression is correlated withrecurrence in patients with breast cancer. YIYA upregulationpromotes tumor growth and glycolysis. Mechanistically, YIYAassociates with cytosolic CDK6 to facilitate the enzymatic activa-tion of CDK6. The presence of YIYA enhanced CDK6-dependentphosphorylation of PFKFB3, which facilitated the catalysis of

1Department of Molecular and Cellular Oncology, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Department of Molecular and CellBiology, Baylor College of Medicine, Houston, Texas. 3Department of MolecularCell Biology and Toxicology, School of Public Health, NanjingMedical University,Nanjing, China. 4Department of Oncology, Yixing People's Hospital, Yixing,China. 5Division of Surgical Science, Department of Surgery, Duke University,School of Medicine, Durham, North Carolina. 6Department of Systems Biology,The University of Texas MD Anderson Cancer Center, Houston, Texas. 7TheGraduate School of Biomedical Sciences, The University of Texas MD AndersonCancer Center, Houston, Texas. 8Graduate Institute of Cancer Biology andCenter for Molecular Medicine, China Medical University, Taichung, Taiwan.9Center for RNA Interference and Non-Coding RNAs, The University of TexasMDAnderson Cancer Center, Houston, Texas. 10Department of Biochemistry andMolecular Biology, The University of Texas Health Science Center at HoustonMcGovern Medical School, Houston, Texas.

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

Z. Xing, Y. Zhang, and K. Liang contributed equally to this article.

Current address for Z. Xing: Sanofi , 270 Albany Street, Cambridge, MA 02139;Y. Zhang: Institute of Immunology, Third Military Medical University, Chongqing,China 400038.

Corresponding Author: Chunru Lin, University of Texas MD Anderson CancerCenter, 1515 Holcombe Blvd, Houston, TX 77030. Phone: 713-795-3226; Fax: 713-745-8100; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0385

�2018 American Association for Cancer Research.

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F-2,6-BP. Thus, YIYA and CDK6 are shown to be required forglycolysis augmentation in breast cancer cells. YIYA KO led toaccumulation of G6P and depletion of F-2,6-BP/F-1,6-BP in cells,as well as decreased cell proliferation, invasion, and tumorgrowth.Our data demonstrated that the dysregulation of lncRNAsin human cancer modulates glycolysis, signifying the importanceof targeting lncRNAs as an antiglycolytic therapy.

Materials and MethodsClinical samples

Two sets of fresh-frozen breast cancer tissues (Yixin-Bre-01 andDuke-Bre-01) were collected from Yixing People's Hospital(Yixing, Jiangsu Province, China) and Duke University, respec-tively. The study protocol was approved by the InstitutionalReview Board of Nanjing Medical University (Nanjing, China)and Duke University Health System. All tissue samples werecollected with written-informed consents. This study was carriedout in accordance with the Declaration of Helsinki of the WorldMedical Association.

Cell culture, transfection, and lentiviral transductionMDA-MB-231, MCF7, BT474, and HEK293T cells, obtained

from the American Type Culture Collection in 2013, were main-tained in DMEM/F12 (Life technologies) with 10% FBS, and 1%penicillin/streptomycin (pen/strep). Routine Mycoplasma testingwas performed by PCR. All of the cell lines were free of myco-plasma contamination tested by vendors using a MycoAlert kitfrom Lonza tested in 2016. No cell lines used in this study arefound in the database of commonly misidentified cell lines (TheInternational Cell Line Authentication Committee and NCBIBiosample) based on short tandem repeats profiling performedby vendors. Cells with relative low-passage numbers (<15) wereused in the study. Plasmid transfections were performed usingLipofectamine 3000 (Invitrogen). Lentiviruses were produced inHEK293T cells with ViraPower Lentiviral Expression System (LifeTechnologies) and used to transduce target cells.

RNAscope assay, immunohistochemistry, and imagequantification

Detection of YIYA expression using RNAScope probe (designedby Advanced Cell Diagnostics) was performed on breast or lungcancer cell lines or microarray of multiple cancer type tissues withRNAScope 2.0 High Definition Assay kit according to the man-ufacturer's instructions (Advanced Cell Diagnostics). Immuno-histochemistry was performed as previously described (17). Theimages were visualized with Zeiss Axioskop2 plus Microscope,and the slides were scanned on the Automated Cellular ImageSystem III (ACIS III) for quantification by digital image analysis asprevious described (18). For survival analysis, the expression ofYIYA was treated as a binary variant and divided into "high" and"low" level. Kaplan–Meier survival curves were compared usingthe log rank test.

In vitro RNA-protein binding assay, RNA pulldown, and massspectrometry analysis

In vitro RNA-protein binding and RNA pulldown assays wereperformed as described previously (17). Briefly, in vitro tran-scribed biotinylated YIYA sense or antisense transcripts were firstcaptured by the magnetic Monoavidin beads (Bioclone Inc.), andthe YIYA-bound beads were further incubated withMDA-MB-231

cell lysates. The YIYA-bound proteins/complexes were in solutiondigested by Immobilized Trypsin (Promega) and subjected toLC-MS/MS analysis at MD Anderson Cancer Center ProteomicsFacility. For identification of binding proteins of F-box And WDrepeat domain containing 7 (FBXW7) and CDK6, FLAG-taggedFBXW7 and CDK6were subjected to affinity pulldown using anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) using FLAG-FBXW7–and FLAG-CDK6–expressing MDA-MB-231 cells. The proteintargets that associated with both FBXW7 and CDK6 were sub-jected to the following studies.

Cell lysis, immunoprecipitation, immunoblotting, and RNAimmunoprecipitation assay

Immunoprecipitation and immunoblotting assays were per-formed as described previously (17). RNA immunoprecipitation(RIP) assay was performed in native conditions as previouslydescribed (17).

In vitro kinase assayRecombinant proteins were incubated with CDK6 þ CCND3

in vitro inkinaseassaybuffer I (SignalChem)containing100mmol/L ATP-gamma-S (ab138911) according to the manufacturer'sinstruction (Promega). Alternatively, the reaction was stopped,separated by SDS-PAGE, and detected byCoomassie Blue stainingor immunoblotting with phospho-specific antibodies.

Three-dimensional spheroid proliferation assay and three-dimensional spheroid BME cell invasion assay

Spheroid growth of MDA-MB-231 parental or YIYA KO cellswas conducted using Cultrex 3-D Spheroid FluorometricProliferation/Viability Assay kit (Trevigen). Three-dimensional(3D) invasion of indicated cells was determined using Cultrex3D Spheroid BMECell Invasion Assay kit (Trevigen). The changesin the area of the spheroid expansion or invasive structures weremeasured using ImageJ software to determine the extent of 3Dculture BME cell invasion for each sample.

Metabolic profiling and metabolite analysis of spent mediaControl, YIYA KO cells, or cells harboring CDK6 shRNA or

PFKFB3 expression vectors in tetraplicate were subjected toglucose-free DMEM supplemented with 10% dialyzed serum for16hours. The cellswere fedbackwithmedia containing glucose or[U13C] glucose (11 mmol/L) for 4 hours. The metabolites wereharvested and determined as previously described (19). Formetabolite analysis of spentmedium, cells were seeded in 12-wellplates in triplicate for 24 hours. Glucose and lactate concentra-tions were measured in fresh and spent medium using a YellowSprings Instruments (YSI) 7100. The concentration of glucose andlactate was normalized to cell numbers.

CRISPR/Cas9-mediated gene editingYIYA KO cell lines were generated using the CRISPR/Cas9

genome editing system. Briefly, YIYA-specific guide RNA (gRNA)expression vectors were generated as described (20). LentiCas9-Blast (plasmid 52962; Addgene) and gRNA cloning vector lenti-Guide-Puro (plasmid 52963; Addgene) were obtained fromAddgene. MBA-MD-231 cells were cotransfected with lenti-Cas9-Blast, gRNA1, and gRNA2 expression vectors. The trans-fected cells were selected using puromycin (0.5 mg/mL) andblasticidin (1 mg/mL). Isolated single colonies were subjected todetection of genomic deletions by PCR. Sequencing validation of

LncRNA-Directed Glycolysis Augmentation in Breast Cancer

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the genomic deletions was performed after cloning the corre-sponding amplicon into pGEM-T-Easy (Promega).

In vivo tumorigenesis studyAll animal experiments were performed in accordance with

protocol approved by the Institutional Animal Care and UseCommittee of MD Anderson. MDA-MB-231 parental or YIYA KOcells (5 � 106) mixed with Matrigel at a 1:1 ratio were orthoto-pically injected to the mammary fat pad of 6-week-old femaleathymic Nu/Nu. Five mice for each group were based on powercalculations, which will allow us to detect an approximately 30%difference in tumor growth and/or glucose uptake between groups

at the 95% confidence level. Tumor size was measured weeklyusing a caliper. Tumor volume was calculated using the standardformula: 0.54� L�W2, where L is the longest diameter, andW isthe shortest diameter.

Data analysis and statistical analysisAnalyses of relative gene expression were determined using

the 2�DDCt method with GAPDH or B2M as the internal referencegenes. Results are reported as mean � SEM of at least threeindependent experiments. Comparisons were performedusing two-tailed paired Student t test (�, P < 0.05; ��, P < 0.01;and ��, P < 0.001).

Figure 1.

The oncogenic role of YIYA in breastcancer. A and B, RNAscope stainingof YIYA in breast tumor or adjacentnormal tissues (n ¼ 35 and 130,respectively; A) or non-TNBC andTNBC tissues (n ¼ 15 and 115,respectively; B). Scale bar, 200 mm(�200). C, Recurrence-free survivalanalysis of YIYA status in patientswith breast cancer (n ¼ 110, log ranktest). D and E, Tumor spheroidformation assay (D) or tumorspheroid invasion assay (E) ofMDA-MB-231 cells harboringindicated sgRNAs. F, Xenografttumor growth of mice harboringMDA-MB-231 with or without YIYAKO (n ¼ 5 animals). Error bar, SEM;n ¼ three independent experiments(� , P < 0.05 and �� , P < 0.01, Studentt test).

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ResultsYIYA is required for cell proliferation and tumor growth

To demonstrate the functional importance of lncRNAs in breastcancer, we first determined the expression status of YIYA in breastcancer tissues with paired adjacent normal tissues using RNA-scope technology, finding that over 50% of patients with breastcancer expressed YIYA and less than 5%of adjacent normal tissuesexhibited detectable YIYA expression (Fig. 1A and B). Patientswith breast cancer with high YIYA expression exhibited poorrecurrence-free survival (Fig. 1C). YIYA is upregulated in breastcancer cells compared with normal mammary gland epithelialcells (Supplementary Fig. S1A). Expression of YIYA regulated thecell proliferation rate, but showedminimal effects on the cell cycle(Supplementary Fig. S1B–S1G), suggesting that YIYA may playadditional roles in promoting breast cancer cell growth that areindependent to cell-cycle regulation.

We generated KO cell lines derived from MDA-MB-231 cellsusing CRISPR/Cas9 technology (21), using two guide RNAs(sgRNA) flanked the transcription body of YIYA (SupplementaryFig. S1H–S1K). YIYA KO significantly reduced the growth andinvasion of 3D tumor spheroids (Fig. 1D and E). Orthotopicinjection of parental or YIYA KO cells into the mammary fat padof mice indicated that the growth of breast tumors was signifi-cantly inhibited when YIYAwas depleted (Fig. 1F). Therefore, ourdata demonstrate the potential oncogenic role of YIYA in breastcancer cells.

YIYA associates with CDK6 and SCF complexTo understand the molecular mechanism by which YIYA pro-

motes breast cancer progression, we identified the binding pro-teins of YIYA using liquid chromatography–mass spectrometry(LC-MS). Compared with beads only and YIYA antisense, YIYAsense associated with S-phase kinase-associated protein 1 (SKP1),FBXW7, and cell division protein kinase 6 (CDK6) with highconfident (Fig. 2A; Supplementary Fig. S2A). CDK6 is regulatedbycyclin-D3 (22). SKP1 and FBXW7 are both key components ofSkp, Cullin, and F-box containing complex (or SCF complex;ref. 23).We confirmed the interactions between CDK6/cyclin-D3,CDK6/SKP1, and CDK6/FBXW7 (Fig. 2B and C; SupplementaryFig. S2B). Coimmunoprecipitation also indicated that FBXW7associated with CDK6 and SKP1, respectively (Fig. 2D and E). Wethen demonstrated that YIYA, CDK6, and cyclin-D3 localized toboth the nucleus and the cytosol, but mostly to the cytosol(Supplementary Fig. S2C and S2D).

YIYA directly associates with CDK6 and FBXW7We confirmed the association between YIYA and YIYA-binding

proteins in cells using an RIP assay (Fig. 3A). Recombinantfull-length CDK6 directly associates with YIYA sense but notthe antisense transcript (Fig. 3B). Furthermore, neither the N-terminus, theC-terminus, nor kinase deadmutant (K43M; ref. 24)of CDK6 exhibited interactions with the YIYA (Fig. 3B and C). TheFBXW7 protein contains a WD40 domain, which has been dem-onstrated to be an atypical RNA-binding domain (17). FBXW7associated with YIYA in vitro in a WD40 domain–dependentmanner (Supplementary Fig. S3).

Immunohistochemistry staining indicated that CDK6 pro-tein is elevated in breast cancer tissues compared with normaladjacent tissues (Fig. 3D). Further, in YIYA-high breast cancertissues, 36 over 42 cases were CDK6 high; and 35 over 63 YIYA-

low breast cancer tissues were CDK6-low, suggesting the cor-relation between the expression of CDK6 and YIYA in breastcancer tissues (Fig. 3E).

CDK6 and FBXW7 coregulate PFKFB3 and STK38To understand how YIYA-associated CDK6/cyclin-D3 and

SKP1/FBXW7 complexes regulate cell growth, we identified theproteins that associated with both CDK6 and FBXW7 usingFLAG-tag pulldown followed by LC-MS (Fig. 4A). The datasuggested that protein arginine methyltransferase 5 (ANM5,also known as PRMT5), methylosome protein 50 (MEP50,also known as WDR77), serine/threonine kinase 38 (STK38),and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3(F263, PFK2, or PFKFB3) commonly associated with both CDK6and FBXW7 (Fig. 4A).

Next, we confirmed the interactions between CDK6, FBXW7with PRMT5, WDR77, PFKFB3, and STK38, respectively (Fig. 4B–E; Supplementary Fig. S4A–S4D). We also detected endogenousCDK6-STK38 and CDK6-PFKFB3 interaction (Fig. 4F and G).FBXW7 is a substrate recognition component of an SCF complex,

Figure 2.

YIYAmediates the interaction betweenCDK6 andFBXW7 complexes.A,Proteinscore identification in beads only, YIYA sense, and YIYA antisense samples byLC-MS. Dash line, score ¼ 200. B–E, Immunoprecipitation followed byimmunoblotting detection using indicated antibodies in MDA-MB-231 cellsexpressing indicated constructs.






which mediates the ubiquitination and subsequent proteasomaldegradation of target proteins (25). Overexpression of FBXW7wild-type (WT) reduced c-Myc protein levels (26, 27), which wereblocked by F-box domain deletion mutant (DF mutant; ref. 25),but not PFKFB3 or STK38 (Supplementary Fig. S5).

YIYA modulates CDK6-dependent phosphorylationWe hypothesized that the CDK6-STK38 and CDK6-PFKFB3

interactions may lead to phosphorylation of STK38

and PFKFB3. Using a quantitative in vitro kinase assay,the recombinant CDK6/cyclin-D3 complex catalyzed theincorporation of ATP to recombinant PFKFB3 and STK38 ina dose-dependent manner (Fig. 5A and B), which wereconfirmed adenosine 50-[g-thio] triphosphate (ATP-g-s;Fig. 5C–E).

LncRNAs have been reported to associate with serine/threonine protein kinases and potentially modulate theirenzymatic activities (28). The presence of YIYA sense tran-script, but not the antisense transcript, enhanced the CDK6-dependent phosphorylation of PFKFB3 and STK38 (Fig. 5Fand G). Using a quantitative in vitro kinase assay, the presenceof YIYA sense significantly enhanced the CDK6-mediatedphosphorylation of PFKFB3 and STK38 (Fig. 5H and I).Therefore, our data suggest that the elevated YIYA expressionlevels may promote CDK6 kinase activity and activate down-stream signaling pathways in breast cancer cells. The enzy-matic activity of CDK6 requires the presence of cyclins (22). Itis possible that the presence of YIYA promotes the associationbetween CDK6 and cyclin-D3, leading to enhanced phosphor-ylation of PFKFB3 and STK38.

Figure 3.

Characterization of YIYA-CDK6 and YIYA-FBXW7 interactions. A, RIP assayfollowed by PCR with RT or without RT using anti-Myc antibody in MDA-MB-231cells expressing indicated expression vectors. B, Streptavidin (Strep.) pulldownfollowedby immunoblottingdetection using recombinant CDK6WT, n-terminus(NT), or c-terminus (CT) in the presence of biotinylated YIYA sense (sen.) orantisense (ant.). C,RIP assay followed by RT-qPCR detection of YIYA using anti-His antibody in MDA-MB-231 cells expressing indicated expression vectors. D,Immunohistochemistry staining of CDK6 in breast cancer and adjacentnormal tissues. Left, representative images. Scale bar, 200 mm. Right, positiverates of CDK6 are shown, n¼ 15 and 105, respectively. E,High or low expressionsof YIYA andCDK6were plotted for correlation in breast cancer tissues. Error bar,SEM; n ¼ three independent experiments (� , P < 0.05; �� , P < 0.01; and �� , P <0.001, Student t test).

Figure 4.

CDK6 and FBXW7 coregulate PFKFB3 and STK38. A, Summary of topbinding proteins associated with FBXW7 or CDK6. The proteins associatedwith both FBXW7 and CDK6 are shown as bold. B–E, Immunoprecipitationfollowed by immunoblotting detection using indicated antibodies inMDA-MB-231 cells expressing FLAG-tagged CDK6 and indicatedconstructs. F and G, Immunoprecipitation followed by immunoblottingdetection using indicated antibodies in cell lysates extracted fromMDA-MB-231.

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The phosphorylation of PFKFB3 promotes glycolysisPFKFB3 catalyzes F-2,6BP (29, 30); the latter allosterically

modulates the enzymatic activity of PFK1, leading toenhanced glycolysis (31). The enzymatic activity of PFKFB3has been reported to be phosphorylated at residue S461,which regulates glycolysis (32). Two phosphorylation sites

(SVT463PLAS467PEPTKK), alone or in combination, were identi-fied by mass spectrometry. To confirm the CDK6-dependentphosphorylation of PFKFB3, we generated stable cell lineswith CDK6 knocked down by shRNAs (Fig. 6A). The phosphor-ylation of Myc-tagged PFKFB3 was reduced upon CDK6 knock-down (Fig. 6B). Expression of Myc-tagged PFKFB3 WT, T463A,

Figure 5.

CDK6 phosphorylates STK38 and PFKFB3. A and B,Phosphorylation incorporation of PFKFB3 (A) orSTK38 (B) in the presence of recombinant CDK6 andcyclin-D3 in dose-dependentmanner. C–E, In vitro kinaseassay using recombinant CDK6/cyclin-D3 and indicatedsubstrates in the presence of ATP-g-S, followed byimmunoblotting using indicated antibodies. F and G,In vitro kinase assay using recombinant CDK6/cyclin-D3,PFKFB3 (F) or STK38 (G) in the presence of invitro–transcribed YIYA (sense or antisense), followed byimmunoblotting using indicated antibodies. H and I,Phosphorylation incorporation of PFKFB3 (H) or STK38(I) using recombinant CDK6/cyclin-D3 in the presence ofin vitro–transcribedYIYA sense (Sen.) or antisense (Ant.).Error bar, SEM (n ¼ three independent experiments;� , P < 0.05; �� , P < 0.01; and �� , P < 0.001, Student t test).n.s., nonsignificant.

Figure 6.

PFKFB3 is regulated by CDK6-dependentphosphorylation. A, Immunoblotting detectionusing indicated antibodies in MDA-MB-231 cellsharboring indicated shRNAs. B and C,Immunoprecipitation followed by immunoblottingdetection of MDA-MB-231 cells harboring indicatedshRNAs (B) or expression vectors (C). D and E,Ratio of G6P-F6P over glucose (D) or GBP-FBP overG6P-F6P (E) in MDA-MD-231 cells expressing blank,PFKFB3 WT, or T463A/S467A mutant. Error bar,SEM (n¼ three independent experiments; �,P <0.05;�� , P < 0.01; and �� , P < 0.001, Student t test). n.s.,nonsignificant.






Figure 7.

YIYA/CDK6 coregulate glucose metabolic reprogramming. A, Graphic illustration of glucose metabolic pathway regulated by YIYA and CDK6. B, Thepercentage of [U-13C] glucose over unlabeled glucose in MDA-MB-231 cells with or without YIYA KO glucose starved overnight, followed with [U-13C]glucose feedback (11 mmol/L, 4 hours). C–F, Ratio of indicated metabolites in MDA-MB-231 cells with or without YIYA KO glucose starved overnight, followedwith [U-13C] glucose feedback (11 mmol/L, 2 hours). G, Medium lactate production detection of MDA-MB-231 cells with or without YIYA KO. H and I, Mediumglucose consumption (H) and lactate production (I) of MCF7 cells expressing indicated constructs (left) or BT474 cells harboring indicated shRNAs (right). J and K,The percentage of [U-13C] glucose over unlabeled glucose (J) or [U-13C] GBP/FBP over unlabeled GBP/FBP (% of pool; K) in MDA-MB-231 cells harboringindicated shRNAs were glucose starved overnight, followed with [U-13C] glucose feedback (11 mmol/L, 4 hours).


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S467A, or T463A/S467Amutants inMDA-MB-231 cells indicatedthat the phosphorylation status of exogenous PFKFB3 wasimpaired when T463 and S467 were both mutated (Fig. 6C).Expression of WT PFKFB3, but not T463A/S467A mutant,enhanced the production of FBP/GBP and reduced thecellular pool of G6P/F6P (Fig. 6D and E). These data suggestedthat CDK6 may regulate glucose metabolism through PFKFB3phosphorylation.

In breast cancer tissues, the expressions of PFKFB3 and CDK6were both elevated in TNBC compared with other biomarkerstatus (Gluck Breast, Oncomine; Supplementary Fig. S6A andS6B). The expression of PFKFB3 significantly correlated withthe expression of CDK6 in breast cancer tissues (Gluck Breast,Oncomine; Supplementary Fig. S6C). Hence, our data sug-gested the molecular linkage between YIYA, CDK6, and PFKFB3in breast cancer.

YIYA and CDK6 coregulate glucose metabolismThe YIYA/CDK6-dependent phosphorylation of PFKFB3 may

play critical roles in regulating glucose metabolism. Therefore,we determined the metabolic flux using a 13C-based strategy(Fig. 7A). The parental and YIYA KO cells were subjected toglucose starvation followed by feedback of [U-13C] glucose.Cellular 13C-glucose uptake was impaired in YIYA KO cellscompared with parental cells (Fig. 7B). Interestingly, in YIYAKO cells, we observed the accumulation of G6P/F6P anddepletion of FBP/GBP, suggesting the inhibition of the con-version from F6P to FBP (Fig. 7C and D). Consistently, deple-tion of YIYA led to impaired production of glycerol 3 phos-phate and pyruvic acid (Fig. 7E and F). The production oflactate was significantly reduced when YIYA was geneticallydeleted (Fig. 7G). Overexpression of YIYA in YIYA-low expres-sing MCF7 cells significantly enhanced glucose consumptionand lactate production (Fig. 7H and I; Supplementary Fig. S7A).In YIYA-high expressing BT474 cells, YIYA knockdownrepressed glucose consumption and lactate production(Fig. 7H and I; Supplementary Fig. S7B). Furthermore, knock-down of CDK6 led to decreased glucose uptake and productionof GBP/FBP (Fig. 7J and K). Taken together, our data indicatedthat lncRNAs may be functionally important in regulatingcancer metabolic reprogramming and tumor growth, makingthese molecules attractive therapeutic targets.

DiscussionThe advance of next-generation sequencing has revealed

large-scale dysregulation of lncRNAs in human diseases, par-ticularly in human cancer. However, the molecular mechan-isms underlying these RNA transcripts and their consequencesremain limited. Our data indicate that YIYA in breast cancermay promote cancer glycolysis augmentation. Mechanistically,YIYA modulates the association between the CDK6 complexand the SCF complex in the cytosol and regulates the CDK6-dependent phosphorylation events. High YIYA expressionlevels promote CDK6-dependent PFKFB3 phosphorylation,leading to the enhanced phosphorylation of F6P to FBP. Con-sequently, expression of YIYA and CDK6 are required to main-tain glycolysis at an elevated state in breast cancer. Our datademonstrate the functional role of lncRNA in breast cancermetabolic reprogramming.

The molecular mechanisms of CDK6 in cancer glycolyticreprogramming have been elusive. Based on our collected data,in the presence of YIYA, CDK6-dependent cytosolic phosphor-ylation events are important for promoting cancer metabolicreprogramming. The significantly elevated rates of glycolysis inbreast cancer situate glycolysis as a valuable component inidentifying new diagnostic strategies and therapeutic targets.Our data suggest that CDK6 inhibitors may block breast cancerglycolysis. Furthermore, antisense oligonucleotides targetinglncRNAs could serve as a new therapeutic strategy. Lockednucleic acids (LNA) or nanoparticle-encapsulated siRNAstargeting lncRNAs are under active development (17, 33).Pilot data indicate that LNAs or nanoparticle-deliveredsiRNAs against lncRNA targets can potently inhibit tumorgrowth, experimental metastasis, and glycolysis in vivo.Therefore, targeting lncRNAs could serve as a promisingtherapeutic strategy in furthering precision medicine in thenear future.

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

Authors' ContributionsConception and design: Z. Xing, Y. Zhang, N. Putluri, J. Zhou, L. Yang, C. LinDevelopment of methodology: Z. Xing, Y. Zhang, K. Liang, C. Li, Q. Hu,N. PutluriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Xing, Y. Zhang, K. Liang, C. Li, F. Jin, V. Putluri,N. Putluri, J. Zhou, J.R. Marks, D.H. HawkeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Xing, Y. Zhang, K. Liang, Y. Xiang, C. Li, V. Putluri,N. Putluri, C. Coarfa, A. Sreekumar, D.H. Hawke, L. Han, C. LinWriting, review, and/or revision of the manuscript: Y. Zhang, C. Li, P.K. Park,T.K. Nguyen, J. Zhou, J.R. Marks, L. Yang, C. LinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Zhang, K. Liang, L. Yan, C. Li, S. Wang,Y. Zhou, H. YingStudy supervision: M.-C. Hung, L. Yang, C. Lin

AcknowledgmentsWe are grateful to Drs. Yin Ye and Junjie Chen for generating YIYA KO cell

lines. We thank Peter K. Park and Tina K. Nguyen for manuscript drafting.This work is partially supported by BCM: Metabolomics Core, (NIHP30CA125123 to A. Sreekumar), DOD W81XWH-12-1-0130 (A. Sreeku-mar), CPRIT Proteomics and Metabolomics Core Facility (N. Putluri;RP170005), Dan L. Duncan Cancer Center, NIH U01 CA167234 (A. Sree-kumar), ACS 127430-RSG-15-105-01-CNE (N. Putluri), R01CA220297 (N.Putluri), and R01CA216426 (N. Putluri). This project was also supported bythe Agilent Technologies Center of Excellence in Mass Spectrometry at BaylorCollege of Medicine. This work was supported in part by CPRIT grantnumber RP130397 and NIH grant number 1S10OD012304-01 to D.H.Hawke. This work was supported by the NCI R00 award(4R00CA166527-02), R01 award (1R01CA218036-01), CPRIT First-timeFaculty Recruitment Award (R1218) and DOD Breakthrough Award(BC151465) to L. Yang and NIDDK R00 award (R00DK094981), NCIR01 award (1 R01 CA218025-01), and The CPRIT individual investigatorresearch award (RP150094 and RP180259) to C. Lin. This research wassupported by the Andrew Sabin Family Fellowship to L. Yang.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 10, 2017; revised August 15, 2017; accepted June 21, 2018;published first July 2, 2018.






References1. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into

functions. Nat Rev Genet 2009;10:155–9.2. Geisler S, Coller J. RNA in unexpected places: long non-coding RNA

functions in diverse cellular contexts. Nat Rev Mol Cell Biol 2013;14:699–712.

3. Zhang F, Lupski JR. Non-coding genetic variants in human disease. HumMol Genet 2015;24:R102–10.

4. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differen-tiation and development. Nat Rev Genet 2014;15:7–21.

5. Yang F, Yi F, Zheng Z, Ling Z, Ding J, Guo J, et al. Characterization of acarcinogenesis-associated long non-coding RNA. RNA Biol 2012;9:110–6.

6. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? NatRev Cancer 2004;4:891–9.

7. Marcus F, Gontero B, Harrsch PB, Rittenhouse J. Amino acid sequencehomology among fructose-1,6-bisphosphatases. Biochem Biophys ResCommun 1986;135:374–81.

8. Storey KB. Metabolic regulation in mammalian hibernation: enzymeand protein adaptations. Comp Biochem Physiol A Physiol 1997;118:1115–24.

9. Shirakihara Y, Evans PR. Crystal structure of the complex of phosphofruc-tokinase from Escherichia coli with its reaction products. J Mol Biol1988;204:973–94.

10. Van Schaftingen E, Lederer B, Bartrons R, Hers HG. A kinetic study ofpyrophosphate: fructose-6-phosphate phosphotransferase from potatotubers. Application to a microassay of fructose 2,6-bisphosphate. Eur JBiochem 1982;129:191–5.

11. Wu C, Khan SA, Peng LJ, Lange AJ. Roles for fructose-2,6-bisphosphate inthe control of fuel metabolism: beyond its allosteric effects on glycolyticand gluconeogenic enzymes. Adv Enzyme Regul 2006;46:72–88.

12. Chesney J, Mitchell R, Benigni F, Bacher M, Spiegel L, Al-Abed Y, et al. Aninducible gene product for 6-phosphofructo-2-kinase with an AU-richinstability element: role in tumor cell glycolysis and the Warburg effect.Proc Natl Acad Sci U S A 1999;96:3047–52.

13. Clem B, Telang S, Clem A, Yalcin A, Meier J, Simmons A, et al. Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses gly-colytic flux and tumor growth. Mol Cancer Ther 2008;7:110–20.

14. Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation.Development 2013;140:3079–93.

15. Scheicher R, Hoelbl-Kovacic A, Bellutti F, Tigan AS, Prchal-Murphy M,HellerG, et al. CDK6as a key regulatorof hematopoietic and leukemic stemcell activation. Blood 2015;125:90–101.

16. ZanuyM, Ramos-MontoyaA, Villaca~nasO,CanelaN,MirandaA, Aguilar E,etal.Cyclin-dependentkinases4and6control tumorprogressionanddirectglucose oxidation in the pentose cycle. Metabolomics 2012;8:454–64.

17. Xing Z, Lin A, Li C, Liang K,Wang S, Liu Y, et al. lncRNA directs cooperativeepigenetic regulation downstream of chemokine signals. Cell 2014;159:1110–25.

18. Li C, Wang S, Xing Z, Lin A, Liang K, Song J, et al. A ROR1-HER3-lncRNAsignalling axis modulates the Hippo-YAP pathway to regulate bonemetas-tasis. Nat Cell Biol 2017;19:106–19.

19. Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al. Oncogenic Kras maintains pancreatic tumors throughregulation of anabolic glucose metabolism. Cell 2012;149:656–70.

20. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al.Genome-scale CRISPR-Cas9 knockout screening in human cells. Science2014;343:84–87.

21. Yang H, Wang H, Jaenisch R. Generating genetically modified mice usingCRISPR/Cas-mediated genome engineering. Nat Protoc 2014;9:1956–68.

22. MeyersonM,HarlowE. IdentificationofG1kinase activity for cdk6, a novelcyclin D partner. Mol Cell Biol 1994;14:2077–86.

23. Cardozo T, Pagano M. The SCF ubiquitin ligase: insights into a molecularmachine. Nat Rev Mol Cell Biol 2004;5:739–51.

24. Schulze-Gahmen U, Kim SH. Structural basis for CDK6 activation by avirus-encoded cyclin. Nat Struct Biol 2002;9:177–81.

25. Suryo Rahmanto A, Savov V, Brunner A, Bolin S, Weishaupt H, MalyukovaA, et al. FBW7 suppression leads to SOX9 stabilization and increasedmalignancy in medulloblastoma. EMBO J 2016;35:2192–212.

26. Moshe Y, Boulaire J, Pagano M, Hershko A. Role of Polo-like kinase in thedegradation of early mitotic inhibitor 1, a regulator of the anaphasepromoting complex/cyclosome. Proc Natl Acad Sci U S A 2004;101:7937–42.

27. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, et al.A signalling pathway controlling c-Myc degradation that impactsoncogenic transformation of human cells. Nat Cell Biol 2004;6:308–18.

28. Liu X, Xiao ZD, Han L, Zhang J, Lee SW, Wang W, et al. LncRNA NBR2engages a metabolic checkpoint by regulating AMPK under energy stress.Nat Cell Biol 2016;18:431–42.

29. Okar DA,ManzanoA,Navarro-Sabat�e A, Riera L, Bartrons R, Lange AJ. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem Sci 2001;26:30–5.

30. Van Schaftingen E. Fructose 2,6-bisphosphate. Adv Enzymol Relat AreasMol Biol 1987;59:315–95.

31. Ros S, Schulze A. Balancing glycolytic flux: the role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases in cancer metabolism. Cancer Metab2013;1:8.

32. Bando H, Atsumi T, Nishio T, Niwa H, Mishima S, Shimizu C, et al.Phosphorylation of the 6-phosphofructo-2-kinase/fructose 2,6-bispho-sphatase/PFKFB3 family of glycolytic regulators in human cancer. ClinCancer Res 2005;11:5784–92.

33. Rupaimoole R, Lee J, Haemmerle M, Ling H, Previs RA, Pradeep S, et al.Long noncoding RNA ceruloplasmin promotes cancer growth by alteringglycolysis. Cell Rep 2015;13:2395–402.


Xing et al.




2018;78:4524-4532. Published OnlineFirst July 2, 2018.Cancer Res Zhen Xing, Yanyan Zhang, Ke Liang, et al. Breast Cancer

Promotes Glycolysis inYIYAExpression of Long Noncoding RNA

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expression of long noncoding rna yiya promotes glycolysis ... · impaired glycolysis and tumor...

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