Nutrient Stress Dysregulated Antisense lncRNA GLS-AS ... · To detect GLS-AS and GLS pre-mRNAs, we...

Molecular Cell Biology

Nutrient Stress–Dysregulated Antisense lncRNAGLS-AS Impairs GLS-Mediated Metabolism andRepresses Pancreatic Cancer ProgressionShi-Jiang Deng1, Heng-Yu Chen1, Zhu Zeng1, Shichang Deng1, Shuai Zhu1, Zeng Ye1,Chi He1, Ming-Liang Liu1, Kang Huang1, Jian-Xin Zhong1, Feng-Yu Xu1, Qiang Li1,Yang Liu1, Chunyou Wang2, and Gang Zhao1

Abstract

© 2018 American Association for Cancer Research

Normal

GLS pre–mRNA

GLS pre–mRNA

AAAAAA

AAAAAA

DegradationDegradation

GLS protein

Myc

ADAR1–dicer

ADAR1–dicer

GLS–ASGLS–AS

GLS–AS gene

GLS–AS gene

Nutrient stress promotes accumulation of Myc. Myc inhibits transcription of the antisense lncRNA of glutaminase (GLS–AS), leadingto GLS elevation and stabilization of Myc.

Nutrient stressCancer cells are known to undergo metabolicreprogramming, such as glycolysis and glutamineaddiction, to sustain rapid proliferation andmetastasis. It remains undefined whether longnoncoding RNAs (lncRNA) coordinate the meta-bolic switch in pancreatic cancer. Here we identifya nuclear-enriched antisense lncRNA of glutamin-ase (GLS-AS) as a critical regulator involved inpancreatic cancer metabolism. GLS-AS was down-regulated in pancreatic cancer tissues comparedwith noncancerous peritumor tissues. Depletionof GLS-AS promoted proliferation and invasion ofpancreatic cancer cells both in vitro and in xenografttumors of nude mice. GLS-AS inhibited GLSexpression at the posttranscriptional level via for-mation of double stranded RNA with GLS pre-mRNA through ADAR/Dicer-dependent RNA interference. GLS-AS expressionwas transcriptionally downregulated by nutrient stress–induced Myc. Conversely, GLS-AS decreased Myc expression byimpairing the GLS-mediated stability of Myc protein. These results imply a reciprocal feedback loop wherein Myc andGLS-AS regulate GLS overexpression during nutrient stress. Ectopic overexpression of GLS-AS inhibited proliferation andinvasion of pancreatic cancer cells by repressing the Myc/GLS pathway. Moreover, expression of GLS-AS and GLS was inverselycorrelated in clinical samples of pancreatic cancer, while low expression of GLS-AS was associated with poor clinical outcomes.Collectively, our study implicates a novel lncRNA-mediated Myc/GLS pathway, which may serve as a metabolic target forpancreatic cancer therapy, and advances our understanding of the coupling role of lncRNA in nutrition stress and tumorigenesis.

Significance: These findings show that lncRNA GLS-AS mediates a feedback loop of Myc and GLS, providing a potentialtherapeutic target for metabolic reprogramming in pancreatic cancer.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1398/F1.large.jpg.See related commentary by Mafra and Dias, p. 1302

IntroductionRecent studies have shown that cancer cells exhibit metabolic

dependencies to distinguish them from normal tissues. One

of these addictions is "Warburg effect" that cancer cells tend totake advantage of glucose via "aerobic glycolysis" pathway,even in the presence of oxygen (1). As an outcome, the pyruvategenerated via the aerobic glycolysis is converted to lactic acid,but not acetyl-CoA. To compensate for the insufficient citricacid cycle, cancer cells often activate glutamine metabolism (2).Therefore, markedly aggravated glucose and glutamine deple-tion may happen in tumor cells as there are inadequaciesbetween vascular supply and metabolic requirement (3). Sucha situation is especially distinct in pancreatic cancer, whereglucose and glutamine metabolism is reprogrammed by onco-genic Kras to support cancer cell growth (4–6). Therefore,the metabolic characteristics and distinct hypovascular ofpancreatic cancer would lead to a dramatic nutrients stressespecially caused by glucose and glutamine depletion (7). Infact, such a paradoxical condition affords pathway to rapidlyproduce the energy and metabolites required for cancer cells'

1Department of Emergency Surgery, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, China. 2Deparment ofPancreatic Surgery, UnionHospital, Tongji Medical College,HuazhongUniversityof Science and Technology, Wuhan, China.

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

S.-J. Deng, H.-Y. Chen, and Z. Zeng contributed equally to this article.

Corresponding Author: Gang Zhao, Department of Emergency Surgery, UnionHospital, Tongji Medical College, Huazhong University of Science and Technol-ogy, Wuhan 430022, China. Phone: 8627-8535-1621; Fax: 8627-8535-1669;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-0419

�2018 American Association for Cancer Research.

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Published OnlineFirst December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-0419

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proliferation, which makes it correlatively resistant to meta-bolic stress including hypoxia and nutrient deprivation (8).Data from Yun and colleagues suggest that glucose deprivationcan drive the acquisition of Kras pathway mutations (9), whichcommonly occurs in pancreatic cancer. The results suggestedthat glucose deprivation increases VEGF mRNA stability, whichmight facilitate tumor angiogenesis (10). Furthermore, resultsfrom Dejure and colleagues showed glutamine deprivationonly halted the proliferation of colon cancer cells, but notkilled them (11). Notably, nutrient deprivation has been cor-related with poor patient survival, suggesting that instead ofkilling the tumor, the scarcity of nutrients can make the cancercell stronger (12). Therefore, it is crucial to investigate themechanisms that are required to accommodate nutrient stressesas an alternative strategy for the therapeutic treatment of pan-creatic cancer.

Long noncoding RNAs (lncRNA) are a major class of tran-scripts, longer than 200 nt, and lack protein-coding potential.Accumulating evidence suggests that lncRNAs are dysregulatedin cancers and involved in the development of cancers (13).Specifically, recent results have demonstrated a link betweenlncRNAs and altered metabolism in cancers. A study reportedthat a glucose starvation–induced lncRNA-NBR2 reciprocallyactivates AMPK pathway in response to energy stress (14).LncRNA-UCA1 promotes glycolysis in bladder cancer cellsby activating the cascade of mTOR-STAT3/miR143-HK2 (15).Results from Ellis and colleagues suggested that insulin/IGFsignaling–repressed lncRNA-CRNDE promotes aerobic glyco-lysis of cancer cells (16). LncRNA-ANRIL is upregulated innasopharyngeal carcinoma and promotes cancer progressionvia increasing glucose uptake for glycolysis (17). In addition,lncR-UCA1 was found to reduce ROS production, and pro-moted mitochondrial glutaminolysis in human bladder can-cer (18). Nevertheless, the specific lncRNAs, which couplenutrient stress and pancreatic cancer, have not been elucidatedyet. In this study, we endeavored to discover a nutrient stress–responsive lncRNA that is involved in the pancreatic cancerprogression.

Glutaminase (GLS) is a phosphate-activated amidohydrolasethat catalyzes the hydrolysis of glutamine to glutamate andammonia to support metabolism homeostasis, bioenergetics,and nitrogen balance (19). Recent studies have revealed GLSis commonly overexpressed in numerous malignant tumorsand acts as an oncogene to support cancer growth (20, 21). It isnoted that GLS is increased in breast cancers compared withsurrounding nontumor tissues and positively correlates to thetumor grade (20). Moreover, GLS couples glutaminolysisof the TCA cycle with elevated glucose uptake and consequentlythe growth of prostate cancer cells (21). Meanwhile, knock-down of GLS significantly blocked the growth and invasiveactivity of various cancer cells (22). Results from Chakrabartiand colleagues demonstrated that GLS is highly upregulated inpancreatic cancer, thereby targeting glutamine metabolism andsensitizing pancreatic cancer cells to PARP-driven metaboliccatastrophe (23). In previous study, we discovered a cluster ofdysregulated lncRNAs in pancreatic cancer (24). Coincidently,one of the significantly downregulated lncRNA, AK123493, isan antisense lncRNA of glutaminase (GLS). Therefore, it drawsour attention whether a nuclear-enriched antisense lncRNA ofglutaminase (GLS-AS) might be involved in the GLS-mediatedmetabolism of pancreatic cancer.

Materials and MethodsPatients and specimens

The clinical tissues were obtained from Pancreatic DiseaseInstitute of Union Hospital from May 2016 to March 2017. Werandomly selected 30 pairs of pancreatic cancer and correspond-ing nontumor tissues from patients without chemotherapy orradiotherapy before operation. Procedures performed on thosepatients included pancreatectomy or palliative surgery includingI125 seed implantation as well as gastroenterostomy and chole-dochojejunostomy according to the National ComprehensiveCancer Network (NCCN 2012) guideline for pancreatic cancer.The samples were obtained from surgical resection of patients orbiopsy of the palliative surgery patients. The study was conductedin accordance with the Declaration of Helsinki. All samples werecollected with the written informed consent of the patients, andthe study was approved by the local Research Ethics Committee atthe Academic Medical Center of Huazhong University of Scienceand Technology (Wuhan, China).

Cell cultureBxPC-3 and PANC-1 cells were obtained fromATCC. Theywere

tested and authenticated for genotypes by DNA fingerprintingwithin 6 months. Cells were cultured in 5% CO2 at 37�C andgrown in complete medium, which was composed of 90%RPMI1640 (Gibco), 10% FBS (Gibco), 100 U/mL penicillin, and100 mg/mL streptomycin. To build nutrition deprivation model,we incubated cells with complete medium without glutamine[Glutamine (�)] or complete medium with 1 mmol/L glucose[Glucose (�)]. RPMI1640 having no glutamine or glucose waspurchased from Gibco.

RNA FISHTo detect GLS-AS and GLS pre-mRNAs, we purchased a kit

named FISH Tag RNA Multicolor Kit from Invitrogen to performFISH. The probe synthesis, labeling, and purification procedureswere performed according to the manufacturer's instructions.The probe-identified GLS pre-mRNA (Probe1) was labeled withgreen fluorescence, and the GLS-AS probe (Probe2) was labeledwith red fluorescence. Cells were fixed in formaldehyde, perme-abilized by Triton X-100, and then hybridization was carried outusing labeled probes in a moist chamber at 42�C overnight. Ifnecessary, the GLS protein immunofluorescence was conductedafter all the FISH procedures were completed.

RNA-binding protein immunoprecipitationTo detect RNA–protein binding complexes, RNA-binding pro-

tein immunoprecipitation (RIP) assays were performed accordingto the instructions of RNA-Binding Protein ImmunoprecipitationKit (Magna RIP, Millipore). First, the cells were lysed in lysis buffercontaining protease inhibitor cocktail and RNase inhibitor.Magnetic beads were preincubated with an anti-ADAR1 antibodyor anti-Dicer for 30minutes at room temperature, and lysates wereimmunoprecipitated with bead-bound antibody at 4�C overnight.Then immobilized magnetic bead–bound antibody–protein com-plexeswere obtained,washedoff unboundmaterials, RNApurifiedfrom RNA–protein complexes, and then analyzed by qPCR.

Northern blotWe purchased a DIG RNA Labeling Kit (Roche) to perform

Northern blot analysis for GLS-AS. First, we prepared GLS-AS–specific DNA template containing T7 promoter sequences from

LncRNA-GLS-AS Inhibits Pancreatic Cancer Progression

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RT-PCR and the DNA template was purified. Then, the DIG-labeled RNA probes were produced according to the kit instruc-tions with the DNA template. DIG-labeled probes were used forhybridization to nylon membrane–blotted total RNA. Thehybridized probes were detected with anti-digoxigenin-AP, andthen were visualized with the chemiluminescence substrateCSPD. The signals were also captured by ChemiDocTm XRSMolecular Imager system (Bio-Rad).

CoimmunoprecipitationFor coimmunoprecipitation (co-IP) analysis, anti-ADAR1,

anti-Dicer, or normal mouse/rabbit IgG were used as the primaryantibodies, and then the antibody–protein complex was incubat-ed with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology).The agarose–antibody–protein complex was collected and thenanalyzed by Western blot.

Chromatin immunoprecipitationThe PCR primers are indicated in Supplementary Table S1. We

conducted chromatin immunoprecipitation (ChIP) assays usingEZ-ChIPTMChromatin Immunoprecipitation Kit (Millipore). Allthe procedures were performed according to the manufacturer'sinstructions. Rabbit anti-Myc (Cell Signaling Technology), anti-RNA polymerase II antibodies (Abcam), and correspondingrabbit-IgG (Cell Signaling Technology) were used as controls.The bound DNA fragments were amplified by PCR reactions, andthen PCR products were analyzed by gel electrophoresis on 2%agarose gel. The PCR primers used were listed in SupplementaryTable S2.

Luciferase activity assayFor GLS-AS promoter activity analysis, BxPC-3 cells were trans-

fected with pGL3 vector wild-type or mutant GLS-AS promoterwith firefly luciferase plasmid while a plasmid pRL-TK carryingRenilla luciferase was used as internal reference. To confirm thenutrition deprivation impact on GLS-AS promoter activity, cellswere cultured under glutamine or glucose deprivation for 24 or 48hours. To investigate the relationship between Myc protein andGLS-AS promoter activity, siMyc or the control siNC was cotrans-fected into the BxPC-3 cells containing luciferase plasmid.The reporter activity was measured using a luciferase assay kit(Promega) and plotted after normalizing with respect to Renillaluciferase activity. Firefly luciferase activity was normalized to thecorresponding Renilla luciferase activity. The data are representedas mean � SD of three independent experiments.

Biotin-RNA pull-down assayThe full or partial length of intron-17 of GLS gene sequences

was amplified by PCR with SP6/T7-containing primer and thentranscribed by MAXIscript SP6/T7 Transcription Kit (ThermoFisher Scientific). The synthetic RNA was biotin-labeled withPierce RNA 30 End Desthiobiotinylation Kit (Thermo FisherScientific). The biotin-labeled RNAs were incubated with cell lysisindividually and the target complexes were precipitated by strep-tavidin-coupled Dynabeads (Invitrogen). Finally, Northern blotanalysis identified whether GLS-AS was pulled down or not.

RNA pull-down by MS2-GSTWe constructed a plasmid expressing GLS-AS tagged with MS2

hairpin loops (GLS-AS-MS2), a plasmid expressingMS2-GST-NSLfusion protein, and a plasmid only expressingMS2 (MS2) RNA as

control. Pancreatic cancer cells in the test and control groups weretransfected with GLS-AS-MS2 and MS2, respectively, along withMS2-GST-NSL fusion protein. After the cotransfection for 48hours, cells were harvested and then RNA pull-down assay wasconducted as described previously (25). The purified proteinswere analyzed byWestern blot analysis while RNAs were detectedwith Northern blot.

Xenograft assayLentivirus containing specific DNA sequences was transfected

into BxPC-3 and PANC-1 cells. Five-week-old BALB/c male nudemice were bought fromHFK Bio-Technology Co. To assess tumorgrowth in vivo, 100 mL RPMI1640 medium without FBS contain-ing 4� 106 cells was suspended and then planted subcutaneouslyinto the nudemice (each group has 6mice). Tumor volumes weremeasured every 4 days according to the formula V ¼ 0.5 � L(length) � W2 (width). Mice were sacrificed at 3 weeks after cellinoculation. Solid tumor tissues were removed and weighed. Toinvestigate tumor metastasis in vivo, mice were injected with1 � 104 tumor cells through the tail vein; visible metastases onliver were counted and then confirmed by hematoxylin andeosin–stained slides after 3 weeks. Care and handling of the micewere approved by the Institutional Animal Care and Use Com-mittee of TongjiMedical College, HuazhongUniversity of Scienceand Technology (Wuhan, China).

Statistical analysesAll results were presented as means � SD. Comparisons

between two groups were performed with Student t test. Thecorrelation between GLS-AS and GLS mRNA or GLS and MycmRNA was revealed by Pearson correlation analysis. The expres-sion of GLS-AS and the clinical characteristics were analyzed byx2 test, while the log-rank test was conducted to survey pancreaticcancer patient survival.Differencewas regarded to be significant at�, P < 0.05 and ��, P < 0.01.

ResultsAK123493.1, a nuclear accumulated antisense lncRNA of GLS(GLS-AS), is downregulated in pancreatic cancer

The microarray analysis showed AK123493.1 was decreased inthe pancreatic cancer tissues compared with noncancerous peri-tumoral (NP) tissues (Fig. 1A). GLS-AS is an intronic antisenselncRNA embedded within intron-17 of the corresponding sensegene GLS (Fig. 1B). In addition, the Northern blot validated theexpression of GLS-AS in BxPC-3 and PANC-1 cells using the RNAprobe (Fig. 1C). Moreover, the expression levels of GLS-AS inBxPC-3 and PANC-1 cells were lower than that in the normalhuman pancreatic duct epithelial cells (HPDE; Fig. 1D). To vali-date the signal specificity, Northern blot analysis was conductedafter the cells were transfected with siGLS-AS. As shown in Fig. 1E,siGLS-AS significantly decreased the expression of GLS-AS. Fur-thermore, the Northern blot results showed that GLS-AS wasobviously lower in pancreatic cancer tissues compared with NP(Fig. 1F). Meanwhile, the FISH assay showed that GLS-AS ismainly accumulated in the nucleus (Fig. 1G), implying GLS-ASmay predominantly function in the nucleus. Similarly, separationof nuclear extract and the cytoplasmic fraction showed thatGLS-AS retained in the nucleus (Fig. 1H). Both Coding PotentialAssessment Tool (26) and Coding Potential Calculator (27) pre-dictedGLS-AS is a noncoding RNA. Furthermore, we blocked newRNA synthesis with RNA polymerase II (Pol II) inhibitor

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Figure 1.

LncRNA-AK123493.1, an antisense of GLS (GLS-AS), is downregulated in pancreatic cancer. A,Microarray analysis demonstrated LncRNA-AK123493.1 wasdistinctly downregulated in two pancreatic cancer (PC) samples compared with paired noncancerous pancreatic (NP) tissues. B, Schematic diagram of GLS-ASand GLS gene location and relationship in genome. Arrows, transcript orientation. Probe-1 labeled with red fluorescence was used to detected GLS-pre-mRNAwhile probe-2 labeled with green fluorescence was for GLS-AS. C,Northern blot analysis of GLS-AS in pancreatic cancer cells BxPC-3 and PANC-1 with RNAprobe. D, Northern blot analysis of GLS-AS in HPDE cells compared with BxPC-3 and PANC-1 cells. E,Northern blot analysis of GLS-AS in BxPC-3 and PANC-1 cellswhen GLS-AS were knocked down by siRNAs. F, Northern blot analysis of GLS-AS in five paired cancer and noncancerous pancreatic tissues. G, FISH analysisshowed the location of GLS-AS in BxPC-3 cells. H, Histogram shows expression level of GLS-AS in the subcellular fractions of BxPC-3 and PANC-1 cells, analyzedby qPCR. RT-PCR products were run on a 2% agarose gel and U6 and GAPDHwere used separately as nuclear and cytoplasmic markers. I, After blocking newRNA synthesis with a-amanitin (50 mmol/L) in BxPC-3 cells, stability of GLS-AS was measured by qPCR compared with time 0. b-Actin was transcribed by RNApolymerase II, while 18s RNAwas a product of RNA polymerase I. J,GLS-AS in 30 pancreatic cancer and corresponding adjacent noncancerous pancreatic tissueswas measured by qPCR. K, The Kaplan–Meier curves for overall survival analysis of patients with pancreatic cancer by GLS-AS expression. Expression levels ofGLS-AS was categorized into "high" and "low" using the median value as the cut-off point. All data are presented as means� SD of at least three independentexperiments. Values are significant at �� , P < 0.01 as indicated.






a-amanitin (50 mmol/L) in BxPC-3 cells and measured the expres-sion of GLS-AS by qPCR relative to time 0. After treating witha-amanitin, the expression of GLS-AS was significantly decreased,while the 18smRNA,which is transcribed byPol I, was not affected.These results indicate that the transcription of GLS-AS is proceededin a Pol II–dependent manner (Fig. 1I). We further validated theGLS-AS expression level in pancreatic cancer tissues and paired NPtissues by qPCR. Results showed that GLS-AS expression in pan-creatic cancer was significantly lower than that in NP (Fig. 1J). Inaddition, the low expression of GLS-AS was associated with largetumor size, lymph node invasion, remote metastasis (Supplemen-tary Table S1), and short overall survival time (Fig. 1K).

Low expression of GLS-AS facilitates proliferation and invasionof pancreatic cancer cells

To understand the roles of GLS-AS downregulation in pancre-atic cancer progression, we depleted GLS-AS expression withsiRNA (siGLS-AS) in pancreatic cancer cells (Fig. 2A; Supplemen-tary Fig. S1A). After downregulation of GLS-AS, the proliferationand colony formation of PANC-1 (Fig. 2B andC) andBxPC-3 cells(Supplementary Fig. S1B and S1C) were significantly enforced.Meanwhile, transwell and wound-healing assays further revealedan enhanced invasion and migration ability in GLS-AS–depletedPANC-1(Fig. 2D and E) and BxPC-3 cells (Supplementary Fig.S1D and S1E). To further confirmwhether reduced GLS-AS affectspancreatic cancer progression in vivo, PANC-1 and BxPC-3 cellswere stably transfected with lentivirus containing siGLS-AS(LV-siGLS-AS) or siNC (LV-NC) transplanted subcutaneouslyinto the mouse, respectively. Compared with LV-NC group, thePANC-1 tumors in the LV-siGLS-AS groupwere larger and heavier(Fig. 2F), withmore visible liver and lungmetastases (Fig. 2G andH). Similarly, the proliferation and metastasis of BxPC-3 tumorwith LV-siGLS-AS were also enhanced (Supplementary Fig. S1F–S1H). These results indicate that the dysregulated GLS-AS expres-sion might contribute to pancreatic cancer development.

GLS is the critical target of GLS-AS to exert function inpancreatic cancer

Because GLS-AS is an antisense lncRNA of GLS, we furtherinvestigated whether GLS is a functional target of GLS-AS. Coin-cidently, knockdown of GLS-AS apparently increased the GLSexpression both in mRNA and protein levels in both PANC-1(Fig. 3A) and BxPC-3 cells (Supplementary Fig. S2A). Coincident-ly, transfectionwith a plasmid containing GLS-AS sequence (GLS-AS) obviously decreased GLS expression both in mRNA andprotein levels of PANC-1 and BxPC-3 cells (Fig. 3B; Supplemen-tary Fig. S2B). Subsequently, co-staining fluorescence of GLS-AStranscription and GLS protein further validated GLS was nega-tively regulated by GLS-AS in PANC-1 and BxPC-3 cells (Fig. 3C;Supplementary Fig. S2C). In agreement, the costaining fluores-cence assay further showed adecreasedGLS-AS accompaniedwithincreased GLS protein expression in pancreatic cancer tissuecompared with NP tissue (Fig. 3D). Meanwhile, Western blotanalysis further validated the downregulation of GLS protein inpancreatic cancer tissues compared with NP tissues (Supplemen-tary Fig. S2D). Meanwhile, depletion of GLS with siGLS remark-able inhibited proliferation, colony formation, invasion, andmigration ability of PANC-1 and BxPC-3 cells, which was rein-forced by siGLS-AS (Supplementary Fig. S3A–S3J). Thus, thesedata implied that the GLS would be a critical target for dysregu-lated GLS-AS to exert its biological function in pancreatic cancer.

GLS-AS inhibits GLS expression in posttranscriptional level byADAR1/Dicer-dependent RNA interference

To further identify whether GLS-AS regulates GLS transcription,BxPC-3 or PANC-1 cells were transfected with pGL3 plasmidcontaining GLS putative promoter region. As shown in Supple-mentary Fig. S4A and S4B, the luciferase reporter assay showedneither knockdown nor overexpression of GLS-AS and did notchange the GLS promoter activity, which implied that GLS-AS didnot regulate GLS expression at the transcriptional level.

The co-RNA FISH assay disclosed both GLS-AS and GLS pre-mRNA hybridized in the same nuclear foci of PANC-1 cells(Fig. 4A), which indicates the formation of dsRNA. To furthervalidate the direct interaction between GLS-AS and GLS pre-mRNA in PANC-1 cells, the RNA–RNA pull-down assay wasperformed with biotin-labeled full or partial length deletion ofintron-17 transcripts (Fig. 4B). As GLS-AS is an antisense lncRNAof GLS pre-mRNA, we further presumed GLS-AS might regulatethe stability of GLS pre-mRNA. To evaluate the stability of GLSpre-mRNA, pancreatic cancer cells were treated with amanitin(50 mmol/L). The expression of GLS pre-mRNA was measured byqPCR at the separated time point. Compared with time 0, thestability was dramatically enhanced by siGLS-AS, but impaired byGLS-AS overexpression in PANC-1 cells (Fig. 4C and D). Researchhad demonstrated that adenosine deaminases acting on RNA(ADAR1) are involved inRNA interference of dsRNAby formationof ADAR1/Dicer heterodimer complexes, a member protein ofRNA-induced silencing complex (RISC; ref. 28). Meanwhile, theco-IP analysis validated the binding between ADAR1 and Dicerprotein in PANC-1 cells (Fig. 4E; Supplementary Fig. S5A). Toconfirm whether the ADAR1/Dicer proteins physically bound toGLS-AS/GLS pre-mRNAdsRNAor not, we performed RNA immu-noprecipitation (RNA-IP) assays in PANC-1 cells. Compared withthe IgG-bound sample, the ADAR1 or Dicer antibody–boundcomplex showed significantly high enrichment of GLS-AS andGLSpre-mRNA inPANC-1 cells (Fig. 4F; Supplementary Fig. S5B).Thus, we wondered whether the GLS-AS and GLS pre-mRNA areregulated by ADAR1/Dicer-mediated RNA silencing. Both siA-DAR1 and siDicer increased the expression of GLS-AS, GLS pre-mRNA, and protein, were remarkably increased in PANC-1 cells(Fig. 4G; Supplementary Fig. S5C), reduced the enrichment ofGLS-AS andGLS pre-mRNA in protein (ADAR1/Dicer)–antibody-bound complex in PANC-1 cells (Fig. 4H; Supplementary Fig.S5D), as well as strengthened the stability of GLS pre-mRNA inPANC-1 cells (Fig. 4I; Supplementary Fig. S5E). Meanwhile, theenrichment of GLS pre-mRNA was reduced by siGLS-AS, butupregulated by GLS-AS overexpression, both by anti-ADAR1(Fig. 4J and K) and anti-Dicer (Supplementary Fig. S5F andS5G) in PANC-1 cells. In addition, both siADAR1 (Fig. 4L) andsiDicer (Supplementary Fig. S5H) could rescue the expression ofGLS mRNA and protein in PANC-1 cells, which was inhibited byGLS-AS overexpression. Furthermore, MS2-tagged RNA affinitypurification analysis was performed to further confirm GLS-AS,GLS-pre-mRNA, and ADAR1/Dicer can form a complex in PANC-1 cells (Fig. 4M). Simultaneously, the experiments describedabove were also conducted in BxPC-3 cells. Results of BxPC-3cells also confirmed an interaction between GLS-pre-mRNA andGLS-AS (Supplementary Fig. S6A and S6B), which could regulatethe stability of GLS-pre-mRNA (Supplementary Fig. S6C andS6D). Moreover, results further displayed that ADAR1 is requiredfor the regulation of GLS-AS on GLS expression in BxPC-3 cells(Supplementary Fig. S6E–S6M). In addition, results also validated

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Figure 2.

Knockdown of GLS-AS facilitates pancreatic cancer proliferation and invasion. A, qPCR analyzed the knockdown efficiency of GLS-AS by the three siRNAs inPANC-1 cells. B, After transfecting with siGLS-AS #2 or siGLS-AS #3, growth rate of the transfected PANC-1 cells was measured by MTT assays for 5 days. C,Colony formation assay was performed in transfected PANC-1 cells. Relative colony number (left) and representative images (right) are shown. D, Transwell assaywas conducted to observe the invasion ability of the transfected PANC-1 cells. The left histogram represents relative cell number while the representative imagesare shown on the right. E, Migration ability of transfected PANC-1 cells was analyzed by wound-healing assay. Representative images (left) and relative wound size(right) are shown. F–H, PANC-1 cells transfected with lentivirus-containing sequence of siGLS-AS (LV-siGLS-AS) or empty lentivirus vector (LV-siNC) weretransplanted subcutaneously into nude mice to observe tumor growth (5 � 106 cells per mouse). F, A photograph of representative nude mice and tumor ispresented after 3 weeks when mice were sacrificed (left). The tumor volumes were measured every 4 days. Two groups of tumor weights were measuredindividually. G, Top, histogram shows number of visible liver metastases per 5 sections in each nude mouse. Bottom, representative images of livers andcorresponding hematoxylin and eosin–stained section. H, Left, histogram shows number of visible lung metastases per 5 sections in each nude mouse. Right,representative hematoxylin and eosin–stained section of lungs with metastases. All data are presented as means � SD of at least three independent experiments.Values are significant at � , P < 0.05 and ��, P < 0.01 as indicated.






that Dicer is necessary for the ADAR1/Dicer-mediated regulationof GLS-AS on GLS expression in BxPC-3 cells (SupplementaryFig. S7A–S7H). Supplementary Figure S8 is a schematic diagramof MS2-tagged RNA affinity purification analysis.

Nutrient stress is responsible for downregulation of GLS-AS inpancreatic cancer

We further investigated whether the GLS-AS downregulation inpancreatic cancer is attributed to metabolism stress includinghypoxia, acidity, or depletion of glucose and glutamine. Interest-

ingly, GLS-ASwas obviously decreased during depletion of glucoseor glutamine, but without significant alteration in BxPC-3 andPANC-1 cells during hypoxia or acidity (Fig. 5A). Moreover, qPCRand FISH assays demonstrated a time-dependent GLS-AS down-regulation during glutamine or glucose deprivation (Fig. 5B–D).Coincidentwith theGLS-ASdownregulation,bothGLSmRNAandprotein expression were elevated during glutamine or glucosedeprivation in a time-dependent manner (Fig. 5E). Nevertheless,the expression of ADAR1 and Dicer showed no obviouschange during glutamine or glucose deprivation in PANC-1

Figure 3.

GLS is the critical target of GLS-ASin pancreatic cancer.A, PANC-1cells were transfected with GLS-ASsiRNA (siGLS-AS) or siRNAnegative control (siNC). The mRNAand protein level of GLS wasanalyzed by qPCR andWesternblot, respectively. B, PANC-1 cellswere transfected with GLS-ASoverexpression vector (GLS-AS) orempty vector as negative control(Vector). The mRNA and proteinlevel of GLS were analyzed by qPCRandWestern blot, respectively. C,Combined immunofluorescence ofGLS protein (red) and RNA-FISHanalysis of GLS-AS (green) inPANC-1 cells transfected withsiGLS-AS or ectopic GLS-AS werecompared with the negative controlcells individually. D, Combinedimmunofluorescence of GLS protein(red) and RNA-FISH analysis ofGLS-AS (green) in pancreaticcancer (PC) and correspondingnoncancerous pancreatic (NP)tissues. All data are presented asmeans� SD of at least threeindependent experiments. Valuesare significant at � , P < 0.05 and�� , P < 0.01 as indicated.

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Figure 4.

GLS-AS inhibits GLS expression via ADAR1/Dicer-dependent RNA silencing in PANC-1 cells.A, Co-RNA-FISH analysis of GLS-AS and GLS-pre-mRNA transcriptswas performed with specific probe, which is against GLS-pre-mRNA (probe1, red) or against GLS-AS (probe2, green) in PANC-1 cells. B, Biotin-labeled RNAscontaining full or partial length of intron-17 were subjected to RNA–RNA pulldown assay and the pull-down GLS-AS was analyzed by Northern blot. C and D,After treating with a-amanitin (50 mmol/L), stability of GLS-pre-mRNA was measured by qPCR compared with time 0 in PANC-1 cells transfected with siGLS-ASor GLS-AS plasmid. E, The representativeWestern blot of the co-IP analysis with anti-ADAR1 or IgG antibody validated the binding between ADAR1 and Dicerprotein. F, RIP assay detected the relative quantification of GLS-AS and GLS pre-mRNA in RIP with ADAR1 or IgG antibodies from cell lysis, measured by qPCRassays.G, After transfecting with siADAR1, the expression of GLS-AS and GLS transcription was detected by qPCR in PANC-1 cells, Western blot assay showedthe expression of GLS and ADAR1 protein of treated PANC-1 cells. H, After knockdown of ADAR1 and Dicer, RIP assay was performed with ADAR1 antibody, andrelative enrichment of GLS-AS and GLS pre-mRNAwas measured by qPCR. I,After transfection with siADAR1, cells were treated with a-amanitin (50 mmol/L),and stability of GLS-pre-mRNAwas measured by qPCR compared with time 0. J and K, After knockdown or overexpression of GLS-AS, RIP assay was performedwith ADAR1 antibody, and relative enrichment of GLS-AS and GLS pre-mRNA was measured by qPCR. L,After cotransfection with GLS-AS or siADAR1, theexpression of GLS and GLS-AS was examined by qPCR orWestern blot, respectively.M,Western blot analysis of Dicer and ADAR1 protein levels in the pull-downcomplex and the GLS-AS and GLS-pre-mRNAmeasured by Northern blot. All data are presented as means� SD of at least three independent experiments.Values are significant at � , P < 0.05 and �� , P < 0.01 as indicated.






(Supplementary Fig. S9A–S9C) and BxPC-3 cells (SupplementaryFig. S9D–S9F), which further confirmed the critical function ofGLS-AS in the regulation of GLS during nutrient stress. Thus, theseresults imply dysregulation of GLS-AS/GLS pathway in pancreaticcancer might, at least partially, be attributed to the nutrient stressincluding glucose or glutamine depletion.

GLS-AS is transcriptionally regulated byMyc under glucose andglutamine deprivation

Myc is amultifunctional transcription factor that is deregulatedin many human cancers and impacts cell proliferation, metabo-lism, and stress responses (29). Specifically, DNA sequence anal-ysis showed GLS-AS promoter region contains potential bindingsites for Myc (Fig. 6A); therefore, we presumed GLS-AS might be

transcriptionally controlled by Myc. As expected, the chromatinimmunoprecipitation (ChIP) assay verified only site 4 locatingfrom �358 to �353 bp on the GLS-AS promoter area could bindto Myc, but not sites 1–3 (Fig. 6B). To further confirm thetranscriptional activity of theputativeGLS-ASpromoter sequence,basic pGL3 plasmid and pGL3 plasmid containing GLS-AS pro-moter was transfected into BxPC-3 and PANC-1 cells. The lucif-erase reporter assay showed the luciferase intensity was enhancedin pGL3-GLS-AS promoter–transfected cells (Fig. 6C), which wasfurther downregulated by siPol II (Fig. 6D). In addition, ChIPanalysis revealed that Pol II could also bind to the binding site ofMyc on GLS-AS promoter (Fig. 6E). Furthermore, GLS-AS pro-moter sequence containing wild-type (WT) or mutant site 4(MUT) was transfected into pancreatic cancer cells. Results

Figure 5.

Nutrient stress is responsible fordownregulation of GLS-AS inpancreatic cancer cells. A, BxPC-3and PANC-1 cells were exposed tostressors including hypoxia,acidosis, glucose, or glutaminestarvation. The GLS-AS level wasmeasured by qPCR and normalizedto GAPDH as an endogenouscontrol. B, qPCR analysis detectedthe GLS-AS expression duringglutamine or glucose deprivationin gradient time. C and D,Representative images of RNA-FISHanalysis displayed the GLS-ASexpression during glutamine orglucose deprivation in gradienttime. E, Expression of GLSmRNAand protein was measured duringglutamine or glucose deprivationconditions, respectively. All data arepresented as means� SD of at leastthree independent experiments.Values are significant at � , P < 0.05as indicated.

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Figure 6.

GLS-AS is transcriptionally inhibited by Myc under glucose and glutamine deprivation. A, Schematic illustration shows the GLS-AS promoter region and the 4sites of potential Myc-binding sites. B, ChIP assay with anti-Myc antibody or IgG was conducted to explore the binding capacity between Myc and GLS-ASpromoter in BxPC-3 and PANC-1 cells. C, Luciferase activity assays were performed in BxPC-3 and PANC-1 cells transfected with pGL3 reporter vector containingGLS-AS promoter or the pGL3 basic vector as control. The luciferase density was measured when cells were transfected for 48 hours. D, Luciferase activityassays of GLS-AS promoter andWestern blot analysis of Pol II were performed in BxPC-3 and PANC-1 cells after knockdown of Pol II. E, ChIP analysis with anti-Pol II antibody or IgG was conducted to reveal the binding capacity between Pol II and site-4 sequences on GLS-AS promoter. F, After overexpression orknockdown of Myc in BxPC-3 and PANC-1 cells, the luciferase activity of BxPC-3 and PANC-1 cells transfected with reporter containing wild-type GLS-ASpromoter (WT) or mutant type (MUT) was measured. The site-4 potential binding sequences were mutated as indicated. G, After knockdown of Myc with siMyc,expression of GLS-AS and GLS mRNA and protein in BxPC-3 and PANC-1 cells were measured by qPCR orWestern blot, respectively.






showed luciferase activity from theWTwasmarkedly repressed byMycoverexpression, but increased after depletionofMyc (Fig. 6F).Coincidently, siMyc substantially increased GLS-AS expression,but decreasedGLS expression (Fig. 6G). These results indicate thatGLS-AS is transcriptionally inhibited byMyc, which consequentlyincreases GLS expression.

Furthermore, both glucose and glutamine deprivation elevatedMyc expression in BxPC-3 and PANC-1 cells (SupplementaryFig. S10A). Specifically, the ChIP assay demonstrated that theenrichment ofGLS-AS promoter byMyc antibodywas remarkablyincreased during glucose and glutamine deprivation (Supplemen-tary Fig. S10B). In addition, the decreased activity of GLS-ASpromoter was noted during glucose or glutamine deprivation(Supplementary Fig. S10C). Moreover, knockdown of Myc couldincrease GLS-AS expression in glutamine or glucose deprivationstress, coupled with GLS downregulation (SupplementaryFig. S10D). Furthermore, Myc-induced upregulation of GLS pro-tein levels can be inhibited by GLS-AS overexpression underglutamine or glucose deprivation (Supplementary Fig. S10E).Together, these results display that the downregulation of GLS-ASin pancreatic cancer might be attributed to energy stress throughMyc-dependent regulation.

GLS mediates a reciprocal feedback between GLS-AS and MycThe results demonstrated that GLS silencing mediates down-

regulation of Myc protein in glioma cells (30). Moreover, resultsfrom Andrew and colleagues showed that GLS inhibitor, CB-839,markedly reduced the protein levels of Myc inmultiple myeloma,acute lymphocytic leukemia, and non-Hodgkin's lymphoma(31). Therefore, we wonder whether Myc can be regulated byGLS-AS/GLS pathway. Interestingly, GLS knockdown andGLS-ASoverexpression significantly inhibited Myc expression at theprotein level (Fig. 7A), but not at themRNA level (SupplementaryFig. S11A and S11B) in BxPC-3 and PANC-1 cells, which indicatesthat GLS might regulate Myc expression at posttranscriptionallevel. To evaluate whether GLS affects stability of Myc protein,Myc protein was measured in the presence of cycloheximide,which blocks de novo protein synthesis. The results showed thestability of Myc protein was decreased by GLS knockdown orGLS-AS overexpression in BxPC-3 and PANC-1 cells (Fig. 7Band C). Besides, the proteasome inhibitor MG132 could rescueMyc protein level from the depression effect of GLS downregu-lation or GLS-AS ectopic expression in BxPC-3 and PANC-1 cells(Fig. 7D). During the glutamine or glucose deprivation, bothGLS knockdown and GLS-AS overexpression obviously inhibitedthe nutrient stress–induced Myc and GLS expression (Fig. 7E).Furthermore, the GLS-AS depletion–induced Myc expressionwas inhibited by siGLS (Fig. 7F) in nutrition-deprived condition.All of these results imply that GLS-AS might regulate Myc expres-sion at a protein level in the proteasome pathway in a GLS-dependent manner.

GLS-AS is conversely correlatedwithMyc andGLS expression inpancreatic cancer

In accordance with the in vitro and in vivo results, the clinicalsamples of pancreatic cancer demonstrated an increased expres-sion of GLS mRNA, which was conversely correlated with GLS-AS(Supplementary Fig. S12A and S12B). In addition, Myc mRNAwas upregulated in pancreatic cancer tissues and associated withGLS mRNA expression (Supplementary Fig. S12C and S12D).Meanwhile, IHC analysis validated the overexpression ofMyc and

GLS in pancreatic cancer tissues (Supplementary Fig. S12E).However, analysis of pancreatic cancer database (QCMG andTCGA) by cBioPortal revealed that the Pearson correlation valueof Myc and GLS mRNA is only �0.007 and �0.049 (32, 33),respectively (Supplementary Fig. S12F and S12G). Similarly,although a positive correlation between Myc and GLS was shownin prostate cancer tissues (34), the similar correlationwas not seenin breast tumors, and c-Jun was shown to drive GLS expres-sion (35). Therefore, these different results indicate that theMyc–GLS correlation is not universal in human tumors, but existsmore strongly in a specific subgroup of tumor samples. Also apossibility, there are multiple mechanisms involved in GLSmRNA regulation causing this complex and diverse scenario.

GLS-AS may be a vital therapeutic target for pancreatic cancertreatment

To further validate the function of GLS-AS in pancreatic cancerdevelopment, BxPC-3 and PANC-1 cells were transfected withGLS-AS overexpression plasmid (GLS-AS) or empty vector (vec-tor), respectively. GLS-AS overexpression effectively inhibitedproliferation as well as invasion and migration ability of BxPC-3 and PANC-1 cells (Supplementary Fig. S13A–S13D). To furthervalidate the functionofGLS-AS in vivo, we transfected PANC-1 andBxPC-3 cells with a lentivirus containing GLS-AS (LV-GLS-AS) orthe control (LV-vector). Then the transfected cells were trans-planted subcutaneously into the nude mouse to investigate thetumor growth and metastasis. Results showed that PANC-1tumors of LV-GLS-AS group were smaller and lighter than LV-vector group (Supplementary Fig. S14A). Moreover, the numberof liver and lung metastases in the LV-GLS-AS group was consid-erably less than that in the LV-vector group (SupplementaryFig. S14B and S14C). Furthermore, the tumor with LV-GLS-ASdisplayed higher GLS-AS expression, coupled with lower expres-sion of GLS mRNA (Supplementary Fig. S14D and S14E).Implanted BxPC-3 cells transfected with LV-GLS-AS also demon-strated impaired proliferation and metastasis in nude mice(Supplementary Fig. S15A–S15E). Together, these results suggestGLS-AS may be a novel metabolic target for therapeutic treat-ment of pancreatic cancer.

DiscussionRecently, accumulative researches have revealed that lncRNAs

play key roles in modulating various aspects of cancer cellularproperties, including proliferation, survival, migration, genomicstability, and metabolism (36). Remarkably, aberrant expressionof lncRNAs is identified in pancreatic cancer; whether the functionof lncRNAs coupling the metabolism and tumorigenesis is farfrom elucidated (37). In our current research, we discovered anovel lncRNA GLS-AS was significantly downregulated in pan-creatic cancer and associated with worse clinical outcomes. Inaddition, the downregulation of GLS-AS dramatically enhancedproliferation and invasion of pancreatic cancer cells both in vitroand in vivo. Therefore, these results intensively indicate that GLS-ASmight function as an inhibitor in the progression of pancreaticcancer.

Antisense lncRNAs are a cluster of lncRNAs transcribed from theopposite DNA strand compared with sense transcripts (38, 39).Recentfindings have shown that antisense lncRNA can regulate theexpression of sense gene by acting as epigenetic regulators of geneexpression and chromatin remodeling. The antisense transcript for

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b-secretase-1 (BACE1-AS) is elevated in Alzheimer's disease, whichincreases BACE1 mRNA stability and generates additional amy-loid-b through a posttranscriptional feed-forward mecha-nism (40). Antisense Uchl1 increases UCHL1 protein synthesis ata posttranscriptional level through an embedded SINEB2repeat (41). In this study, both GLSmRNA and protein expressionwere inhibited or increased by GLS-AS overexpression or down-regulation. Moreover, GLS knockdown significantly decreased

proliferation and invasion of pancreatic cancer cells, which waspromoted by downregulation of GLS-AS. Furthermore, the clinicalsamples demonstrated a reversed correlation betweenGLS-AS andGLS expression. Therefore, our findings indicate GLS is a criticaltarget for GLS-AS exerting inhibition effects on pancreatic cancer.

ADAR is a family of enzymes with double stranded RNA(dsRNA)-binding domains that converts adenosine residues intoinosine (A-to-I RNA editing) specifically in dsRNA (28, 42). To

Figure 7.

GLSmediates a reciprocal feedback between GLS-AS andMyc in PANC-1 cells. A,Western blot analysis of Myc protein in BxPC-3 and PANC-1 cells upon GLSknockdown or GLS-AS overexpression. B and C, After being treated with siGLS or GLS-AS overexpression, the stability of Myc protein in BxPC-3 and PANC-1 cellswas compared with time 0 for periods of time with treatment of cycloheximide (CHX; 50 mg/mL). D, Expression of Myc protein in BxPC-3 and PANC-1 cells, whichwere treated with the proteasome inhibitor MG132 (20 mmol/L) and simultaneously transfected with siGLS or GLS-AS. E, The expression of Myc and GLS proteinswas analyzed in BxPC-3 and PANC-1 cells treated with siGLS or GLS-AS overexpression during glucose or glutamine deprivation, respectively. F, BxPC-3 andPANC-1 cells were cotransfected with siGLS-AS and siGLS or the paired NC, and then cultured in glutamine or glucose deprivation medium for 48 hours. Westernblot analysis of GLS and Myc in those cells were conducted.






date, three ADAR gene family members (ADAR1–3) have beendiscovered in mammals (43). ADAR1 differentiates its functionsin RNA editing and RNAi by formation of either ADAR1/ADAR1homodimer or heterodimer complexes with Dicer (28). Resultsshowed that PCA3, an antisense intronic lncRNA of PRUNE2,forms a dsRNA that undergoes ADAR-dependent RNA editing todownregulate PRUNE2 level (44). However, genome-widescreening has revealed numerous RNA editing sites withininverted Alu repeats in introns and untranslated regions (43).ADAR1 promotes pre-miRNA cleavage and siRNA process byforming a Dicer/ADAR1 complex (28). Meanwhile, the FISHassay showed a colocalization of GLS-AS and GLS pre-mRNA.Moreover, we found that GLS-AS did not affect transcription ofGLS, but impaired the stability of GLS pre-mRNA. Moreover, RIPassay further identified both ADAR and Dicer could bind to GLS-AS and GLS pre-mRNA simultaneously. Nevertheless, downregu-lation of ADAR1 or Dicer increased GLS expression, and alsorescued the GLS-AS–induced inhibition of GLS. Therefore, theseresults intensively imply that GLS-AS inhibits GLS expression at aposttranscriptional level via ADAR1/Dicer-dependent RNAinterference.

Recent research showed that a part of lncRNA was dysregu-lated in cancer due to nutrient stress including glucose de-privation, hypoxia, and so on (14, 24, 45, 46). Therefore, wefurther investigated whether the GLS-AS downregulation isattributed to nutrient stress including deprivation of glucoseand glutamine, hypoxia, and acidity. Interestingly, only depri-vation of glutamine and glucose dramatically decreased GLS-ASexpression, but increased GLS expression. Nevertheless, over-expression of GLS-AS dramatically inhibited the survival andinvasion of pancreatic cancer cells in nutrient stress. Theseresults imply the dysregulated GLS-AS/GLS pathway is anadaption to nutrient stress and is required for the pancreaticcancer progression.

We further explored the mechanism for downregulation ofGLS-AS during nutrient stress. The Myc oncogene is a "masterregulator," which controls glucose and glutamine metabolism tomaintain growth and proliferation of cancer cells (47). Researchdemonstrated deprivation of glucose or glutamine dramaticallyelevated Myc expression and further activated serine biosynthesispathway (48). Results from Wu and colleagues also showedglucose deprivation upregulates Myc protein in BxPC-3 andPANC-1 cells (49). Meanwhile, a study demonstrated that Myc-induced mouse liver tumors significantly increase both glucoseand glutamine catabolism with GLS upregulation (50). Resultsindicated thatMyc is a dual-function transcription factor thatmayactivate or repress coding or noncoding RNA expression. Hart andcolleagues showed that 534 lncRNAs were either up- or down-regulated in response to Myc overexpression in P493-6 human Bcells (51). Zhang and colleagues showed that a Myc-inducedlncRNA-MIF inhibits aerobic glycolysis and tumorigenesis (52).On the contrary, Gao and colleagues reported that Myc transcrip-tionally represses miR-23a and miR-23b, resulting in greaterexpression of their target protein, GLS (34). Interestingly, thebioinformatics analysis demonstrated a putativeMyc-binding sitein the promoter area of GLS-AS gene. Moreover, the ChIP andluciferase reporter assays verified the binding and transcriptionalinhibition ofMyc onGLS-AS promoter. Coincidently,Myc knock-down significantly increased GLS-AS expression, but inhibitedGLS expression. In addition, the deprivation of glucose andglutamine dramatically induced Myc expression and its transcrip-

tional inhibition on GLS-AS. Consistently, our data showedknockdown of Myc dramatically increased GLS-AS expressionduring nutrient stress. Furthermore,Myc expressionwas increasedand reversely correlated with GLS expression in pancreatic cancer.Therefore, these data indicate that GLS-AS might be transcrip-tionally inhibited by Myc, leading to GLS upregulation inresponse to nutrient deprivation. Different from a recent studythat reported that GLS expression was regulated by miR-23a/b inlymphoma cells and PC3 prostate cancer cells (34), we found alncRNA-dependent regulation of GLS expression in pancreaticcancer cells.

Interestingly, recent results reminded a potential feedbackbetween Myc and GLS. As elevated GLS activity is under regu-latory control of Myc (50, 53), research observed that knock-down of GLS decreased Myc protein expression in gliomacells (30). Recently, Madlen and colleagues demonstrated thatglutamine depletion with GLS inhibitor is reflected by rapid lossof Myc protein, which is dependent on proteasomal activi-ty (54). Similarly, our results showed that downregulation ofGLS dramatically inhibited Myc protein expression by impairingits stability. Coincidently, the Myc protein during nutrient stresswas also inhibited by GLS-AS overexpression. In addition,siGLS-AS dramatically increased Myc expression, but decreasedby siMyc. Therefore, our data provide further evidence for areciprocal feedback of Myc and GLS-AS, which regulates GLSexpression at a posttranscriptional level during nutrient depri-vation. Given the regulatory mechanism for Myc is complex, theprecise mechanism for the regulation of GLS on Myc proteinstability needs investigation in the further research.

In summary, our study implicates a nutrient stress–repressedlncRNA GLS-AS is involved in the progression of pancreaticcancer through mediating reciprocal feedback of Myc and GLS.Furthermore, our findings suggest that the Myc/GLS-AS/GLSaxis may be promising molecular targets for the nutrient-restricted treatment of pancreatic cancer.

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

Authors' ContributionsConception and design: G. ZhaoDevelopment of methodology: S.-J. Deng, H.-Y. Chen, Z. Zeng, C. HeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Zhu, Z. Ye, M.-L. Liu, K. HuangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Deng, J.-X. Zhong, F.-Y. Xu, Q. Li, Y. LiuWriting, review, and/or revision of the manuscript: G. ZhaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. WangStudy supervision: G. Zhao

AcknowledgmentsThis study was supported from the National Science Foundation Committee

(NSFC) of China (grant nos: 81372666, 81672406, and 81872030 to G. Zhao).

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 March 12, 2018; revised September 25, 2018; accepted December14, 2018; published first December 18, 2018.

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References1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell

2011;144:646–74.2. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in

cancer. Trends Biochem Sci 2010;35:427–33.3. Ledoux S, Yang R, Friedlander G, Laouari D. Glucose depletion

enhances P-glycoprotein expression in hepatoma cells: role ofendoplasmic reticulum stress response. Cancer Res 2003;63:7284–90.

4. Son J, Lyssiotis CA, Ying H, Wang X, Hua S, Ligorio M, et al. Glutaminesupports pancreatic cancer growth through a KRAS-regulated metabolicpathway. Nature 2013;496:101–05.

5. Sousa CM, Kimmelman AC. The complex landscape of pancreatic cancermetabolism. Carcinogenesis 2014;35:1441–50.

6. Gaglio D, Metallo CM, Gameiro PA, Hiller K, Danna LS, Balestrieri C, et al.Oncogenic K-Ras decouples glucose and glutaminemetabolism to supportcancer cell growth. Mol Syst Biol 2011;7:523.

7. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, et al.Human pancreatic cancer tumors are nutrient poor and tumor cells activelyscavenge extracellular protein. Cancer Res 2015;75:544–53.

8. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the war-burg effect: the metabolic requirements of cell proliferation. Science 2009;324:1029–33.

9. Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, et al.Glucose deprivation contributes to the development of KRAS pathwaymutations in tumor cells. Science 2009;325:1555–59.

10. Yun H, Lee M, Kim SS, Ha J. Glucose deprivation increases mRNA stabilityof vascular endothelial growth factor through activation of AMP-activatedprotein kinase in DU145 prostate carcinoma. J Biol Chem 2005;280:9963–72.

11. Dejure FR, Royla N, Herold S, Kalb J, Walz S, Ade CP, et al. TheMYCmRNA3'-UTR couples RNA polymerase II function to glutamine and ribonucle-otide levels. EMBO J 2017;36:1854–68.

12. Moscat J, Richardson A, Diaz-Meco MT. Nutrient stress revamps cancer cellmetabolism. Cell Res 2015;25:537–38.

13. Cheetham SW, Gruhl F, Mattick JS, Dinger ME. Long noncoding RNAsand the genetics of cancer. Br J Cancer 2013;108:2419–25.

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

15. Li Z, Li X, Wu S, Xue M, Chen W. Long non-coding RNA UCA1 promotesglycolysis by upregulating hexokinase 2 through themTOR–STAT3/micro-RNA143 pathway. Cancer Sci 2014;105:951–55.

16. Ellis BC, Graham LD, Molloy PL. CRNDE, a long non-coding RNAresponsive to insulin/IGF signaling, regulates genes involved in centralmetabolism. Biochim Biophys Acta 2014;1843:372–86.

17. Wei ZouZ,MaC,Medoro L, Chen L,Wang B,Gupta R, et al. LncRNAANRILis up-regulated in nasopharyngeal carcinoma and promotes the cancerprogression via increasing proliferation, reprograming cell glucose metab-olism and inducing side-population stem-like cancer cells. Oncotarget2016;7:61741–54.

18. Li H, Li X, Pang H, Pan J, Xie X, Chen W. Long non-coding RNA UCA1promotes glutamine metabolism by targeting miR-16 in human bladdercancer. Jpn J Clin Oncol 2015;45:1055–63.

19. Tennant DA, Dur�an RV, Gottlieb E. Targetingmetabolic transformation forcancer therapy. Nat Rev Cancer 2010;10:267–77.

20. Cassago A, Ferreira APS, Ferreira IM, Fornezari C, Gomes ERM, Greene KS,et al. Mitochondrial localization and structure-based phosphate activationmechanism of Glutaminase C with implications for cancer metabolism.Proc Natl Acad Sci U S A 2012;109:1092–97.

21. Pan T, Gao L, Wu G, Shen G, Xie S, Wen H, et al. Elevated expression ofglutaminase confers glucose utilization via glutaminolysis in prostatecancer. Biochem Biophys Res Commun 2015;456:452–58.

22. Erickson JW, Cerione RA. Glutaminase: a hot spot for regulation of cancercell metabolism? Oncotarget 2010;1:734–40.

23. Chakrabarti G, Moore ZR, Luo X, Ilcheva M, Ali A, Padanad M, et al.Targeting glutamine metabolism sensitizes pancreatic cancer to PARP-driven metabolic catastrophe induced by ß-lapachone. Cancer Metab2015;3:12.

24. Li X, Deng SJ, Zhu S, Jin Y, Cui SP, Chen JY, et al. Hypoxia-inducedlncRNA-NUTF2P3-001 contributes to tumorigenesis of pancreatic can-

cer by derepressing the miR-3923/KRAS pathway. Oncotarget 2016;7:6000–14.

25. Yoon J, Srikantan S, Gorospe M. MS2-TRAP (MS2-tagged RNA affinitypurification): Tagging RNA to identify associated miRNAs. Methods 2012;58:81–87.

26. Wang L, Park HJ, Dasari S, Wang S, Kocher J, Li W. CPAT: Coding-PotentialAssessment Tool using an alignment-free logistic regression model.Nucleic Acids Res 2013;41:e74.

27. Kong L, Zhang Y, Ye Z, Liu X, Zhao S, Wei L, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vectormachine. Nucleic Acids Res 2007;35:W345–49.

28. Ota H, Sakurai M, Gupta R, Valente L, Wulff B, Ariyoshi K, et al. ADAR1forms a complex with dicer to promote microRNA processing and RNA-induced gene silencing. Cell 2013;153:575–89.

29. Li B, Simon MC. Molecular pathways: targeting MYC-induced metabolicreprogramming and oncogenic stress in cancer. Clin Cancer Res 2013;19:5835–41.

30. Martin-Rufian M, Nascimento-Gomes R, Higuero A, Crisma AR, Campos-Sandoval JA, Gomez-Garcia MC, et al. Both GLS silencing and GLS2overexpression synergize with oxidative stress against proliferation ofglioma cells. J Mol Med 2014;92:277–90.

31. MackinnonA,BennettM, RodriguezM, Parlati F. Biomarkers of response tothe glutaminase inhibitor CB-839 inmultiple myeloma cells. Blood 2014;124:3429.

32. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.Integrative analysis of complex cancer genomics and clinical profiles usingthe cBioPortal. Sci Signal 2013;6:l1.

33. Cerami E,Gao J,DogrusozU,Gross BE, Sumer SO, Aksoy BA, et al. The cBiocancer genomics portal: an open platform for exploring multidimensionalcancer genomics data: figure 1. Cancer Discov 2012;2:401–04.

34. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Mycsuppression of miR-23a/b enhances mitochondrial glutaminase expres-sion and glutamine metabolism. Nature 2009;458:762–65.

35. Lukey MJ, Greene KS, Erickson JW, Wilson KF, Cerione RA. The oncogenictranscription factor c-Jun regulates glutaminase expression and sensitizescells to glutaminase-targeted therapy. Nat Commun 2016;7:11321.

36. Huarte M. The emerging role of lncRNAs in cancer. Nat Med 2015;21:1253–61.

37. Huang X, Zhi X, Gao Y, Ta N, Jiang H, Zheng J. LncRNAs in pancreaticcancer. Oncotarget 2016;7:57379–90.

38. FaghihiMA,Wahlestedt C. Regulatory roles of natural antisense transcripts.Nat Rev Mol Cell Bio 2009;10:637–43.

39. Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M,et al. Antisense transcription in the mammalian transcriptome. Science2005;309:1564–66.

40. Faghihi MA, Modarresi F, Khalil AM, Wood DE, Sahagan BG, Morgan TE,et al. Expression of a noncoding RNA is elevated in Alzheimer's disease anddrives rapid feed-forward regulation of b-secretase. Nat Med 2008;14:723–30.

41. Carrieri C, Cimatti L, BiagioliM, Beugnet A, Zucchelli S, Fedele S, et al. Longnon-coding antisense RNA controls Uchl1 translation through an embed-ded SINEB2 repeat. Nature 2012;491:454–57.

42. Nishikura K. Functions and regulation of RNA editing by ADAR deami-nases. Annu Rev Biochem 2010;79:321–49.

43. Nishikura K. Editormeets silencer: crosstalk between RNA editing andRNAinterference. Nat Rev Mol Cell Bio 2006;7:919–31.

44. Salameh A, Lee AK, Cardo-Vila M, Nunes DN, Efstathiou E, Staquicini FI,et al. PRUNE2 is a human prostate cancer suppressor regulated by theintronic long noncoding RNA PCA3. Proc Natl Acad Sci U S A 2015;112:8403–08.

45. Yang F, Zhang H, Mei Y, Wu M. Reciprocal regulation of HIF-1alpha andlincRNA-p21 modulates the Warburg effect. Mol Cell 2014;53:88–100.

46. Xiao Z, Han L, Lee H, Zhuang L, Zhang Y, Baddour J, et al. Energy stress-induced lncRNA FILNC1 represses c-Myc-mediated energy metabolismand inhibits renal tumor development. Nat Commun 2017;8.

47. Miller DM, Thomas SD, Islam A, Muench D, Sedoris K. c-Myc and cancermetabolism. Clin Cancer Res 2012;18:5546–53.

48. Sun L, Song L, WanQ,WuG, Li X, Wang Y, et al. cMyc-mediated activationof serine biosynthesis pathway is critical for cancer progression undernutrient deprivation conditions. Cell Res 2015;25:429–44.






49. Wu S, Yin X, Fang X, Zheng J, Li L, Liu X, et al. c-MYC responds to glucosedeprivation in a cell-type-dependent manner. Cell Death Discov 2015;1:15057.

50. Yuneva MO, Fan TWM, Allen TD, Higashi RM, Ferraris DV, Tsukamoto T,et al. The metabolic profile of tumors depends on both the responsiblegenetic lesion and tissue type. Cell Metab 2012;15:157–70.

51. Hart JR, Roberts TC, Weinberg MS, Morris KV, Vogt PK. MYC regulates thenon-coding transcriptome. Oncotarget 2014;5:12543–54.

52. Zhang P, Cao L, Fan P, Mei Y, Wu M. LncRNA-MIF, a c-Myc-activated longnon-coding RNA, suppresses glycolysis by promoting Fbxw7-mediatedc-Myc degradation. England 2016;17:1204–20.

53. Dang CV.MYC,microRNAs and glutamine addiction in cancers. Cell Cycle2009;8:3243–45.

54. Effenberger M, Bommert KS, Kunz V, Kruk J, Leich E, Rudelius M, et al.Glutaminase inhibition in multiple myeloma induces apoptosis via MYCdegradation. Oncotarget 2017;8:85858–67.


Deng et al.




2019;79:1398-1412. Published OnlineFirst December 18, 2018.Cancer Res Shi-Jiang Deng, Heng-Yu Chen, Zhu Zeng, et al. ProgressionGLS-Mediated Metabolism and Represses Pancreatic Cancer

Dysregulated Antisense lncRNA GLS-AS Impairs−Nutrient Stress

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