Nutrient Stress–Dysregulated Antisense Lncrna GLS-AS Impairs

Published OnlineFirst December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-0419 Cancer Molecular Cell Biology Research

Nutrient Stress–Dysregulated Antisense lncRNA GLS-AS Impairs GLS-Mediated Metabolism and Represses Pancreatic Cancer Progression Shi-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

Cancer cells are known to undergo metabolic Normal Nutrient stress reprogramming, such as glycolysis and glutamine AAAAAA addiction, to sustain rapid proliferation and AAAAAA metastasis. It remains undefined whether long GLS pre–mRNA noncoding RNAs (lncRNA) coordinate the metabolic switch in pancreatic cancer. Here we identify GLS pre–mRNA GLS protein ADAR1–dicer a nuclear-enriched antisense lncRNA of glutamin- Degradation ADAR1–dicer Degradation GLS–AS ase (GLS-AS) as a critical regulator involved in GLS–AS pancreatic cancer metabolism. GLS-AS was down- GLS–AS gene Myc regulated in pancreatic cancer tissues compared GLS–AS gene with noncancerous peritumor tissues. Depletion Nutrient stress promotes accumulation of Myc. Myc inhibits transcription of the antisense lncRNA of glutaminase (GLS–AS), leading of GLS-AS promoted proliferation and invasion of to GLS elevation and stabilization of Myc. pancreatic cancer cells both in vitro and in xenograft © 2018 American Association for Cancer Research tumors of nude mice. GLS-AS inhibited GLS expression at the posttranscriptional level via formation of double stranded RNA with GLS pre-mRNA through ADAR/Dicer-dependent RNA interference. GLS-AS expression was transcriptionally downregulated by nutrient stress–induced Myc. Conversely, GLS-AS decreased Myc expression by impairing the GLS-mediated stability of Myc protein. These results imply a reciprocal feedback loop wherein Myc and GLS-AS regulate GLS overexpression during nutrient stress. Ectopic overexpression of GLS-AS inhibited proliferation and invasion of pancreatic cancer cells by repressing the Myc/GLS pathway. Moreover, expression of GLS-AS and GLS was inversely correlated 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 for pancreatic 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 potential therapeutic 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. XXX

Introduction of these addictions is "Warburg effect" that cancer cells tend to take advantage of glucose via "aerobic glycolysis" pathway, Recent studies have shown that cancer cells exhibit metabolic eveninthepresenceofoxygen(1).Asanoutcome,thepyruvate dependencies to distinguish them from normal tissues. One generated via the aerobic glycolysis is converted to lactic acid, but not acetyl-CoA. To compensate for the insufﬁcient citric 1Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 2Deparment of acid cycle, cancer cells often activate glutamine metabolism (2). Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University Therefore, markedly aggravated glucose and glutamine deple- of Science and Technology, Wuhan, China. tion may happen in tumor cells as there are inadequacies Note: Supplementary data for this article are available at Cancer Research between vascular supply and metabolic requirement (3). Such Online (http://cancerres.aacrjournals.org/). a situation is especially distinct in pancreatic cancer, where S.-J. Deng, H.-Y. Chen, and Z. Zeng contributed equally to this article. glucose and glutamine metabolism is reprogrammed by onco- – Corresponding Author: Gang Zhao, Department of Emergency Surgery, Union genic Kras to support cancer cell growth (4 6). Therefore, Hospital, Tongji Medical College, Huazhong University of Science and Technol- the metabolic characteristics and distinct hypovascular of ogy, Wuhan 430022, China. Phone: 8627-8535-1621; Fax: 8627-8535-1669; pancreatic cancer would lead to a dramatic nutrients stress E-mail: [email protected] especially caused by glucose and glutamine depletion (7). In doi: 10.1158/0008-5472.CAN-18-0419 fact, such a paradoxical condition affords pathway to rapidly 2018 American Association for Cancer Research. produce the energy and metabolites required for cancer cells'

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proliferation, which makes it correlatively resistant to meta- Materials and Methods bolic stress including hypoxia and nutrient deprivation (8). Patients and specimens Data from Yun and colleagues suggest that glucose deprivation The clinical tissues were obtained from Pancreatic Disease can drive the acquisition of Kras pathway mutations (9), which Institute of Union Hospital from May 2016 to March 2017. We commonly occurs in pancreatic cancer. The results suggested randomly selected 30 pairs of pancreatic cancer and correspond- that glucose deprivation increases VEGF mRNA stability, which ing nontumor tissues from patients without chemotherapy or might facilitate tumor angiogenesis (10). Furthermore, results radiotherapy before operation. Procedures performed on those from Dejure and colleagues showed glutamine deprivation patients included pancreatectomy or palliative surgery including only halted the proliferation of colon cancer cells, but not I125 seed implantation as well as gastroenterostomy and chole- killed them (11). Notably, nutrient deprivation has been cor- dochojejunostomy according to the National Comprehensive related with poor patient survival, suggesting that instead of Cancer Network (NCCN 2012) guideline for pancreatic cancer. killing the tumor, the scarcity of nutrients can make the cancer The samples were obtained from surgical resection of patients or cell stronger (12). Therefore, it is crucial to investigate the biopsy of the palliative surgery patients. The study was conducted mechanisms that are required to accommodate nutrient stresses in accordance with the Declaration of Helsinki. All samples were as an alternative strategy for the therapeutic treatment of pan- collected with the written informed consent of the patients, and creatic cancer. the study was approved by the local Research Ethics Committee at Long noncoding RNAs (lncRNA) are a major class of tran- the Academic Medical Center of Huazhong University of Science scripts, longer than 200 nt, and lack protein-coding potential. and Technology (Wuhan, China). Accumulating evidence suggests that lncRNAs are dysregulated in cancers and involved in the development of cancers (13). Cell culture fi Speci cally, recent results have demonstrated a link between BxPC-3 and PANC-1 cells were obtained from ATCC. They were lncRNAs and altered metabolism in cancers. A study reported tested and authenticated for genotypes by DNA fingerprinting that a glucose starvation–induced lncRNA-NBR2 reciprocally within 6 months. Cells were cultured in 5% CO2 at 37 C and activates AMPK pathway in response to energy stress (14). grown in complete medium, which was composed of 90% LncRNA-UCA1 promotes glycolysis in bladder cancer cells RPMI1640 (Gibco), 10% FBS (Gibco), 100 U/mL penicillin, and by activating the cascade of mTOR-STAT3/miR143-HK2 (15). 100 mg/mL streptomycin. To build nutrition deprivation model, Results from Ellis and colleagues suggested that insulin/IGF we incubated cells with complete medium without glutamine – signaling repressed lncRNA-CRNDE promotes aerobic glyco- [Glutamine ()] or complete medium with 1 mmol/L glucose lysis of cancer cells (16). LncRNA-ANRIL is upregulated in [Glucose ()]. RPMI1640 having no glutamine or glucose was nasopharyngeal carcinoma and promotes cancer progression purchased from Gibco. via increasing glucose uptake for glycolysis (17). In addition, lncR-UCA1 was found to reduce ROS production, and pro- RNA FISH moted mitochondrial glutaminolysis in human bladder can- To detect GLS-AS and GLS pre-mRNAs, we purchased a kit fi cer (18). Nevertheless, the speci c lncRNAs, which couple named FISH Tag RNA Multicolor Kit from Invitrogen to perform nutrient stress and pancreatic cancer, have not been elucidated FISH. The probe synthesis, labeling, and purification procedures – yet. In this study, we endeavored to discover a nutrient stress were performed according to the manufacturer's instructions. responsive lncRNA that is involved in the pancreatic cancer The probe-identified GLS pre-mRNA (Probe1) was labeled with progression. green fluorescence, and the GLS-AS probe (Probe2) was labeled Glutaminase (GLS) is a phosphate-activated amidohydrolase with red fluorescence. Cells were fixed in formaldehyde, perme- that catalyzes the hydrolysis of glutamine to glutamate and abilized by Triton X-100, and then hybridization was carried out ammonia to support metabolism homeostasis, bioenergetics, using labeled probes in a moist chamber at 42C overnight. If and nitrogen balance (19). Recent studies have revealed GLS necessary, the GLS protein immunofluorescence was conducted is commonly overexpressed in numerous malignant tumors after all the FISH procedures were completed. and acts as an oncogene to support cancer growth (20, 21). It is noted that GLS is increased in breast cancers compared with RNA-binding protein immunoprecipitation surrounding nontumor tissues and positively correlates to the To detect RNA–protein binding complexes, RNA-binding pro- tumor grade (20). Moreover, GLS couples glutaminolysis tein immunoprecipitation (RIP) assays were performed according of the TCA cycle with elevated glucose uptake and consequently to the instructions of RNA-Binding Protein Immunoprecipitation the growth of prostate cancer cells (21). Meanwhile, knock- Kit (Magna RIP, Millipore). First, the cells were lysed in lysis buffer down of GLS significantly blocked the growth and invasive containing protease inhibitor cocktail and RNase inhibitor. activity of various cancer cells (22). Results from Chakrabarti Magnetic beads were preincubated with an anti-ADAR1 antibody and colleagues demonstrated that GLS is highly upregulated in or anti-Dicer for 30 minutes at room temperature, and lysates were pancreatic cancer, thereby targeting glutamine metabolism and immunoprecipitated with bead-bound antibody at 4Covernight. sensitizing pancreatic cancer cells to PARP-driven metabolic Then immobilized magnetic bead–bound antibody–protein com- catastrophe (23). In previous study, we discovered a cluster of plexes were obtained, washed off unbound materials, RNA purified dysregulated lncRNAs in pancreatic cancer (24). Coincidently, from RNA–protein complexes, and then analyzed by qPCR. one of the significantly downregulated lncRNA, AK123493, is an antisense lncRNA of glutaminase (GLS). Therefore, it draws Northern blot our attention whether a nuclear-enriched antisense lncRNA of We purchased a DIG RNA Labeling Kit (Roche) to perform glutaminase (GLS-AS) might be involved in the GLS-mediated Northern blot analysis for GLS-AS. First, we prepared GLS-AS– metabolism of pancreatic cancer. specific DNA template containing T7 promoter sequences from

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RT-PCR and the DNA template was purified. Then, the DIG- control. Pancreatic cancer cells in the test and control groups were labeled RNA probes were produced according to the kit instruc- transfected with GLS-AS-MS2 and MS2, respectively, along with tions with the DNA template. DIG-labeled probes were used for MS2-GST-NSL fusion protein. After the cotransfection for 48 hybridization to nylon membrane–blotted total RNA. The hours, cells were harvested and then RNA pull-down assay was hybridized probes were detected with anti-digoxigenin-AP, and conducted as described previously (25). The purified proteins then were visualized with the chemiluminescence substrate were analyzed by Western blot analysis while RNAs were detected CSPD. The signals were also captured by ChemiDocTm XRS with Northern blot. Molecular Imager system (Bio-Rad). Xenograft assay fi Coimmunoprecipitation Lentivirus containing speci c DNA sequences was transfected For coimmunoprecipitation (co-IP) analysis, anti-ADAR1, into BxPC-3 and PANC-1 cells. Five-week-old BALB/c male nude anti-Dicer, or normal mouse/rabbit IgG were used as the primary mice were bought from HFK Bio-Technology Co. To assess tumor in vivo m antibodies, and then the antibody–protein complex was incubat- growth , 100 L RPMI1640 medium without FBS contain- 6 ed with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). ing 4 10 cells was suspended and then planted subcutaneously The agarose–antibody–protein complex was collected and then into the nude mice (each group has 6 mice). Tumor volumes were V ¼ L analyzed by Western blot. measured every 4 days according to the formula 0.5 (length) W2 (width). Mice were sacrificed at 3 weeks after cell Chromatin immunoprecipitation inoculation. Solid tumor tissues were removed and weighed. To in vivo The PCR primers are indicated in Supplementary Table S1. We investigate tumor metastasis , mice were injected with 4 conducted chromatin immunoprecipitation (ChIP) assays using 1 10 tumor cells through the tail vein; visible metastases on fi EZ-ChIPTM Chromatin Immunoprecipitation Kit (Millipore). All liver were counted and then con rmed by hematoxylin and – the procedures were performed according to the manufacturer's eosin stained slides after 3 weeks. Care and handling of the mice instructions. Rabbit anti-Myc (Cell Signaling Technology), anti- were approved by the Institutional Animal Care and Use Com- RNA polymerase II antibodies (Abcam), and corresponding mittee of Tongji Medical College, Huazhong University of Science rabbit-IgG (Cell Signaling Technology) were used as controls. and Technology (Wuhan, China). The bound DNA fragments were amplified by PCR reactions, and Statistical analyses then PCR products were analyzed by gel electrophoresis on 2% All results were presented as means SD. Comparisons agarose gel. The PCR primers used were listed in Supplementary between two groups were performed with Student t test. The Table S2. correlation between GLS-AS and GLS mRNA or GLS and Myc mRNA was revealed by Pearson correlation analysis. The expres- Luciferase activity assay sion of GLS-AS and the clinical characteristics were analyzed by For GLS-AS promoter activity analysis, BxPC-3 cells were trans- x2 test, while the log-rank test was conducted to survey pancreatic fected with pGL3 vector wild-type or mutant GLS-AS promoter cancer patient survival. Difference was regarded to be significant at with firefly luciferase plasmid while a plasmid pRL-TK carrying , P < 0.05 and , P < 0.01. Renilla luciferase was used as internal reference. To confirm the nutrition deprivation impact on GLS-AS promoter activity, cells were cultured under glutamine or glucose deprivation for 24 or 48 Results hours. To investigate the relationship between Myc protein and AK123493.1, a nuclear accumulated antisense lncRNA of GLS GLS-AS promoter activity, siMyc or the control siNC was cotrans- (GLS-AS), is downregulated in pancreatic cancer fected into the BxPC-3 cells containing luciferase plasmid. The microarray analysis showed AK123493.1 was decreased in The reporter activity was measured using a luciferase assay kit the pancreatic cancer tissues compared with noncancerous peri- (Promega) and plotted after normalizing with respect to Renilla tumoral (NP) tissues (Fig. 1A). GLS-AS is an intronic antisense luciferase activity. Firefly luciferase activity was normalized to the lncRNA embedded within intron-17 of the corresponding sense corresponding Renilla luciferase activity. The data are represented gene GLS (Fig. 1B). In addition, the Northern blot validated the as mean SD of three independent experiments. expression of GLS-AS in BxPC-3 and PANC-1 cells using the RNA probe (Fig. 1C). Moreover, the expression levels of GLS-AS in Biotin-RNA pull-down assay BxPC-3 and PANC-1 cells were lower than that in the normal The full or partial length of intron-17 of GLS gene sequences human pancreatic duct epithelial cells (HPDE; Fig. 1D). To vali- was amplified by PCR with SP6/T7-containing primer and then date the signal specificity, Northern blot analysis was conducted transcribed by MAXIscript SP6/T7 Transcription Kit (Thermo after the cells were transfected with siGLS-AS. As shown in Fig. 1E, Fisher Scientific). The synthetic RNA was biotin-labeled with siGLS-AS significantly decreased the expression of GLS-AS. Fur- Pierce RNA 30 End Desthiobiotinylation Kit (Thermo Fisher thermore, the Northern blot results showed that GLS-AS was Scientific). The biotin-labeled RNAs were incubated with cell lysis obviously lower in pancreatic cancer tissues compared with NP individually and the target complexes were precipitated by strep- (Fig. 1F). Meanwhile, the FISH assay showed that GLS-AS is tavidin-coupled Dynabeads (Invitrogen). Finally, Northern blot mainly accumulated in the nucleus (Fig. 1G), implying GLS-AS analysis identified whether GLS-AS was pulled down or not. may predominantly function in the nucleus. Similarly, separation of nuclear extract and the cytoplasmic fraction showed that RNA pull-down by MS2-GST GLS-AS retained in the nucleus (Fig. 1H). Both Coding Potential We constructed a plasmid expressing GLS-AS tagged with MS2 Assessment Tool (26) and Coding Potential Calculator (27) pre- hairpin loops (GLS-AS-MS2), a plasmid expressing MS2-GST-NSL dicted GLS-AS is a noncoding RNA. Furthermore, we blocked new fusion protein, and a plasmid only expressing MS2 (MS2) RNA as RNA 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 was distinctly downregulated in two pancreatic cancer (PC) samples compared with paired noncancerous pancreatic (NP) tissues. B, Schematic diagram of GLS-AS and GLS gene location and relationship in genome. Arrows, transcript orientation. Probe-1 labeled with red fluorescence was used to detected GLS-pre-mRNA while 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 RNA probe. 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 cells when GLS-AS were knocked down by siRNAs. F, Northern blot analysis of GLS-AS in five paired cancer and noncancerous pancreatic tissues. G, FISH analysis showed 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, analyzed by qPCR. RT-PCR products were run on a 2% agarose gel and U6 and GAPDH were used separately as nuclear and cytoplasmic markers. I, After blocking new RNA 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 RNA polymerase II, while 18s RNA was a product of RNA polymerase I. J, GLS-AS in 30 pancreatic cancer and corresponding adjacent noncancerous pancreatic tissues was measured by qPCR. K, The Kaplan–Meier curves for overall survival analysis of patients with pancreatic cancer by GLS-AS expression. Expression levels of GLS-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 independent experiments. Values are significant at , P < 0.01 as indicated.

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a-amanitin (50 mmol/L) in BxPC-3 cells and measured the expres- GLS-AS inhibits GLS expression in posttranscriptional level by sion of GLS-AS by qPCR relative to time 0. After treating with ADAR1/Dicer-dependent RNA interference a-amanitin, the expression of GLS-AS was significantly decreased, To further identify whether GLS-AS regulates GLS transcription, while the 18s mRNA, which is transcribed by Pol I, was not affected. BxPC-3 or PANC-1 cells were transfected with pGL3 plasmid These results indicate that the transcription of GLS-AS is proceeded containing GLS putative promoter region. As shown in Supple- in a Pol II–dependent manner (Fig. 1I). We further validated the mentary Fig. S4A and S4B, the luciferase reporter assay showed GLS-AS expression level in pancreatic cancer tissues and paired NP neither knockdown nor overexpression of GLS-AS and did not tissues by qPCR. Results showed that GLS-AS expression in pan- change the GLS promoter activity, which implied that GLS-AS did creatic cancer was significantly lower than that in NP (Fig. 1J). In not regulate GLS expression at the transcriptional level. addition, the low expression of GLS-AS was associated with large The co-RNA FISH assay disclosed both GLS-AS and GLS pre- tumor size, lymph node invasion, remote metastasis (Supplemen- mRNA hybridized in the same nuclear foci of PANC-1 cells tary Table S1), and short overall survival time (Fig. 1K). (Fig. 4A), which indicates the formation of dsRNA. To further validate the direct interaction between GLS-AS and GLS pre- Low expression of GLS-AS facilitates proliferation and invasion mRNA in PANC-1 cells, the RNA–RNA pull-down assay was of pancreatic cancer cells performed with biotin-labeled full or partial length deletion of To understand the roles of GLS-AS downregulation in pancre- intron-17 transcripts (Fig. 4B). As GLS-AS is an antisense lncRNA atic cancer progression, we depleted GLS-AS expression with of GLS pre-mRNA, we further presumed GLS-AS might regulate siRNA (siGLS-AS) in pancreatic cancer cells (Fig. 2A; Supplemen- the stability of GLS pre-mRNA. To evaluate the stability of GLS tary Fig. S1A). After downregulation of GLS-AS, the proliferation pre-mRNA, pancreatic cancer cells were treated with amanitin and colony formation of PANC-1 (Fig. 2B and C) and BxPC-3 cells (50 mmol/L). The expression of GLS pre-mRNA was measured by (Supplementary Fig. S1B and S1C) were significantly enforced. qPCR at the separated time point. Compared with time 0, the Meanwhile, transwell and wound-healing assays further revealed stability was dramatically enhanced by siGLS-AS, but impaired by an enhanced invasion and migration ability in GLS-AS–depleted GLS-AS overexpression in PANC-1 cells (Fig. 4C and D). Research PANC-1(Fig. 2D and E) and BxPC-3 cells (Supplementary Fig. had demonstrated that adenosine deaminases acting on RNA S1D and S1E). To further confirm whether reduced GLS-AS affects (ADAR1) are involved in RNA interference of dsRNA by formation pancreatic cancer progression in vivo, PANC-1 and BxPC-3 cells of ADAR1/Dicer heterodimer complexes, a member protein of were stably transfected with lentivirus containing siGLS-AS RNA-induced silencing complex (RISC; ref. 28). Meanwhile, the (LV-siGLS-AS) or siNC (LV-NC) transplanted subcutaneously co-IP analysis validated the binding between ADAR1 and Dicer into the mouse, respectively. Compared with LV-NC group, the protein in PANC-1 cells (Fig. 4E; Supplementary Fig. S5A). To PANC-1 tumors in the LV-siGLS-AS group were larger and heavier confirm whether the ADAR1/Dicer proteins physically bound to (Fig. 2F), with more visible liver and lung metastases (Fig. 2G and GLS-AS/GLS pre-mRNA dsRNA or not, we performed RNA immu- H). Similarly, the proliferation and metastasis of BxPC-3 tumor noprecipitation (RNA-IP) assays in PANC-1 cells. Compared with with LV-siGLS-AS were also enhanced (Supplementary Fig. S1F– the IgG-bound sample, the ADAR1 or Dicer antibody–bound S1H). These results indicate that the dysregulated GLS-AS expres- complex showed significantly high enrichment of GLS-AS and sion might contribute to pancreatic cancer development. GLS pre-mRNA in PANC-1 cells (Fig. 4F; Supplementary Fig. S5B). Thus, we wondered whether the GLS-AS and GLS pre-mRNA are GLS is the critical target of GLS-AS to exert function in regulated by ADAR1/Dicer-mediated RNA silencing. Both siA- pancreatic cancer DAR1 and siDicer increased the expression of GLS-AS, GLS pre- Because GLS-AS is an antisense lncRNA of GLS, we further mRNA, and protein, were remarkably increased in PANC-1 cells investigated whether GLS is a functional target of GLS-AS. Coin- (Fig. 4G; Supplementary Fig. S5C), reduced the enrichment of cidently, knockdown of GLS-AS apparently increased the GLS GLS-AS and GLS pre-mRNA in protein (ADAR1/Dicer)–antibody- expression both in mRNA and protein levels in both PANC-1 bound complex in PANC-1 cells (Fig. 4H; Supplementary Fig. (Fig. 3A) and BxPC-3 cells (Supplementary Fig. S2A). Coincident- S5D), as well as strengthened the stability of GLS pre-mRNA in ly, transfection with a plasmid containing GLS-AS sequence (GLS- PANC-1 cells (Fig. 4I; Supplementary Fig. S5E). Meanwhile, the AS) obviously decreased GLS expression both in mRNA and enrichment of GLS pre-mRNA was reduced by siGLS-AS, but protein levels of PANC-1 and BxPC-3 cells (Fig. 3B; Supplemen- upregulated by GLS-AS overexpression, both by anti-ADAR1 tary Fig. S2B). Subsequently, co-staining fluorescence of GLS-AS (Fig. 4J and K) and anti-Dicer (Supplementary Fig. S5F and transcription and GLS protein further validated GLS was nega- S5G) in PANC-1 cells. In addition, both siADAR1 (Fig. 4L) and tively regulated by GLS-AS in PANC-1 and BxPC-3 cells (Fig. 3C; siDicer (Supplementary Fig. S5H) could rescue the expression of Supplementary Fig. S2C). In agreement, the costaining fluores- GLS mRNA and protein in PANC-1 cells, which was inhibited by cence assay further showed a decreased GLS-AS accompanied with GLS-AS overexpression. Furthermore, MS2-tagged RNA affinity increased GLS protein expression in pancreatic cancer tissue purification analysis was performed to further confirm GLS-AS, compared with NP tissue (Fig. 3D). Meanwhile, Western blot GLS-pre-mRNA, and ADAR1/Dicer can form a complex in PANC- analysis further validated the downregulation of GLS protein in 1 cells (Fig. 4M). Simultaneously, the experiments described pancreatic cancer tissues compared with NP tissues (Supplemen- above were also conducted in BxPC-3 cells. Results of BxPC-3 tary Fig. S2D). Meanwhile, depletion of GLS with siGLS remark- cells also confirmed an interaction between GLS-pre-mRNA and able inhibited proliferation, colony formation, invasion, and GLS-AS (Supplementary Fig. S6A and S6B), which could regulate migration ability of PANC-1 and BxPC-3 cells, which was rein- the stability of GLS-pre-mRNA (Supplementary Fig. S6C and forced by siGLS-AS (Supplementary Fig. S3A–S3J). Thus, these S6D). Moreover, results further displayed that ADAR1 is required data implied that the GLS would be a critical target for dysregu- for the regulation of GLS-AS on GLS expression in BxPC-3 cells lated GLS-AS to exert its biological function in pancreatic cancer. (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 in PANC-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 assay was conducted to observe the invasion ability of the transfected PANC-1 cells. The left histogram represents relative cell number while the representative images areshownontheright.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) were transplanted subcutaneously into nude mice to observe tumor growth (5 106 cells per mouse). F, A photograph of representative nude mice and tumor is presented after 3 weekswhenmiceweresacrificed (left). The tumor volumes were measured every 4 days. Two groups of tumor weights were measured individually. G, Top, histogram shows number of visible liver metastases per 5 sections in each nude mouse. Bottom, representative images of livers and corresponding 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.

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Figure 3. GLS is the critical target of GLS-AS in pancreatic cancer. A, PANC-1 cells were transfected with GLS-AS siRNA (siGLS-AS) or siRNA negative control (siNC). The mRNA and protein level of GLS was analyzed by qPCR and Western blot, respectively. B, PANC-1 cells were transfected with GLS-AS overexpression vector (GLS-AS) or empty vector as negative control (Vector). The mRNA and protein level of GLS were analyzed by qPCR and Western blot, respectively. C, Combined immunofluorescence of GLS protein (red) and RNA-FISH analysis of GLS-AS (green) in PANC-1 cells transfected with siGLS-AS or ectopic GLS-AS were compared with the negative control cells individually. D, Combined immunofluorescence of GLS protein (red) and RNA-FISH analysis of GLS-AS (green) in pancreatic cancer (PC) and corresponding noncancerous pancreatic (NP) tissues. 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 regulation ingly, GLS-AS was obviously decreased during depletion of glucose of GLS-AS on GLS expression in BxPC-3 cells (Supplementary or glutamine, but without significant alteration in BxPC-3 and Fig. S7A–S7H). Supplementary Figure S8 is a schematic diagram PANC-1 cells during hypoxia or acidity (Fig. 5A). Moreover, qPCR of MS2-tagged RNA affinity purification analysis. and FISH assays demonstrated a time-dependent GLS-AS downregulation during glutamine or glucose deprivation (Fig. 5B–D). Nutrient stress is responsible for downregulation of GLS-AS in Coincident with the GLS-AS downregulation, both GLS mRNA and pancreatic cancer protein expression were elevated during glutamine or glucose We further investigated whether the GLS-AS downregulation in deprivation in a time-dependent manner (Fig. 5E). Nevertheless, pancreatic cancer is attributed to metabolism stress including the expression of ADAR1 and Dicer showed no obvious hypoxia, acidity, or depletion of glucose and glutamine. Interest- change during glutamine or glucose deprivation in PANC-1

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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 transcripts was 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 RNAs containing 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-AS or GLS-AS plasmid. E, The representative Western blot of the co-IP analysis with anti-ADAR1 or IgG antibody validated the binding between ADAR1 and Dicer protein. 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 qPCR assays. G, After transfecting with siADAR1, the expression of GLS-AS and GLS transcription was detected by qPCR in PANC-1 cells, Western blot assay showed the 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, and relative enrichment of GLS-AS and GLS pre-mRNA was measured by qPCR. I, After transfection with siADAR1, cells were treated with a-amanitin (50 mmol/L), and stability of GLS-pre-mRNA was measured by qPCR compared with time 0. J and K, After knockdown or overexpression of GLS-AS, RIP assay was performed with ADAR1 antibody, and relative enrichment of GLS-AS and GLS pre-mRNA was measured by qPCR. L, After cotransfection with GLS-AS or siADAR1, the expression of GLS and GLS-AS was examined by qPCR or Western blot, respectively. M, Western blot analysis of Dicer and ADAR1 protein levels in the pull-down complex and the GLS-AS and GLS-pre-mRNA measured 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.

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Figure 5. Nutrient stress is responsible for downregulation of GLS-AS in pancreatic cancer cells. A, BxPC-3 and PANC-1 cells were exposed to stressors including hypoxia, acidosis, glucose, or glutamine starvation. The GLS-AS level was measured by qPCR and normalized to GAPDH as an endogenous control. B, qPCR analysis detected the GLS-AS expression during glutamine or glucose deprivation in gradient time. C and D, Representative images of RNA-FISH analysis displayed the GLS-AS expression during glutamine or glucose deprivation in gradient time. E, Expression of GLS mRNA and protein was measured during glutamine or glucose deprivation conditions, respectively. All data are presented as means SD of at least three independent experiments. Values are signiﬁcant at , P < 0.05 as indicated.

(Supplementary Fig. S9A–S9C) and BxPC-3 cells (Supplementary transcriptionally controlled by Myc. As expected, the chromatin Fig. S9D–S9F), which further confirmed the critical function of immunoprecipitation (ChIP) assay verified only site 4 locating GLS-AS in the regulation of GLS during nutrient stress. Thus, these from 358 to 353 bp on the GLS-AS promoter area could bind results imply dysregulation of GLS-AS/GLS pathway in pancreatic to Myc, but not sites 1–3 (Fig. 6B). To further confirm the cancer might, at least partially, be attributed to the nutrient stress transcriptional activity of the putative GLS-AS promoter sequence, including glucose or glutamine depletion. basic pGL3 plasmid and pGL3 plasmid containing GLS-AS promoter was transfected into BxPC-3 and PANC-1 cells. The lucif- GLS-AS is transcriptionally regulated by Myc under glucose and erase reporter assay showed the luciferase intensity was enhanced glutamine deprivation in pGL3-GLS-AS promoter–transfected cells (Fig. 6C), which was Myc is a multifunctional transcription factor that is deregulated further downregulated by siPol II (Fig. 6D). In addition, ChIP in many human cancers and impacts cell proliferation, metabo- analysis revealed that Pol II could also bind to the binding site of lism, and stress responses (29). Specifically, DNA sequence anal- Myc on GLS-AS promoter (Fig. 6E). Furthermore, GLS-AS pro- ysis showed GLS-AS promoter region contains potential binding moter sequence containing wild-type (WT) or mutant site 4 sites for Myc (Fig. 6A); therefore, we presumed GLS-AS might be (MUT) was transfected into pancreatic cancer cells. Results

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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 4 sites 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-AS promoter 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 containing GLS-AS promoter or the pGL3 basic vector as control. The luciferase density was measured when cells were transfected for 48 hours. D, Luciferase activity assays of GLS-AS promoter and Western 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 or knockdown 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-AS promoter (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, GLS mRNA, and protein in BxPC-3 and PANC-1 cells were measured by qPCR or Western blot, respectively.

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showed luciferase activity from the WT was markedly repressed by GLS in pancreatic cancer tissues (Supplementary Fig. S12E). Myc overexpression, but increased after depletion of Myc (Fig. 6F). However, analysis of pancreatic cancer database (QCMG and Coincidently, siMyc substantially increased GLS-AS expression, TCGA) by cBioPortal revealed that the Pearson correlation value but decreased GLS expression (Fig. 6G). These results indicate that of Myc and GLS mRNA is only 0.007 and 0.049 (32, 33), GLS-AS is transcriptionally inhibited by Myc, which consequently respectively (Supplementary Fig. S12F and S12G). Similarly, increases GLS expression. although a positive correlation between Myc and GLS was shown Furthermore, both glucose and glutamine deprivation elevated in prostate cancer tissues (34), the similar correlation was not seen Myc expression in BxPC-3 and PANC-1 cells (Supplementary in breast tumors, and c-Jun was shown to drive GLS expres- Fig. S10A). Specifically, the ChIP assay demonstrated that the sion (35). Therefore, these different results indicate that the enrichment of GLS-AS promoter by Myc antibody was remarkably Myc–GLS correlation is not universal in human tumors, but exists increased during glucose and glutamine deprivation (Supplemen- more strongly in a specific subgroup of tumor samples. Also a tary Fig. S10B). In addition, the decreased activity of GLS-AS possibility, there are multiple mechanisms involved in GLS promoter was noted during glucose or glutamine deprivation mRNA regulation causing this complex and diverse scenario. (Supplementary Fig. S10C). Moreover, knockdown of Myc could increase GLS-AS expression in glutamine or glucose deprivation GLS-AS may be a vital therapeutic target for pancreatic cancer stress, coupled with GLS downregulation (Supplementary treatment Fig. S10D). Furthermore, Myc-induced upregulation of GLS pro- To further validate the function of GLS-AS in pancreatic cancer tein levels can be inhibited by GLS-AS overexpression under development, BxPC-3 and PANC-1 cells were transfected with glutamine or glucose deprivation (Supplementary Fig. S10E). GLS-AS overexpression plasmid (GLS-AS) or empty vector (vec- Together, these results display that the downregulation of GLS-AS tor), respectively. GLS-AS overexpression effectively inhibited in pancreatic cancer might be attributed to energy stress through proliferation as well as invasion and migration ability of BxPC- Myc-dependent regulation. 3 and PANC-1 cells (Supplementary Fig. S13A–S13D). To further validate the function of GLS-AS in vivo, we transfected PANC-1 and GLS mediates a reciprocal feedback between GLS-AS and Myc BxPC-3 cells with a lentivirus containing GLS-AS (LV-GLS-AS) or The results demonstrated that GLS silencing mediates down- the control (LV-vector). Then the transfected cells were trans- regulation of Myc protein in glioma cells (30). Moreover, results planted subcutaneously into the nude mouse to investigate the from Andrew and colleagues showed that GLS inhibitor, CB-839, tumor growth and metastasis. Results showed that PANC-1 markedly reduced the protein levels of Myc in multiple myeloma, tumors of LV-GLS-AS group were smaller and lighter than LV- acute lymphocytic leukemia, and non-Hodgkin's lymphoma vector group (Supplementary Fig. S14A). Moreover, the number (31). Therefore, we wonder whether Myc can be regulated by of liver and lung metastases in the LV-GLS-AS group was consid- GLS-AS/GLS pathway. Interestingly, GLS knockdown and GLS-AS erably less than that in the LV-vector group (Supplementary overexpression significantly inhibited Myc expression at the Fig. S14B and S14C). Furthermore, the tumor with LV-GLS-AS protein level (Fig. 7A), but not at the mRNA level (Supplementary displayed higher GLS-AS expression, coupled with lower expres- Fig. S11A and S11B) in BxPC-3 and PANC-1 cells, which indicates sion of GLS mRNA (Supplementary Fig. S14D and S14E). that GLS might regulate Myc expression at posttranscriptional Implanted BxPC-3 cells transfected with LV-GLS-AS also demon- level. To evaluate whether GLS affects stability of Myc protein, strated impaired proliferation and metastasis in nude mice Myc protein was measured in the presence of cycloheximide, (Supplementary Fig. S15A–S15E). Together, these results suggest which blocks de novo protein synthesis. The results showed the GLS-AS may be a novel metabolic target for therapeutic treat- stability of Myc protein was decreased by GLS knockdown or ment of pancreatic cancer. GLS-AS overexpression in BxPC-3 and PANC-1 cells (Fig. 7B and C). Besides, the proteasome inhibitor MG132 could rescue Myc protein level from the depression effect of GLS downregu- Discussion lation or GLS-AS ectopic expression in BxPC-3 and PANC-1 cells Recently, accumulative researches have revealed that lncRNAs (Fig. 7D). During the glutamine or glucose deprivation, both play key roles in modulating various aspects of cancer cellular GLS knockdown and GLS-AS overexpression obviously inhibited properties, including proliferation, survival, migration, genomic the nutrient stress–induced Myc and GLS expression (Fig. 7E). stability, and metabolism (36). Remarkably, aberrant expression Furthermore, the GLS-AS depletion–induced Myc expression of lncRNAs is identified in pancreatic cancer; whether the function was inhibited by siGLS (Fig. 7F) in nutrition-deprived condition. of lncRNAs coupling the metabolism and tumorigenesis is far All of these results imply that GLS-AS might regulate Myc expres- from elucidated (37). In our current research, we discovered a sion at a protein level in the proteasome pathway in a GLS- novel lncRNA GLS-AS was significantly downregulated in pan- dependent manner. creatic cancer and associated with worse clinical outcomes. In addition, the downregulation of GLS-AS dramatically enhanced GLS-AS is conversely correlated with Myc and GLS expression in proliferation and invasion of pancreatic cancer cells both in vitro pancreatic cancer and in vivo. Therefore, these results intensively indicate that GLS- In accordance with the in vitro and in vivo results, the clinical AS might function as an inhibitor in the progression of pancreatic samples of pancreatic cancer demonstrated an increased expres- cancer. sion of GLS mRNA, which was conversely correlated with GLS-AS Antisense lncRNAs are a cluster of lncRNAs transcribed from the (Supplementary Fig. S12A and S12B). In addition, Myc mRNA opposite DNA strand compared with sense transcripts (38, 39). was upregulated in pancreatic cancer tissues and associated with Recent findings have shown that antisense lncRNA can regulate the GLS mRNA expression (Supplementary Fig. S12C and S12D). expression of sense gene by acting as epigenetic regulators of gene Meanwhile, IHC analysis validated the overexpression of Myc and expression and chromatin remodeling. The antisense transcript for

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Figure 7. GLS mediates a reciprocal feedback between GLS-AS and Myc in PANC-1 cells. A, Western blot analysis of Myc protein in BxPC-3 and PANC-1 cells upon GLS knockdown 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 cells was 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, which were treated with the proteasome inhibitor MG132 (20 mmol/L) and simultaneously transfected with siGLS or GLS-AS. E, The expression of Myc and GLS proteins was analyzed in BxPC-3 and PANC-1 cells treated with siGLS or GLS-AS overexpression during glucose or glutamine deprivation, respectively. F, BxPC-3 and PANC-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. Western blot analysis of GLS and Myc in those cells were conducted.

b-secretase-1 (BACE1-AS) is elevated in Alzheimer's disease, which proliferation and invasion of pancreatic cancer cells, which was increases BACE1 mRNA stability and generates additional amy- promoted by downregulation of GLS-AS. Furthermore, the clinical loid-b through a posttranscriptional feed-forward mecha- samples demonstrated a reversed correlation between GLS-AS and nism (40). Antisense Uchl1 increases UCHL1 protein synthesis at GLS expression. Therefore, our findings indicate GLS is a critical a posttranscriptional level through an embedded SINEB2 target for GLS-AS exerting inhibition effects on pancreatic cancer. repeat (41). In this study, both GLS mRNA and protein expression ADAR is a family of enzymes with double stranded RNA were inhibited or increased by GLS-AS overexpression or down- (dsRNA)-binding domains that converts adenosine residues into regulation. Moreover, GLS knockdown significantly decreased inosine (A-to-I RNA editing) specifically in dsRNA (28, 42). To

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date, three ADAR gene family members (ADAR1–3) have been tional inhibition on GLS-AS. Consistently, our data showed discovered in mammals (43). ADAR1 differentiates its functions knockdown of Myc dramatically increased GLS-AS expression in RNA editing and RNAi by formation of either ADAR1/ADAR1 during nutrient stress. Furthermore, Myc expression was increased homodimer or heterodimer complexes with Dicer (28). Results and reversely correlated with GLS expression in pancreatic cancer. showed that PCA3, an antisense intronic lncRNA of PRUNE2, Therefore, these data indicate that GLS-AS might be transcrip- forms a dsRNA that undergoes ADAR-dependent RNA editing to tionally inhibited by Myc, leading to GLS upregulation in downregulate PRUNE2 level (44). However, genome-wide response to nutrient deprivation. Different from a recent study screening has revealed numerous RNA editing sites within that reported that GLS expression was regulated by miR-23a/b in inverted Alu repeats in introns and untranslated regions (43). lymphoma cells and PC3 prostate cancer cells (34), we found a ADAR1 promotes pre-miRNA cleavage and siRNA process by lncRNA-dependent regulation of GLS expression in pancreatic forming a Dicer/ADAR1 complex (28). Meanwhile, the FISH cancer cells. assay showed a colocalization of GLS-AS and GLS pre-mRNA. Interestingly, recent results reminded a potential feedback Moreover, we found that GLS-AS did not affect transcription of between Myc and GLS. As elevated GLS activity is under regu- GLS, but impaired the stability of GLS pre-mRNA. Moreover, RIP latory control of Myc (50, 53), research observed that knock- assay further identified both ADAR and Dicer could bind to GLS- down of GLS decreased Myc protein expression in glioma AS and GLS pre-mRNA simultaneously. Nevertheless, downregu- cells (30). Recently, Madlen and colleagues demonstrated that lation of ADAR1 or Dicer increased GLS expression, and also glutamine depletion with GLS inhibitor is reflected by rapid loss rescued the GLS-AS–induced inhibition of GLS. Therefore, these of Myc protein, which is dependent on proteasomal activi- results intensively imply that GLS-AS inhibits GLS expression at a ty (54). Similarly, our results showed that downregulation of posttranscriptional level via ADAR1/Dicer-dependent RNA GLS dramatically inhibited Myc protein expression by impairing interference. its stability. Coincidently, the Myc protein during nutrient stress Recent research showed that a part of lncRNA was dysregu- was also inhibited by GLS-AS overexpression. In addition, lated in cancer due to nutrient stress including glucose de- siGLS-AS dramatically increased Myc expression, but decreased privation, hypoxia, and so on (14, 24, 45, 46). Therefore, we by siMyc. Therefore, our data provide further evidence for a further investigated whether the GLS-AS downregulation is reciprocal feedback of Myc and GLS-AS, which regulates GLS attributed to nutrient stress including deprivation of glucose expression at a posttranscriptional level during nutrient depri- and glutamine, hypoxia, and acidity. Interestingly, only deprivation. Given the regulatory mechanism for Myc is complex, the vation of glutamine and glucose dramatically decreased GLS-AS precise mechanism for the regulation of GLS on Myc protein expression, but increased GLS expression. Nevertheless, over- stability needs investigation in the further research. expression of GLS-AS dramatically inhibited the survival and In summary, our study implicates a nutrient stress–repressed invasion of pancreatic cancer cells in nutrient stress. These lncRNA GLS-AS is involved in the progression of pancreatic results imply the dysregulated GLS-AS/GLS pathway is an cancer through mediating reciprocal feedback of Myc and GLS. adaption to nutrient stress and is required for the pancreatic Furthermore, our findings suggest that the Myc/GLS-AS/GLS cancer progression. axis may be promising molecular targets for the nutrient- We further explored the mechanism for downregulation of restricted treatment of pancreatic cancer. GLS-AS during nutrient stress. The Myc oncogene is a "master regulator," which controls glucose and glutamine metabolism to Disclosure of Potential Conflicts of Interest maintain growth and proliferation of cancer cells (47). Research No potential conflicts of interest were disclosed. demonstrated deprivation of glucose or glutamine dramatically elevated Myc expression and further activated serine biosynthesis pathway (48). Results from Wu and colleagues also showed Authors' Contributions glucose deprivation upregulates Myc protein in BxPC-3 and Conception and design: G. Zhao PANC-1 cells (49). Meanwhile, a study demonstrated that Myc- Development of methodology: S.-J. Deng, H.-Y. Chen, Z. Zeng, C. He fi Acquisition of data (provided animals, acquired and managed patients, induced mouse liver tumors signi cantly increase both glucose provided facilities, etc.): S. Zhu, Z. Ye, M.-L. Liu, K. Huang and glutamine catabolism with GLS upregulation (50). Results Analysis and interpretation of data (e.g., statistical analysis, biostatistics, indicated that Myc is a dual-function transcription factor that may computational analysis): S. Deng, J.-X. Zhong, F.-Y. Xu, Q. Li, Y. Liu activate or repress coding or noncoding RNA expression. Hart and Writing, review, and/or revision of the manuscript: G. Zhao colleagues showed that 534 lncRNAs were either up- or down- Administrative, technical, or material support (i.e., reporting or organizing regulated in response to Myc overexpression in P493-6 human B data, constructing databases): C. Wang Study supervision: G. Zhao cells (51). Zhang and colleagues showed that a Myc-induced lncRNA-MIF inhibits aerobic glycolysis and tumorigenesis (52). Acknowledgments On the contrary, Gao and colleagues reported that Myc transcrip- This study was supported from the National Science Foundation Committee tionally represses miR-23a and miR-23b, resulting in greater (NSFC) of China (grant nos: 81372666, 81672406, and 81872030 to G. Zhao). expression of their target protein, GLS (34). Interestingly, the bioinformatics analysis demonstrated a putative Myc-binding site The costs of publication of this article were defrayed in part by the in the promoter area of GLS-AS gene. Moreover, the ChIP and payment of page charges. This article must therefore be hereby marked luciferase reporter assays verified the binding and transcriptional advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate inhibition of Myc on GLS-AS promoter. Coincidently, Myc knock- this fact. down significantly increased GLS-AS expression, but inhibited GLS expression. In addition, the deprivation of glucose and Received March 12, 2018; revised September 25, 2018; accepted December glutamine dramatically induced Myc expression and its transcrip- 14, 2018; published first December 18, 2018.

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Nutrient Stress−Dysregulated Antisense lncRNA GLS-AS Impairs GLS-Mediated Metabolism and Represses Pancreatic Cancer Progression

Shi-Jiang Deng, Heng-Yu Chen, Zhu Zeng, et al.

Cancer Res Published OnlineFirst December 18, 2018.

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