Promotes Pancreatic Tumor Growth

Published OnlineFirst October 20, 2016; DOI: 10.1158/0008-5472.CAN-16-1666

Cancer Tumor and Stem Cell Biology Research

Glucose Metabolism Reprogrammed by Overexpression of IKK« Promotes Pancreatic Tumor Growth Haseeb Zubair1, Shafquat Azim1, Sanjeev Kumar Srivastava1, Aamir Ahmad1, Arun Bhardwaj1, Mohammad Aslam Khan1, Girijesh Kumar Patel1, Sumit Arora1, James Elliot Carter2, Seema Singh1,3, and Ajay Pratap Singh1,3

Abstract

Aberrant expression of the kinase IKKe in pancreatic ductal tion rate. IKKe silencing also attenuated c-Myc in a manner adenocarcinoma (PDAC) has been associated with poor prog- associated with diminished signaling through an AKT/GSK3b/ nosis. In this study, we define a pathobiologic function for c-MYC phosphorylation cascade that promoted MYC nuclear IKKe in reprogramming glucose metabolism and driving pro- accumulation. In an orthotopic mouse model, IKKe-silenced gression in PDAC. Silencing IKKe in PDAC cells, which over- PDAC exhibited a relative reduction in glucose uptake, tumor- expressed it endogenously, was sufficient to reduce malignant igenicity, and metastasis. Overall, our findings offer a preclin- cell growth, clonogenic potential, glucose consumption, lac- ical mechanistic rationale to target IKKe to improve the ther- tate secretion, and expression of genes involved in glucose apeutic management of PDAC in patients. Cancer Res; 76(24); metabolism, without impacting the basal oxygen consump- 7254–64. 2016 AACR.

Introduction regulatory factor (IRF)-dependent gene transcription of proin- flammatory cytokines and interferons (4). It is expressed at basal Pancreatic ductal adenocarcinoma (PDAC) is one of the most levels in a subset of tissues involved in immune function, and can lethal malignancies with a 5-year survival rate of about 8% after be readily induced in a variety of cell- and tissue types upon initial diagnosis (1). It is expected to overtake breast malignancy external stimuli (5). Interestingly, IKKe has also been shown to this year as the third leading cause of cancer-related deaths in regulate energy balance in high-fat diet–induced obesity (6) and the United States with an estimated 53,070 new diagnoses and recognized to possess oncogenic properties in breast cancer (7) 41,780 deaths, and may actually become the second by 2020 if with some later reports in other cancers as well (8, 9). In many similar trends continue (1, 2). Over the years, significant progress cancer cases, including PDAC, an upregulation of IKKe, even in the has been made in our understanding of the genetics of PDAC (3); absence of gene amplification, has been reported and associated however, these seminal advancements have not helped much in with poor clinical outcome (7, 9, 10). However, we lack direct the development of an effective treatment strategy for this lethal evidence for its oncogenic activity in PDAC along with complete malignancy. As a consequence, search for novel, functionally lack of an in-depth understanding of involved molecular relevant molecular targets continues, so that effective, mecha- pathways. nism-based approaches for its therapy and management can be Cancer cells remain under constant demand for energy and formulated. building blocks to ensure their continued, rapidly proliferative Inhibitor of kappa kinase subunit-epsilon (IKKe) is an impor- development. As a result, they adapt to glycolytic metabolism, tant member of the IKK family along with four other, distinct yet even when oxygen is not a limiting factor, to meet their demands closely related, members (IKKa, IKKb, IKKg, and NAK). IKKe plays for quick energy (ATPs) and metabolic intermediates that serve as a central role in innate immunity by inducing NF-kB- and IFN building blocks for rapidly dividing cancer cells (11). This shift provides added advantage to the tumor cells, that is, the ability to 1Department of Oncologic Sciences, Mitchell Cancer Institute, University of thrive independently of oxygen diffusion that would otherwise be South Alabama, Mobile, Alabama. 2Department of Pathology, College of Med- a limiting factor for rapidly growing tumors (12). Indeed, mount- 3 icine, University of South Alabama, Mobile, Alabama. Department of Biochem- ing evidence continues to associate enhanced aerobic glycolysis to istry and Molecular Biology, College of Medicine, University of South Alabama, the etiology and malignant progression of several cancers, includ- Mobile, Alabama. ing pancreatic malignancy (13). This metabolic shift, in general, is Note: Supplementary data for this article are available at Cancer Research mediated through aberrant activation of oncogenic transcription Online (http://cancerres.aacrjournals.org/). factors, leading to altered expression of genes involved in glucose Corresponding Author: Ajay Pratap Singh, University of South Alabama, 1660 import and metabolism (14). c-MYC serves as a "master regula- Springhill Avenue, Mobile, AL 36604-1405. Phone: 251-445-9843; Fax: 251-460- tor" of growth and cellular metabolism pathways, and its aberrant 6994; E-mail: [email protected] activation is facilitated at multiple levels (15–17). Emerging doi: 10.1158/0008-5472.CAN-16-1666 clinical and experimental data also support its role in PDAC 2016 American Association for Cancer Research. pathobiology (10).

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This study provides first evidence for a link between IKKe and Growth kinetics assay c-MYC oncoprotein. We demonstrate that IKKe regulates nuclear Growth rate and population-doubling time (PDT) were deter- retention and stabilization of c-MYC through a cascade of signal- mined by counting number of viable cells using the Trypan blue ing events. We further identify a novel role of IKKe in regulating dye exclusion on the Countess Automated Cell Counter (Life glucose metabolism in PDAC, at least in part, through its c-MYC– Technologies) every day for 8 days, as described previously (18). mediated regulation of metabolic gene expression. IKKe overexpression is also shown to promote growth and metastasis of Clonogenicity assays PDAC cells, thus establishing it as an important molecular target Anchorage-dependent and anchorage-independent clonogeni- for clinical management. city assays were carried out as described previously (20). Materials and Methods qPCR and Ingenuity Pathway Analysis RNA isolation, cDNA synthesis, and qPCR were performed as Cell lines and tissue samples described previously (20) using primers listed in Supplementary The human pancreatic cell lines were obtained and maintained Table S2. The altered genes (fold-change 1.5; P 0.05) were as previously described (18). All the cell lines were tested inter- subjected to Ingenuity Pathway Analysis (IPA) to identify putative mittently and determined to be free from mycoplasma, and upstream regulator. authenticated by either in-house or commercial (Genetica DNA Laboratories) short-tandem repeats genotyping. Normal and Luciferase assay tumor pancreatic tissue specimens were obtained through the Control or IKKe-silenced PDAC cells were transfected with Southern Division of Cooperative Human Tissue Network under either a negative control or c-MYC–responsive luciferase-promot- – an Institutional Review Board approved protocol. er-reporter plasmid (Cignal MYC Reporter Assay Kit, SABios- ciences), and assayed as per the manufacturer's protocol. Antibodies Antibodies used were: anti-IKKe, -c-MYC (rabbit monoclonal), S62 T58 Nuclear and cytoplasmic fractionation -phospho-c-MYC , -phospho-c-MYC (rabbit polyclonal), Cytoplasmic and nuclear extracts were prepared using Nucle- -ubiquitin (mouse monoclonal; Abcam); -phospho-AktT308 (rab- Ser473 ar-Extract Kit (Active Motif) following the manufacturer's bit monoclonal), -phospho-Akt (rabbit polyclonal; Cell instructions. Signaling Technologies); -GSK3b, -phospho-GSK3bSer9, -Akt (rabbit monoclonal; Epitomics); -LaminA, (mouse monoclonal), Coimmunoprecipitation analysis -a-tubulin (rabbit polyclonal; Santa Cruz Biotechnology). Anti- Coimmunoprecipitation was performed using c-MYC–specific – – b-actin HRP conjugated (mouse monoclonal) antibody was antibody as described previously (21). from Sigma-Aldrich. All secondary antibodies were from Santa Cruz Biotechnology. Glucose uptake and lactate production assays Glucose and lactate concentration in the culture media was Transfections and treatments determined using the Glucose and Lactate Assay Kit (Biovision) as Generation of stable IKKe-knockdown and control cell lines was per manufacturer's instructions. To measure glucose uptake, cells done in IKKe-overexpressing MiaPaCa and Colo357 cells by were first incubated in glucose-free, FBS-free media for 6 hours transfection of IKBKE-shRNA-pGFP-B-RS or the control-plasmid, followed by incubation with a glucose-free DMEM supplemented Scr-shRNA-pGFP-B-RS (Origene), respectively, using X-treme- with 100 mmol/L of fluorescent D-glucose derivative, 2-NBDG GENE HP DNA Transfection Reagent (Roche) as per the manu- (Invitrogen) for 3 hours, and analyzed by fluorescence imaging or facturer's instructions. Transfectants were selected using blastici- flow cytometry using FACS AriaII (BD Biosciences). din (2 mg/mL MiaPaCa and 20 mg/mL Colo357) and assessed for IKKe expression using immunoblotting. For transient knock- Measurement of extracellular acidification rate and oxygen down, cells were cultured in 6-well plates and transfected with consumption rate 100 nmol/L of nontarget or ON-TARGETplus SMARTpool IKBKE- Basal rate of glycolysis and oxidative phosphorylation was targeting siRNAs (Dharmacon) using DharmaFECT (Dharmacon) determined by measuring the extracellular acidification (ECAR) according to the manufacturer's instruction. Cycloheximide and the oxygen consumption rates (OCR) using the Seahorse- (50 mmol/L; Sigma-Aldrich) and MG132 (10 mmol/L; Sigma- XF24 Analyzer (Seahorse Bioscience) as per the manufacturer's Aldrich) were used to inhibit protein synthesis or proteasome- instructions. Briefly, 4 104 cells per well were seeded in XF24 cell degradation machinery, respectively. Cells were treated with GSK3b culture microplates in a two-step process and incubated at 37C inhibitor LiCl (40 mmol/L; Sigma-Aldrich) for 6 hours or tran- for 24 hours. Subsequently, culture media was changed to XF siently transfected with constitutively active Akt (pcDNA3-HA PKB Assay Medium (supplemented with 5 mmol/L glucose), and T308D S473D, plasmid number 14751; Addgene) mutant or con- plates loaded into the XF24 analyzer to record the data. trol plasmids to dissect the roles of GSK3b and Akt, respectively. Assessment of tumorigenicity and glucose uptake in vivo Immunoblot analysis Animal studies were conducted under protocol approved by Total protein from PDAC cells was isolated in NP-40 lysis buffer Institutional Animal Care and Use Committee. Luciferase-tagged supplemented with phosphatase and protease inhibitors (Roche), IKKe-knockdown or control cells (1 106 cells/50 mL) were and estimated using DC Protein-Assay Kit (Bio-Rad). Protein injected into the pancreas of immunocompromised mice (Harlan samples (60–80 mg, unless noted) were resolved on SDS-PAGE Laboratories; 10 mice/group). After 1 week, tumor growth was and subjected to immunoblot analysis as described previously monitored every third day by palpation and weekly by noninva- (18, 19). Band intensities were quantitated using ImageJ software. sive in vivo imaging. For glucose uptake, mice were injected

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A BC MiaPaCa Colo357 250 MiaPaCa-SCR 600 Colo357-SCR MiaPaCa-ShIKKe-1 Colo357-ShIKKe-1 e e ShIKKe ShIKKe ) MiaPaCa-ShIKK -3 Colo357-ShIKK -3 4 MiaPaCa-ShIKKe ) Colo357-ShIKKe

4 500 Scr 1 2 3Scr 1 2 3 200 IKKe Scr ShIKKe (1+3)Scr ShIKKe (1+3) 400 IKKe 150 b-Actin b-Actin 300 MiaPaCa Colo357 100 1.0 200 1.0 50

Number of cells (×10 100

0.5 0.5 Number of cells (×10 0 0 0 2 4 6 8 0 2 4 6 8 Relative band intensity 0.0

Relative band intensity 0.0 Scr Sh1 Sh2 Sh3 Scr Sh1 Sh2 Sh3 Day Day MiaPaCa Colo357

DEe Scr ShIKK -3 e ShIKKe-1 ShIKKe e e Scr ShIKK -3 500 Scr ShIKKe-1 ShIKK -3 ShIKK ShIKKe-1 ShIKKe 100 400 80 300 MiaPaCa 60 200 40 100

(10 random fields) 20 Colo357 Total number of colonies number Total

0 of colonies number Total MiaPaCa Colo357 0 MiaPaCa Colo357

Figure 1. IKKe supports the growth and clonogenicity of PDAC cells. A, Total protein isolated from stably transfected PDAC cells was examined for IKKe expression by immunoblotting. B, Clonal population of transfectants emerging from IKKe-targeting shRNA constructs #1 and #3, which consistently produced maximum silencing of IKKe, were pooled (ShIKKe), propagated, and monitored for IKKe expression by immunoblot assay. b-Actin was used as loading control. C, Growth kinetics of IKKe-silenced clonal and pooled population was studied relative to their controls for 8 days using the Trypan blue exclusion assay. D, Plating efﬁciency was measured by seeding the PDAC cells at low density. E, Anchorage-independent colony formation was measured as an in vitro measure of tumorigenic ability of control and IKKe-silenced PDAC cells. Data are presented as mean SD, n ¼ 3; , P 0.05.

intraperitoneally with 100 mL of XenoLight RediJect 2-DeoxyGlu- expression was detected in non-neoplastic cases (Supplementary cosone (2-DG; Perkin Elmer) 24 hours prior to final imaging (28 Fig. S1A). In search of model cell lines for functional studies, we days postimplantation) and epi-fluorescence recorded using analyzed IKKe expression in a panel of 14 PDAC cell lines, of Xenogen-IVIS-cooled CCD optical system (IVIS Spectrum) next which 12 expressed high-to-moderate levels, whereas 2 had low/ day. To image tumors, mice were given intraperitoneal injections null expression (Supplementary Fig. S1B). Considering that IKKe- of D-Luciferin (150 mg/kg body weight) and bioluminescence overexpressing cells would likely have IKKe-dependent growth recorded using the Xenogen-IVIS-cooled CCD optical system. mechanisms, we selected MiaPaCa and Colo357 cells that carry mut mut After euthanasia, primary tumors were resected and mice imaged most common PDAC genetic aberrations (KRAS , TP53 , del/inactive for the detection of metastases. Final measurements of tumor CDKN2A ) and have been well characterized for their weight and volumes were also made from resected xenografts. aggressiveness and metastatic potential (22, 23) for functional studies. IKKe expression was silenced by stable transfection of IKBKE WST-1 proliferation assay three different -targeted shRNA expression constructs – Cells (3 103/well) were seeded in 96-well plates and growth (ShIKKe#1 3). Control cells were generated by stable transfec- examined using WST-1 Assay Kit (Roche) and analyzed as tions with nontargeted scrambled sequence (Scr) expression described previously (19). construct. Stable silencing of IKKe was analyzed by immunoblotting in polyclonal populations from individual constructs Statistical analysis (Fig. 1A). As ShIKKe plasmid #1 and #3 transfectants exhibited The experiments were conducted in triplicates and repeated at most potent reduction in IKKe, compared with control, they were least three times. Wherever appropriate, the data were also sub- combined to generate pooled population (ShIKKe; Fig. 1B). IKKe fi jected to unpaired two-tailed Student t test. P 0.05 was con- inhibition resulted in signi cant growth reduction in MiaPaCa sidered significant. ( 45.4%) and Colo357 ( 42.4%) cells on the eighth day of growth kinetics (Fig. 1C) due to increase in their PDT calculated during exponential growth phase (96–144 hours). Silencing of Results IKKe prolonged PDT from approximately 36.46 and 21.14 hours IKK« is overexpressed and associated with increased growth to approximately 45.71 and 30.37 hours in MiaPaCa–ShIKKe and clonogenicity of PDAC cells and Colo357–ShIKKe cells, respectively, relative to their controls We first analyzed IKKe expression in a set of malignant (n ¼ 21) (Supplementary Table S2). Moreover, when seeded at low density, and non-neoplastic (n ¼ 7) pancreas. Similar to a published we observed a significant decrease (P < 0.05) in the plating report (10), we observed an overexpression of IKKe in 81% cases efficiency of MiaPaCa–ShIKKe (2.8-fold) and Colo357–ShIKKe of malignant pancreas, whereas no (n ¼ 5) or very low (n ¼ 2) (3.2-fold) cells as compared with their controls (Fig. 1D). A

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IKKe Reprograms Glucose Metabolism in Pancreatic Cancer

Figure 2. IKKe silencing suppresses glucose uptake and consumption in PDAC cells. A and B, Glucose uptake (A) and lactate efflux (B) was measured in the used culture media and normalized to cell counts. The data are depicted as percent change in IKKe-silenced cells relative to their respective controls. C and D, PDAC cells were transfected with nontargeting control (NT) or IKBKE- targeting (SiIKKe) siRNAs. After 72 hours, cells were cultured in glucose-free FBS-free media for 6 hours and further incubated with glucose-free media supplemented with 100 mmol/L 2-NBDG for 3 hours. Thereafter, either the cells were visualized under fluorescent microscope and photographed (C) or subjected to flow cytometry analysis (D). Representative images are from independent experiments. E and F, To examine basal ECAR and OCR, 4 104 cells per well were seeded in XF24 cell culture microplates and incubated at 37C overnight. Next day, culture media was replaced with XF Assay Medium (supplemented with 5 mmol/L glucose) and plates loaded into the XF24 analyzer. Data are presented as mean SD, n ¼ 3; , P 0.05.

significant (P < 0.05) decrease (3.5- and 3.9-fold, respectively) carried out after transient silencing of IKKe in MiaPaCa and was also recorded in anchorage-independent clonogenic poten- Colo357 cell lines using ON-TARGETplus SMARTpool IKBKE- tial (an in vitro measure of tumorigenecity) of MiaPaCa–ShIKKe targeting siRNAs or nontargeting control siRNAs, as stable and Colo357–ShIKKe cells relative to their controls (Fig. 1E). To lines expressed GFP, which could hinder 2-NBDG signal detec- further confirm the role of IKKe in pancreatic cancer cell growth, tion. IKKe silencing by siRNA was determined at 72 hours by we also ectopically overexpressed IKKe in BxPC3 cells that has its immunoblotting (Supplementary Fig. S3). In line with the low endogenous expression (Supplementary Fig. S2A). IKKe over- above observation, cellular accumulation of 2-NBDG was sig- expression led to increase in growth of BxPC3 cells by approxi- nificantly less in IKKe-silenced PDAC cells compared with their mately 37.94% on the eighth day of growth kinetics due to control cells (Fig. 2C) as quantitative analysis by flow cytome- decrease in doubling time (Supplementary Fig. S2B). try revealed a reduction in the mean fluorescence intensity by 23.62% and 33.32% in MiaPaCa–SiIKKe and Colo357–SiIKKe Silencing of IKK« inhibits glycolytic metabolism in prostate cells, respectively, relative to their controls (Fig. 2D). To get cancer further insight into altered metabolic phenotype, we monitored As a defined hallmark, tumor cells sustain their rapid growth basal glycolytic metabolism and oxidative phosphorylation by by shifting to glycolytic metabolism to ensure quick and extracellular acidification rate (ECAR) and OCR using Seahorse- sufficient supply of energy and macromolecular precursors XF Extracellular-Flux Analyzer. ECAR is a surrogate measure of (11, 14). Therefore, we evaluated the influence of IKKe silenc- glycolysis and an alternate measure for lactate secretion, where- ing on glucose metabolism in PDAC cells. First, consumption as OCR measures the changes in dissolved oxygen concentra- of glucose and the lactate release was measured from the used tion. We observed a significant decrease in the glycolytic activity culture media of control and IKKe-silenced PDAC cells, which of IKKe-silenced cells relative to control cells; however, no demonstrated substantial attenuation of glucose consumption substantial change in oxygen consumption was recorded (Fig. 2A) and lactate secretion (Fig. 2B) upon IKKe silencing. (Fig.2E and F). In concordance, overexpression of IKKe in The influence on glucose uptake in a cell-autonomous manner BxPC3 cells enhanced glucose uptake and lactate efflux, which in control and IKKe-silenced cells was further investigated using was also reflected in elevated ECAR (Supplementary Fig. S4). the fluorescent glucose analogue 2-NBDG, which was imaged This suggests that IKKe silencing impacts glycolytic phenotype by fluorescence microscopy or quantified using flow cytometry only without having noticeable effect on the electron transport upon incorporation into the cells. These experiments were system machinery.

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Figure 3. IKKe alters the expression of genes encoding glucose-metabolizing enzymes and regulates c-MYC expression and subcellular localization. A, Expression of genes involved in glucose metabolism was measured by qRT-PCR. Data shown as fold change in IKKe-silenced cells relative to control. B, Differential gene expression dataset was subjected to IPA that predicted c-MYC as a potential upstream regulator. C, Transcriptional activity of c-MYC was measured using luciferase promoter-reporter assay. Data (mean SD, n ¼ 3) are presented as fold change in normalized luciferase activity; , P 0.05. Expression of c-MYC in IKKe knockdown and control cells was examined at protein (D) and transcript levels (E) by immunoblot and qRT-PCR assays, respectively. ACTB (for mRNA) and b-actin (for protein) were used as internal controls. F, Expression of c-MYC in cytoplasmic and nuclear fractions was examined by immunoblot analysis. Lamin A and a-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively.

IKK« alters the expression of genes of glucose metabolic predicted inhibition of c-MYC, we examined its transcriptional pathway that converge at c-MYC activity using a c-MYC–responsive promoter reporter system To identify the altered gene expression associated with shift andobservedsignificant abrogation (79% and 63%, P < 0.05) in glucose metabolism, we employed a qPCR-based custom in luciferase activity of IKKe-silenced MiaPaCa and Colo357 array that included 80 genes involved in glucose metabolism. A cells, respectively, relative to their controls (Fig. 3C). This total of 33 genes were found to be dysregulated upon IKKe correlated with a reduction in total c-MYC protein (Fig. 3D) silencing including those involved in glucose transport, glycol- without any significant change in its transcripts levels (Fig. 3E). ysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, Interestingly, subcellular fractionation demonstrated greater and lactate transport (Fig. 3A). Gene expression dataset was nuclear accumulation of c-MYC in IKKe-expressing control subjected to the IPA software in search for a putative upstream cells, while relatively greater cytoplasmic levels of c-MYC were transcriptional regulator mediating the effect of IKKe silencing. detected in IKKe-silenced cells (Fig. 3F). In line with these In silico analysis suggested c-MYC to be a candidate upstream observations, ectopic expression of IKKe in BxPC3 cells also regulator of altered gene expression (Fig. 3B). To confirm the elevated c-MYC protein level along with a greater nuclear

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Figure 4. IKKe-induced c-MYC expression and localization is controlled through its inhibition of phosphorylation-mediated nuclear export and subsequent degradation. A, Phosphorylated c-MYCS62 and c-MYCT58 were analyzed by immunoblot using speciﬁc antibodies. B, Cells were treated with the proteasome inhibitor (MG132, 10 mmol/L) for the indicated time period, total protein isolated, and effect on c-MYC expression determined by immunoblot analysis. We used 60 and 25 mg protein from IKKe-silenced and control cells, respectively, to correct for differences in initial protein levels. b-Actin was used as loading control. C, Equal amount (500 mg) of protein from Scr and ShIKKe PDAC cells untreated or treated with MG132 (10 mmol/L, 30 minutes) was subjected to immunoprecipitation with anti-c-MYC antibody, followed by immunoblot with anti-Ub antibody. D, To monitor the turnover of c-MYC protein, cells were treated with cycloheximide (CHX; 50 mmol/L), neo-protein synthesis inhibitor, for the indicated time intervals. Thereafter, total protein was isolated and changes in c-MYC expression monitored by immunoblotting. Considering differences in c-MYC levels between control and IKKe-silenced cells, we used different amounts (60 and 150 mg, respectively) to keep initial signal at near-similar intensity. &, the rate of total c-MYC accumulation; &, the rate of c-MYC degradation based on the densitometry of the data presented.

accumulation (Supplementary Fig. S5). These cells, however, likely due to diminished total c-MYC protein content. However, sustained significant c-MYC in the cytoplasm, thus suggesting despite reduced levels of total c-MYC, IKKe-silenced PDAC cells the involvement of additional cell type and/or context-depen- had significantly increased levels of phospho-c-MYCT58, whereas dent mechanism(s) in regulation of c-MYC by IKKe. negligible c-MYCT58 phosphorylation was detected in control cells of both PDAC lines (Fig. 4A). A similar correlation between c-MYC downregulation upon IKK« silencing is caused by its differential c-MYC phosphorylation and IKKe was also observed nuclear export and subsequent proteasomal degradation upon forced IKKe expression in BxPC3 cells (Supplementary Fig. Differential subcellular distribution of c-MYC besides its over- S5). Because c-MYCT58 phosphorylation is known to promote its all repression at the protein level prompted us to investigate the nuclear export followed by ubiquitin (Ub)-mediated proteasomal underlying molecular mechanism(s). Phosphorylation at con- degradation (15–17), we next examined the effect of MG-132 served serine-62 (S62) residue of c-MYC governs the activation (proteasome inhibitor) treatment on c-MYC levels. To correct the and nuclear import of c-MYC and subsequent phosphorylation of differences in initial protein levels, we adjusted protein loading as threonine-58 (T58) residue leads to its nuclear export followed by mentioned in the figure legend. IKKe-silenced PDAC cells exhib- degradation (15–17). Therefore, we examined their levels in IKKe- ited a time-dependent increase in total c-MYC levels upon MG- overexpressing and -silenced PDAC cells, which demonstrated a 132 treatment (Fig. 4B, top). Similar observation was also decrease in phospho-c-MYCS62 in IKKe-silenced cells (Fig. 4A) recorded in MG-132–treated control cells (Fig. 4B, bottom);

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Figure 5. Subcellular localization and stabilization of c-MYC is governed through IKKe/Akt/GSK3b axis. A, GSK3bS9 phosphoryation was determined by immunoblotting using speciﬁc antibodies. B, PDAC cells were treated with GSK3b inhibitor, LiCl (40 mmol/L, 6 hours), followed by total protein isolation and immunoblotting (top). Subsequently, cytoplasmic and nuclear levels of c-MYC were also measured following LiCl treatment (bottom). C, Determination of phospho-AktT308/S473 by immunoblotting suggested their reduced expression in IKKe-silenced PDAC cells. D, Cells were transfected with constitutively active-Akt (CA-Akt) or control plasmid, followed by measurement of c-MYC, c-MYCT58, pGSK3bS9, and total GSK3b by immunoblotting. E, In parallel experiments, effect of LiCl treatment and CA-Akt transfection was investigated on mRNA levels of HK2, LDHA, SLC2A3,andSLC16A1 by qRT-PCR. F, Glucose uptake (top) and lactate efﬂux (middle), and growth recovery on day 3 and 5 by WST1 assay (bottom). Bars, mean SD (n ¼ 3); , P 0.05. b-Actin was used as a loading control for total protein, and Lamin A and a-tubulin were used as controls for nuclear and cytoplasmic fractions, respectively. qPCR data were normalized with ACTB expression.

however, the rate of c-MYC accumulation in these cells was IKKe-silenced MiaPaCa and Colo357 cells, respectively (Supple- relatively slower compared with IKKe-silenced cells. To confirm mentary Fig. S6). that c-MYC stabilization was a result of decreased degradation of ubiquitinated-c-MYC, we performed immunoprecipitation using c-MYC destabilization upon IKK« silencing is mediated anti-c-MYC antibody followed by immunoprobing with anti-Ub through Akt repression–induced GSK3b activation antibody. An accumulation of Ub-c-MYC upon MG132 treatment Considering a role of GSK3b in c-MYCT58 phosphorylation was observed in both control and IKKe-silenced cells; however, (15), we examined its activation status in IKKe-expressing and the latter exhibited elevated levels (Fig. 4C). To measure the -silenced PDAC cells. GSK3bS9 phosphorylation, which is known overall impact of IKKe silencing on the stability of c-MYC, we to cause its inactivation (24), was decreased in both IKKe-silenced measured its turn-over after blocking protein synthesis by cyclo- PDAC cell lines, without any appreciable changes in total GSK3b heximide (CHX; Fig. 4D). Differences in initial protein levels were (Fig. 5A). When IKKe-silenced PDAC cells were treated with LiCl, corrected by adjusting protein loading as mentioned in the figure an inhibitor of GSK3b (25), it restored total c-MYC levels to legend. Subsequent analyses based on the densitometry measure- significant extent, which correlated with decreased phospo-c- ments of c-MYC signals predicted half-life of c-MYC to be about MYCT58 and enhanced phospo-GSK3bS9 levels (Fig. 5B). Further- 41 and 30 minutes in MiaPaCa and Colo357 cells, respectively, more, LiCl treatment also enhanced the nuclear retention of whereas it was decreased to approximately 21 and 16 minutes in c-MYC in IKKe-silenced PDAC cells (Fig. 5B). We next investigated

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whether IKKe was directly able to phosphorylate GSK3b through understanding the molecular causes of PDAC aggressiveness as its Ser/Thr kinase activity as reported for GSK3a (26). However, no well as on formulating strategies that could target this very feature interaction between IKKe and GSK3b was observed in reciprocal of pancreatic malignancy. It is now being widely recognized that coimmunoprecipitation assays (data not shown). Hence, we cancer cells rewire their metabolic state to fulfill the enhanced focused our attention to Akt, which is known to inactivate GSK3b requirements of bioenergetics and biomass production, so that through its S9 phosphorylation (27). Moreover, IKKe can also they can maintain their rapid proliferation (11, 13). Glucose, a directly phosphorylate Akt at both T308 and S473, independent of major source of energy, gets metabolized through a combination PI3K and mTORC2 activities, respectively (28, 29). A significant of anaerobic glycolysis and oxidative phosphorylation in normal reduction in AktT308/S473 levels was observed in immunoblot cells. However, cancer cells reprogram the glucose metabolism to analyses in IKKe-silenced PDAC cells (Fig. 5C). Similarly, ectopic support their fast growth by shifting their dependence of ATP expression of IKKe in BxPC3 cells promoted Akt activation as production from oxidative phosphorylation to "aerobic" glycol- evidenced by phosphorylation at Thr-308 and Ser-473 sites, and ysis—an observation referred to as "Warburg effect" (11, 13, 14). thus resulting in Ser-9 phosphorylation of GSK3b (Supplemen- Moreover, altered glucose metabolism in cancer cells has also tary Fig. S7). Thus, to further explore the role of activated Akt, we been associated with their metastatic potential and therapeutic expressed its constitutively active mutant form (T308D-S473D; resistance (32, 33). In these contexts, our observation of IKKe- CA-Akt) in IKKe-silenced cells through transient transfection. mediated regulation of glucose metabolism is highly significant Upon ectopic activation of Akt, significant upregulation of c-MYC and suggests an important role of IKKe in molecular pathogenesis protein was detected that correlated with phospho-GSK3bS9 and of PDAC. This is even more interesting considering a role of reduced c-MYCT58 levels (Fig. 5D). Thereafter, we ascertained a deregulated glucose metabolism in inflammation (34). In fact, role of IKKe/Akt/GSK3b signaling axis in glucose metabolism and chronic inflammation is known to exacerbate glucose metabolism expression of involved gene targets. First, the effect of LiCl and CA- and also promote cancer progression (35). Thus, these vicious Akt on relative mRNA expression of some randomly selected genes connections of altered glucose metabolism, inflammation, and (HK2, LDHA, SLC2A3, and SLC16A1) was examined followed by cancer, and suggested a role of IKKe in these phenotypes makes glucose uptake measurements. LiCl and CA-Akt activity rescued our findings even more noteworthy for future therapeutic and the gene expression either partially (SLC2A3 and SLC16A1)or preventive intervention points. almost completely (HK2 and LDHA) in IKKe-silenced cells relative From the mechanistic standpoint, we observed dysregulation to that in control cells (Fig. 5E). Similarly, a significant increase in of several genes involved in glucose metabolism upon IKKe glucose uptake and lactate efflux was also observed upon LiCl silencing. This involved downregulation of genes associated with treatment and CA-Akt expression in IKKe-silenced cells, which glucose and lactate transport (GLUT1, GLUT3, MCT1,andMCT4) correlated with their enhanced growth (Fig. 5F). and glycolysis (ALDOC, ENO3, GALM, GCK, HK2, HK3, etc.). As per the published data, PDAC cells are known to overexpress IKK« promotes glucose uptake and tumorigenicity of PDAC GLUT1, whereas the upregulation of GLUT3 along with GLUT1 cells in an orthotopic mouse model has been shown in many other cancer types and correlated with To assess the functional relevance of IKKe in vivo, we injected poor prognosis (36–38). Similarly, expression and activity of HK2 luciferase-tagged MiaPaCa–Scr/MiaPaCa–ShIKKe cells directly into has been observed to be upregulated in nearly all types of cancers the pancreas of mice, and monitored tumor growth every alternate (32). Notably, enhanced activity of HK2 is required for the day by palpation and weekly by noninvasive bioluminescence initiation and maintenance of K-Ras–driven cancers, and its imaging. A significantly greater fluorescent signal was detected in inhibition is shown to reduce tumor growth both in vitro and all mice from MiaPaCa–Scr group as compared with that from in vivo (39). There is also evidence to suggest the utility of HK2 as a MiaPaCa–ShIKKe group, indicating higher glucose uptake by the prognostic marker in PDAC patients (40). The strong reliance tumors (Fig. 6A). To normalize the glucose uptake with tumor size, toward aerobic glycolysis by cancer cells leads to the production of we also performed bioluminescent imaging of tumors following lactate by the enzyme LDHA, which is then exported to the tumor D-luciferin injection (Fig. 6B). Normalized fluorescence signal also microenvironment by lactate transporters (MCT1–4) to have a suggested significantly greater glucose uptake in IKKe-overexpres- multitude of functions in cancer growth and metastasis (41). sing MiaPaCa–Scr cells compared with IKKe-silenced MiaPaCa– Furthermore, in a majority of pancreatic tumors that express high ShIKKe cells (Fig. 6C). End-point measurement also confirmed levels of MCT4, stromal cells have also been reported to have high significant decrease in tumor growth in IKKe-silenced group. Aver- MCT4 expression, indicating a putative co-relation between these age weight and volume of the tumors developed in control group two events (42). Interestingly, although knockdown of IKKe led to were recorded to be 1.71 0.05 g and 866 64.86 mm3 as the reduction of lactate efflux in the culture media, we did not compared with 0.303 0.01 g and 259.9 19.42 mm3,respec- observe any change in basal OCR in our study. This suggests that tively, in IKKe-silenced group (Fig. 6D). Imaging of mice after although IKKe knockdown hampers glycolytic metabolism, it resection of primary tumors exhibited strong bioluminescence does not affect oxidative phosphorylation. Furthermore, signals in various organs of control group mice only, suggesting observed reduction in lactate levels could also be due to the drop distant metastasis (Fig. 6E), which was further confirmed by ex vivo in LDHA and lactate transporters, MCT1 and MCT4. This is imaging of resected organs (spleen, liver, and lung; Fig. 6F). significant as the accumulation of lactate within the cell could otherwise alter intracellular pH and subsequently induce meta- Discussion bolic feedback inhibitions, ultimately hampering ATP production by glycolysis (43). PDACs are highly aggressive, exhibiting rapid growth and Our findings also identified c-MYC as an important mediator in metastasis, which is one important reason for their high lethality potentiating the effect of IKKe on glucose metabolism. This is very in patients (2, 30, 31). As a result, efforts have focused on significant, as c-MYC is frequently deregulated not only in PDAC,

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Zubair et al.

Figure 6. IKKe downregulation suppresses glucose uptake, tumor growth, and metastasis in orthotopic PDAC xenografts. Luciferase-tagged control or IKKe-silenced MiaPaCa cells were implanted into the pancreas of athymic nude mice (n ¼ 10 per group). A, A day prior to endpoint, mice were injected with fluorescent analogue of glucose, 2-DG (100 mL) intraperitoneally and epifluorescence measured after 24 hours using the IVIS imaging system. B, Prior to sacrificing the mice, D-luciferin (150 mg/kg body weight) was injected intraperitoneally and bioluminescence imaging data recorded using the IVIS imaging system. C, In vivo glucose uptake normalized with bioluminescent tumor measurements at the end time point and shown as relative 2-DG uptake. D and E, After sacrifice, tumors were resected and measured for weight and volume (D), and mice imaged to visualize metastases (E). F, To further confirm metastases, livers, lungs, and spleens were carefully removed from the mice and imaged separately. Representative images of mice and organs from individual groups are presented. Bars, mean SD (n ¼ 10 mice); , P 0.05.

but several other cancers as well (44, 45). c-MYC is shown to IKKe could replace Myr-Akt, leading to transformation of integrate cellular metabolism with survival and proliferation of immortalized human mammary epithelial cells (7). It was later cancer cells through the regulation of a number of genes (46). confirmed that IKKe can directly phosphorylate and enhance Under normal conditions, c-MYC transcriptional activity is con- AktT308/S473 levels even in the absence of external stimuli, and trolled at several levels involving transcriptional and posttran- independent of other regulators (48). Our findings also dem- scriptional regulation, posttranslational modification, subcellular onstrated significant downregulation of Akt phosphorylation at distribution, and protein turn-over (16). Moreover, interaction of both Thr-308 and Ser-473 sites in IKKe-silenced cells, which c-MYC with other nuclear proteins has also been suggested to alter correlated with the decrease in inhibitory GSK3bS9 phosphor- its genomic occupancy and transcriptional activation. Our data ylation. Interestingly, despite regained Akt activation and demonstrate that IKKe does not alter mRNA levels of c-MYC in GSK3b inactivation in IKKe-silenced PDAC cells, it did not lead PDAC cells, but supports its nuclear retention and total protein to complete restoration of c-MYC expression, metabolic shift, and stability. Moreover, we show that IKKe stabilizes c-MYC through tumor growth. This may be due to technical limitation or more inactivation of GSK3b, which is known to promote c-MYCT58 likely due to other IKKe responsive, cross-talking signaling net- phosphorylation (25). Despite a previous report demonstrating works that are involved in potentiating its pathobiologic func- direct interaction of IKKe with GSK3a (26), we did not observe its tions. It is possible that IKKe silencing may modulate the activity similar interaction with GSK3b; its phosphorylation was rather of other molecular targets, such as PP2A, which are known to alter dependent on IKKe-mediated Akt activation. Activity of GSK3b c-MYC expression and/or its activation (49, 50). has been demonstrated to be diminished by extracellular signals In summary, we have defined a novel role of IKKe in PDAC upon GSK3b-Ser-9 phosphorylation mediated by Akt and other pathogenesis through its regulation of glycolytic phenotype. We kinases (47). Initial studies identified that constitutively activated have also demonstrated that IKKe acts as a novel regulator of

7262 Cancer Res; 76(24) December 15, 2016 Cancer Research

IKKe Reprograms Glucose Metabolism in Pancreatic Cancer

Figure 7. Schematic representation of IKKe signaling in PDAC. IKKe promotes glycolytic metabolism and pancreatic tumor growth through its regulation of Akt/GSK3b/c-MYC axis. c-MYC is an important mediator of IKKe signaling, whose nuclear retention and stabilization is controlled by IKKe through a chain of events that include Akt activation, resulting in inhibitory phosphorylation of GSK3b and the escape of c-MYC from GSK3b-mediated nuclear efflux and subsequent degradation. Once stabilized, c-MYC activates multiple factors of glycolytic pathway with concomitant increase in glucose uptake and lactate efflux, leading to quick and efficient energy production to serve as prelude for synthesis of downstream molecules/factors involved in pancreatic tumor growth and metastasis.

c-MYC by promoting its nuclear retention and stabilization. c- Administrative, technical, or material support (i.e., reporting or organizing MYC stability is afforded by IKKe-mediated Akt activation, which data, constructing databases): J.E. Carter, A.P. Singh leads to phosphorylation-mediated inhibition of GSK3b, and, in Study supervision: A.P. Singh turn, promotes nuclear retention and increase in overall transcriptional activity (Fig. 7). Our results, thus, provide strong Acknowledgments rationale for further testing of IKKe as a novel molecular target We would like to thank Mr. Steven McClellan, Manager, Flow Cytometry Core at the USA Mitchell Cancer Institute for his assistance with flow cytometry. to counter PDAC progression and its therapeutic management. We also thank Ms. Barbara Putnam (USAMCI) for careful reading of the Disclosure of Potential Conflicts of Interest manuscript. No potential conflicts of interest were disclosed. Grant Support Authors' Contributions This work was supported in part by NIH grants (R01CA175772 and Conception and design: H. Zubair, S. Azim, S. Arora, A.P. Singh U01CA185490 to A.P. Singh). The laboratory of S. Singh is supported by NIH Development of methodology: H. Zubair, S. Azim, S. Singh, A.P. Singh grants (R01CA204801 and R03CA186223). Their laboratories also receive Acquisition of data (provided animals, acquired and managed patients, funding and resource support from the University of South Alabama Mitchell provided facilities, etc.): H. Zubair, S. Azim, S.K. Srivastava, M. Aslam Khan, Cancer Institute. G.K. Patel, E. Carter, S. Singh, A.P. Singh The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, payment of page charges. This article must therefore be hereby marked advertisement computational analysis): H. Zubair, S. Azim, S.K. Srivastava, A. Ahmad, in accordance with 18 U.S.C. Section 1734 solely to indicate A. Bhardwaj, M. Aslam Khan, G.K. Patel, J.E. Carter, S. Singh, A.P. Singh this fact. Writing, review, and/or revision of the manuscript: H. Zubair, S. Azim, S.K. Srivastava, A. Ahmad, A. Bhardwaj, M. Aslam Khan, G.K. Patel, S. Singh, Received June 17, 2016; revised September 19, 2016; accepted October 4, A.P. Singh 2016; published OnlineFirst October 20, 2016.

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7264 Cancer Res; 76(24) December 15, 2016 Cancer Research

Glucose Metabolism Reprogrammed by Overexpression of IKKε Promotes Pancreatic Tumor Growth

Haseeb Zubair, Shafquat Azim, Sanjeev Kumar Srivastava, et al.

Cancer Res 2016;76:7254-7264. Published OnlineFirst October 20, 2016.

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