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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. Ó2016 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 Ó2016 American Association for Cancer Research. pathobiology (10). 7254 Cancer Res; 76(24) December 15, 2016 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 20, 2016; DOI: 10.1158/0008-5472.CAN-16-1666 IKKe Reprograms Glucose Metabolism in Pancreatic Cancer 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 over- expression 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 (rab- bit 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
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