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Nupr1-Aurora Kinase a Pathway Provides Protection Against Metabolic Stress-Mediated Autophagic-Associated Cell Death

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Nupr1-Aurora Kinase a Pathway Provides Protection Against Metabolic Stress-Mediated Autophagic-Associated Cell Death Published OnlineFirst August 16, 2012; DOI: 10.1158/1078-0432.CCR-12-0026 Clinical Cancer Human Cancer Biology Research Nupr1-Aurora Kinase A Pathway Provides Protection against Metabolic Stress-Mediated Autophagic-Associated Cell Death Tewfik Hamidi1, Carla E. Cano1, Daniel Grasso1, Maria Noe Garcia1, Maria Jose Sandi1, Ezequiel L. Calvo2, Jean-Charles Dagorn1, Gwen Lomberk3, Raul Urrutia3, Sandro Goruppi4, Arkaitz Carracedo5,6, Guillermo Velasco7,8, and Juan L. Iovanna1 Abstract Purpose: The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. Experimental Design: Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle–associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. Results: We showed that both Nupr1 and HIF1a are coexpressed in human pancreatic ductal adeno- carcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage–dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. Conclusions: Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress. Clin Cancer Res; 1–13. Ó2012 AACR. Introduction cases in the United States and 65,000 in Europe every year. The incidence of pancreatic ductal adenocarcinoma After years of research, we are now beginning to unveil the (PDAC) is increasing with more than 38,000 predicted new genetic determinants of premalignant lesions of PDAC. PDAC arises from precursor lesions known as pancreatic intraepithelial neoplasia (PanIN) and, less frequently, from Authors' Affiliations: 1INSERM U.624, Stress Cellulaire, Parc Scientifique 2 types of cystic tumors, the mucinous cystic neoplasms 2 et Technologique de Luminy, Marseille, France; Molecular Endocrinology (MCN) and the intraductal papillary mucinous neoplasms and Oncology Research Center, CHUL Research Center, Quebec City, Quebec, Canada; 3Laboratory of Epigenetics and Chromatin Dynamics, (IPMN). Recurrent genetic alterations associated with these Gastroenterology Research Unit, Departments of Biochemistry and Molec- lesions have been identified, including the mutational acti- ular Biology, Biophysics, and Medicine, Mayo Clinic, Rochester, New York; 4MCRI, Tufts Medical Center and Department of Medicine, Tufts University vation of the KRAS proto-oncogene, present in virtually all 5 School of Medicine, Boston, Massachusetts; CIC bioGUNE, Bizkaia cases (1), and loss-of-function mutations of the G1 cyclin– Technology Park, Derio; 6IKERBASQUE, Basque Foundation for Science; dependent kinase inhibitor INK4A/ADP ribosylation factor 7Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University; and 8Instituto de Investigacion Sanitaria del (ARF; 80% of cases), the SMAD4/DPC4 (50%), and the Hospital Clínico San Carlos (IdISSC), Madrid, Spain TP53 (50%) tumor suppressor genes (2). In turn, this Note: Supplementary data for this article are available at Clinical Cancer research has increased our knowledge of the genetic altera- Research Online (http://clincancerres.aacrjournals.org/). tions required for pancreatic cancer development. Never- Corresponding Author: Juan L. Iovanna, INSERM U.624, Stress Cellulaire, theless, the irreversibility of these mutations and absence 163 Avenue de Luminy, CP 915, 13288 Marseille Cedex 9, France. Phone: of drugs that would counteract their consequences has 33-491-828803; Fax: 33-491-826083; E-mail: [email protected] hindered the development of effective therapies. doi: 10.1158/1078-0432.CCR-12-0026 Surgery, the most effective treatment of pancreatic cancer Ó2012 American Association for Cancer Research. to date, results in a mean life expectancy of only 15 to 18 www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 16, 2012; DOI: 10.1158/1078-0432.CCR-12-0026 Hamidi et al. endogenous peroxidase activity using standard conditions. Translational Relevance Then, slides were incubated with primary antibody diluted In this work, we showed that both Nupr1 and HIF1a in phosphate saline buffer. Antibodies were revealed using are coexpressed in human pancreatic ductal adenocar- the avidin–biotin peroxidase complex method (Dako) fol- cinoma (PDAC) samples and negatively correlate with lowing recommendation of the manufacturer and counter- survival time, and therefore could be used as prognosis stained with hematoxylin. Finally, slides were dehydrated, markers. In addition, we also showed that PDAC cells cleared, and mounted. HIF1a antibody (Bethyl) was used at become resistant to the metabolic stress by activating an 1:100 dilution and the monoclonal anti-Nupr1 (home- efficient intracellular defense pathway involving a made) at 1:200. Nupr1-dependent AURKA activation pathway. These findings pointed at a strong protumoral function of Cell culture AURKA and, in consequence, at the potential efficacy MiaPaCa2, Panc1, and BxPC-3 cells were obtained from of inhibitors of AURKA to treat cancers. Such a rationale the American Type Culture Collection and maintained in was based on the effect of AURKA on mitosis, and Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) data from our study provide an additional argument for supplemented with 10% FBS at 37 C with 5% CO2. To avoid its targeting. We suggest that inhibition of AURKA will any supplementary stress, all media were preheated at 37C sensitize preferentially intrapancreatic tumor cells locat- before rinsing or changing media. Glucose starvation was ed near hypovascularized regions, which should corre- obtained by cultivating cells with DMEM (no glucose) spond to the most resistant cells because they have been (Invitrogen, Ref# 11966) supplemented with 10% FBS. selected by exposure to an adverse microenvironment, Hypoxia experiments were carried out using C-Shuttle Glove and also to the areas of the tumor in which the anticancer Box coupled hypoxia chamber (BioSpherix). Nupr1-defi- drug results are less accessible. cient and Nupr1-restored mouse embryonic fibroblasts (MEF) were previously reported (13). N-acetyl-L-cysteine (NAC; Sigma–Aldrich) was used at 15 mmol/L. months in the 15% to 20% of patients who harbor resect- Plasmid transfection able tumors. Moreover, in the fraction of patients who pCS2-Myc and pCS2-Myc-Aurka expression vectors were qualify for surgery, chemotherapy/radiotherapy–resistant kindly given by Dr. Paolo Sassone-Corsi (Department of metastases often arise after surgical resection of the primary Biological Chemistry, University of California, Irvine, tumor. The resistance to chemo and radiotherapy is thought Irvine, CA; ref.14). MiaPaCa2 cells were seeded a day to be associated both with the intrinsic nature of pancreatic before transfection using Lipofectamine2000 reagent (Invi- cancer cells (3) and with the abundant fibrotic stroma of the trogen), according to the manufacturer’s protocol. tumors, which favors rapid tumor progression and creates a physical barrier preventing drug delivery and immune cell siRNA transfection infiltration (4, 5). In addition to the importance of fibrotic Cells were plated at 70% confluence in 100 mm dishes. stroma, PDAC histolopathology is characterized by poor Nupr1, Atg5, Beclin 1, and AURKA were knocked down vascularization indicating that pancreatic cancer cells must using 140 ng of specific siRNA. Scrambled siRNA targeting cope with an adverse microenvironment with nutrient and no known gene sequence was used as negative control. oxygen shortage. However, the mechanism underlying the INTERFErin reagent (POLYPLUS transfection) was used to resistance to such metabolic stress remains obscure. The conduct siRNA transfection according to the manufacturer’s limited supply of oxygen and nutrients is thought to result protocol. The sequences of the siRNA used are shown in in rigorous selection of cells that will eventually form the Supplementary Table S1A. tumor. Nupr1 is a stress response protein systematically DNA microarray overexpressed in pancreatic cancer and associated with bad Total RNA (15 mg) was isolated and reverse transcribed prognosis (6, 7). Nupr1 regulates TGF-b responsiveness, for hybridization to the human oligonuleotide array U133 inhibits apoptosis, and promotes cancer cell resistance to Plus 2.0 (Genechip, Affymetrix) as described
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