View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 989–994 Hypoxia increases cytoplasmic expression of NDRG1, but is insufficient for its membrane localization in human hepatocellular carcinoma Sonja Sibolda, Vincent Roha, Adrian Keogha, Peter Studera,Ce´line Tiffona, Eliane Angsta, Stephan A. Vorburgera, Rosemarie Weimannb, Daniel Candinasa, Deborah Strokaa,* a Department of Clinical Research, Visceral and Transplantation Surgery, University of Bern, Murtenstrasse 35, 3010 Bern, Switzerland b Institute of Pathology, University of Bern, Bern, Switzerland Received 20 November 2006; revised 22 January 2007; accepted 29 January 2007 Available online 7 February 2007 Edited by Vladimir Skulachev NDRG1 is a physiological substrate for serum- and gluco- Abstract NDRG1 is a hypoxia-inducible protein, whose modu- lated expression is associated with the progression of human can- corticoid-induced kinase 1 (SGK1) and glycogen synthase ki- cers. Here, we reveal that NDRG1 is markedly upregulated in nase 3 (GSK3) [20]. It is phosphorylated on several residues the cytoplasm and on the membrane in human hepatocellular within three 10-amino acid tandem repeats located near its carcinoma (HCC). We demonstrate further that hypoxic stress C-terminus. The expression of NDRG1 can be altered by var- increases the cytoplasmic expression of NDRG1 in vitro, but ious physiological conditions and external stimuli, in particu- does not result in its localization on the plasma membrane. How- lar, hypoxia is a key stimulus for its increased expression ever, grown within an HCC-xenograft in vivo, cells express [10,11]. Central to the regulation of hypoxia-inducible genes NDRG1 in the cytoplasm and on the plasma membrane. In con- is the heterodimeric transcription factor, hypoxia-inducible clusion, hypoxia is a potent inducer of NDRG1 in HCCs, albeit factor (HIF)-1 [12]. HIF-1 is negatively-regulated at a normal requiring additional stimuli within the tumour microenvironment oxygen concentrations, whereas under hypoxic conditions, its for its recruitment to the membrane. Ó 2007 Federation of European Biochemical Societies. Published alpha-subunit is stabilized and is translocated to the nucleus, by Elsevier B.V. All rights reserved. where it binds to its b-subunit and activates the transcription of its target genes [13–15]. Keywords: N-myc downstream-regulated gene 1; Hypoxia; Hypoxia is a common characteristic and a key stimulus in HIF-1; Carcinoma, hepatocellular the pathophysiology of many solid tumours including the highly-aggressive, chemoresistant hepatocellular carcinomas (HCCs) [16–18]. In the present study, we investigated the influ- ence of hypoxia on the expression and subcellular localization of NDRG1 and phosphorylated-NDRG1 in HCC. 1. Introduction The N-myc downstream-regulated gene-1 (NDRG1) is a no- 2. Materials and methods vel protein initially described as a stress-responsive protein and as being involved in cellular differentiation events [1–3]. Cur- 2.1. Tissue samples rent studies indicate that its putative biological function may Resected tissues were collected from patients at the Inselspital in be as a tumour suppressor protein, and more specifically as Bern, Switzerland in accordance with standard procedures which were approved by an Institutional Review Board (approval no.: an inhibitor of tumour metastasis [3–8]. Paradoxically, findings 1.05.01.30.À17) and with the patient’s written consent. HCC samples relating to the expression of NDRG1 are not always consistent were graded 1–3 according to Edmondson and Steiner and according with this tenet. NDRG1 is commonly found to be expressed at to the classifications of the World Health Organization (n = 11). Hepa- a higher level in cancerous than non-cancerous tissue of the tic tissue taken from tumour-free resection margins served as normal same origin, suggesting that the modulated expression of controls (n = 6). NDRG1 may be tissue- or cell-type specific [9]. 2.2. Antibodies, immuno-histochemistry and -fluorescence Antibodies were obtained from the following companies: NDRG1 and phosphorylated-NDRG1 (p3xThr 346, 356 and 366 ) (Kinasource Limited, Scotland, UK), Sp1 and IjBa (both from Santa Cruz, USA), E-cadherin (BD Biosciences), b-actin (Sigma) and HIF-1a (Novus Bio- logicals). Formalin fixed tissues sections (5-lm-thick) were pressure *Corresponding author. Fax: +41 31 382 4508. cooked for 15 min in ChemMateä Target Retrieval Solution (Dako- E-mail address: [email protected] (D. Stroka). Cytomation). Endogenous peroxidase was blocked with 3% H2O2 for 10 min and blocked with 1% normal rabbit serum (NRS). Sections Abbreviations: NDRG1, N-myc downstream regulated gene-1; HIF-1, were incubated overnight at 4 °C with the primary antibody and 0 hypoxia-inducible factor-1; HCC, hepatocellular carcinoma; O2, oxy- immunoreactivity was detected using ABC kits (Vectastain) and 3,3 - gen; siRNA, short-interfering RNA; DMOG, dimethyloxallyl glycine; diaminobenzidine as the peroxidase substrate or a FITC-conjugated GSK3, glycogen synthase kinase 3; SGK-1, serum- and glucocorticoid- secondary antibody was used for fluorescence microscopy. Cells were induced kinase 1 grown on glass cover slips, fixed in 4% PBS-buffered formaldehyde, 0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.080 990 S. Sibold et al. / FEBS Letters 581 (2007) 989–994 permeabilized in methanol for 1 min at À20 °C. Samples were exam- using the BioRad Protein Assay (BioRad). Fifty micrograms of protein ined with a Leica DMRB microscope and documented using IM50 were separated by SDS–PAGE, transferred to a nitrocellulose mem- Leica imaging software. brane and analyzed by Western blotting using standard protocols. Sig- nals were detected by enhanced chemiluminescence (LiteAblot, 2.3. Cells and culture conditions Euroclone). For patient sample analysis, the average of three indepen- The human HCC cell line, Hep3B, was purchased from the Euro- dent protein concentration measurements was used for as an accurate pean Collection of Cell Cultures and cultured according to suggested loading control, given that the expression of many housekeeping genes guidelines. Cells were incubated under either normoxic conditions are modified in cancerous tissues. (21% O2), using a standard CO2 incubator, or hypoxic conditions (1.5% O ), using a hypoxia workstation (Ruskinn Technology Limited, 2 2.5. Real-time RT-PCR West Yorkshire, UK). For a non-hypoxic stabilization of HIF-1a, the Total RNA was isolated with Trizol (Invitrogen) and 1 lg of total cells were incubated in 125 lM dimethyloxallyl glycine [(DMOG), RNA was reverse transcribed (Qiagen). Primers and FAM-labelled Alexis Biochemicals] for 24 h at 21% O . RNA oligonucleotides spe- 2 probes were obtained from the Assay-on-Demand for human NDRG1 cific for HIF cDNA were designed with the aid of an online tool and HIF-1a and quantitative PCR was performed using an ABI 7700 (www.ambion.com/techlib/misc/silencer_siRNA_template.html). Dou- Sequence Detection System (Applied Biosystems). The C values for ble-stranded short-interfering RNA (siRNA) was transfected into t each target genes were standardized against ribosomal RNA (18S). Hep3B cells using the supplied Transfection Reagent. The efficiency DDC values were calculated by subtracting the DC values of cells un- of RNA interference was determined by real-time PCR. t t der normoxia from the DCt value of cells under hypoxia. Fold in- creases were calculated using the formula 2ÀDDCt. All reactions were 2.4. Protein isolation and Western blot analysis performed in triplicate, and each experiment was conducted on at least For the detection of NDRG1, the total protein content of cells and three independent occasions. tissues was extracted using a RIPA lysis buffer and 1:100 dilution of a protease inhibitor cocktail (Sigma). For the detection of HIF-1a in cells and tissues, enriched nuclear–protein extracts were prepared as 2.6. Analysis of the NDRG1 sequence previously described [19]. For cell fractionation, the ProteoExtract The domain structure of NDRG1 (Accession No. NP_006087) was Subcellular Proteome Extraction kit (Calbiochem) was used in accor- determined using the Predict Protein database (http://www.predictpro- dance with the manufacture’s instructions. All proteins were quantified tein.org). Subcellular localization of NDRG4 (Accession No. A B C Normal Liver HCC HCC D E F NDRG1 HIF-1α Negative Control Normal G Liver HCC HIF-1α NDRG1 Fig. 1. Expression of NDRG1 and HIF-1a in HCCs by immunohistochemistry. (A–C) NDRG1 expression in (A) normal liver, arrow indicates a weak staining of NDRG1 in biliary cells, (B) cytoplasmic and membrane localization of NDRG1 in a HCC tumour (trabecular type, grade 3) and (C) membrane localization of NDRG1 in a HCC tumour (mixed trabecular and acinar type, grade 2). (D–E) Serial sections of a HCC tumour (mixed trabecular and acinar type, grade 2) for (D) cytoplasmic and membrane staining of NDRG1, (E) nuclear localization of HIF-1a, and (F) a negative control to illustrate the specificity of the reaction (200· magnification). (G) Western blot analysis of HIF-1a and NDRG1 expression in HCC compared to normal liver tissue. S. Sibold et al. / FEBS Letters 581 (2007) 989–994 991 AAH11795) was predicted from pTARGET database (http://bioinfor- 3. Results matics.albany.edu/~ptarget/). Sequence homology for NDRG1 and NDRG4 were determined using the SIM alignment tool for proteins 3.1. Expression and cellular localization of NDRG1 and HIF-1a (http://www.expasy.org/tools/sim-prot.html). in human HCC 2.7. Hep3B liver-xenograft model In normal liver tissue, NDRG1 protein was found to be Experiments were performed according to the National Institute of weakly expressed in the biliary epithelial cells, whereas the Health’s Guidelines for the Care and Use of Laboratory Animals and hepatocytes were negative (Fig. 1A). In contrast, the cells of with the approval of the local Animal Ethics Committee. Liver-tumour HCCs were highly immunoreactive (Fig.