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In Pancreatic Carcinoma Modern Pathology (2012) 25, 758–766 758 & 2012 USCAP, Inc. All rights reserved 0893-3952/12 $32.00 Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma Yeon Tae Chung1,3, Kristina A Matkowskyj1,3, Haonan Li1, Han Bai1, Wanying Zhang1, Ming-Sound Tsao2, Jie Liao1 and Guang-Yu Yang1 1Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA and 2Ontario Cancer Institute, Princess Margaret Hospital, Toronto, ON, Canada Aldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), and metabolizing these lipid substrates has a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohisto- chemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 overexpressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared with a normal pancreatic ductal epithelial cell (HPDE6E7), all of the six cultured pancreatic adenocarcinoma cell lines had an overexpression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in (1) increased cell apoptosis, (2) increased non-farnesyled HDJ2 protein and (3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation. Modern Pathology (2012) 25, 758–766; doi:10.1038/modpathol.2011.191; published online 6 January 2012 Keywords: AKR1B10; immunohistochemistry; pancreatic adenocarcinoma; prenylation; smoking Aldo-keto reductases (AKRs) are a gene superfamily with AKR1B1. AKR1B1 is globally expressed in that are monomeric, soluble, NADPH-dependent human tissues, whereas AKR1B10 is highly oxidoreductases that give functionality to the carbo- expressed in the small and large intestine as well nyl groups present on aldehydes and ketones to as the adrenal glands, and very low levels of expres- form alcohols in eliminating reactions. AKRs consist sion have been found in the liver. Expression of of 14 families, with the AKR1 family composed of AKR1B10 in the adrenal glands is consistent with its many isoforms. The AKR1B subfamily is a group of role in steroid metabolism and in the small and large aldolase reductases, and aldo-keto reductase family intestine for the detoxification of aldehydes within 1B10 (AKR1B10) shares 70% sequence homology the digestive tract.1,2 Overexpression of AKR1B10 has been identified in hepatocellular carcinoma and non-small cell lung carcinomas. Further studies Correspondence: Dr G-Y Yang, MD, PhD, Department of Patho- suggest that AKR1B10 is a unique biomarker of logy, Northwestern University, Feinberg School of Medicine, 303 smoking-related cancer and that AKR1B10 can be East Chicago Avenue, Ward 6-118, Chicago, IL 60611, USA. activated by tobacco-related carcinogenic transcrip- E-mail: [email protected] tion factors.3–5 As smoking is the most clearly 3These authors contributed equally to this work. Received 12 August 2011; revised 27 September 2011; accepted established risk factor for pancreatic cancer and is 27 September 2011; published online 6 January 2012 associated with KRAS mutations,6 it is reasonable to www.modernpathology.org AKR1B10 in pancreatic carcinogenesis YT Chung et al 759 postulate the AKR1B10 is upregulated in human groups in some anticancer drugs are converted by pancreatic cancer. AKR1B10 to their corresponding alcohols and that AKR1B10 exhibits more restricted substrate speci- this carbonyl reducing activity decreases the antic- ficity than that of general human aldo-ketoreduc- ancer drug’s effectiveness.22 tases; only farnesal, geranylgeranyl, retinal and The aims of the present studies are to determine carbonyls are its specific substrates.7–11 Metaboliz- whether the AKR1B10 expresses in human pancrea- ing these specific substrates has a crucial role in tic adenocarcinomas and what is its potential promoting carcinogenesis. The reduced products functional role in pancreatic carcinoma cells. Using of farnesal and geranylgeranyl are intermediates of immunohistochemical and western blot approaches cholesterol synthesis involved in protein prenyla- as well enzyme activity assay, we evaluated tion.12 Protein prenylation is a process of lipid AKR1B10 expression and enzyme activity in vitro modification, which involves the covalent addition and ex vivo human pancreatic adenocarcinoma of either farnesyl (15-carbon) or geranylgeranyl samples. To investigate the oncogenic role of (20-carbon) isoprenoids to C-terminal cysteines of AKR1B10 in pancreatic cancer, we further analyzed the target protein. This post-translational modifica- if knockdown of AKR1B10 expression via a siRNA tion allows the prenylated protein to localize to approach resulted in the reduction of protein prenyl- cellular membranes, aid in protein-protein inter- ation and the induction of cell apoptosis in pancreatic actions and exert its effects on a wide range of signal carcinoma cells overexpressing AKR1B10. transduction pathways, some of which include cell growth, differentiation, maintenance of the cellular cytoskeleton and vesicle trafficking.12–15 Prenylation Materials and methods appears to be a crucial event in carcinogenesis. Proteins that undergo prenylation include Ras and Cell Lines Ras-related GTP-binding proteins (G proteins), pro- Six human pancreatic adenocarcinoma cell lines tein kinases, fungal mating factors and nuclear (MiaPANC, Panc10.05, CAPAN-2, CAPAN-1, CD18 13,14 lamins. More than 95% of human pancreatic and Panc2.03) and the human hepatocellular carci- cancers carry a KRAS gene mutation and the KRAS noma (HepG2) cell line were obtained from ATCC 16 protein requires prenylation for its activity. Recent (Manassas, VA). The immortalized human pancrea- studies demonstrate that conversion of the protein– tic duct epithelial cell line (called HPDE6E6E7) was cysteine acceptor site in oncogenic Ras prevents established in Dr Ming-Sound Tsao’s lab (Ontario prenylation and abolishes its malignant transform- Cancer Institute; Ontario, Canada). All cell lines 17 ing ability. Many reports have demonstrated the were cultured in the appropriate medium obtained central role of Ras activation in pancreatic cancer, from Mediatech (Manassas, VA) containing 10% yet the role of AKR1B10 expression in protein fetal bovine serum (ThermoScientific, Waltham, prenylation in pancreatic cancer have not been MA) and maintained at 37 1C and 5% CO2. studied to date. With respect to retinal, AKR1B10 is the most effi- 7,9,18–20 cient retinal reductase. The conversion to Protein Extraction and Immunoblot Analysis retinol results in suppression of the conversion of retinal to retinoic acid—the major active anti-neoplas- Cultured cells representing normal and adenocarci- tic metabolite. Retinol can be reversibly transformed to noma were washed with 1 Â DPBS buffer, scraped retinal, which in turn is irreversibly oxidized to with sterile cell lifters and pelleted using a table-top retinoic acid. Retinoic acid is an important signaling centrifuge at 1000 rpm for 5 min. Cells were then molecule affecting cell differentiation. Several recent lysed on ice by vortexing every 5 min for 30 min studies have demonstrated that AKR1B10 can influ- using RIPA buffer containing 10 mM Tris (pH 7.4), ence carcinogenesis by controlling the retinoid signal- 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, 0.5% ing pathway.7,8,18 An increased level of AKR1B10 sodium deoxycholate, 0.1% SDS, 0.004% sodium expression in neoplastic cells results in an enzymatic azide, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1% reduction of retinal to retinol, a concomitant decrease phosphatase inhibitor cocktail 1 and 2 (Sigma, St in retinoic acid, and a shift in signaling from Louis, MO), 2% protease inhibitor cocktail and 1% differentiation to unopposed cellular proliferation.7,8,18 PMSF (Sigma). Membrane proteins from cultured With respect to carbonyl metabolism, conversion cells were extracted for detecting the GTP-bound/ of highly reactive aldehyde and ketone groups into active form prenylated KRAS protein (anchored hydroxyl groups by AKR1B10 in neoplastic cells on the membrane) using Mem-PER Eukaryotic protects these neoplastic cells against highly active Membrane Protein Extraction Reagent Kit (Thermo carbonyl-induced apoptosis. Recent data demon- Scientific, IL) according to manufacturer’s instruc- strates that AKR1B10 affects cell survival through tions. The extracts were collected by centrifugation modulating lipid synthesis, mitochondrial func- at 10 000 rpm for 3 min
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