Proteomic Identification of Aldo-Keto Reductase AKR1B10 Induction After Treatment of Colorectal Cancer Cells with the Proteasome Inhibitor Bortezomib

Published OnlineFirst June 30, 2009; DOI: 10.1158/1535-7163.MCT-08-0987

1995

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Judith Loeffler-Ragg,1 Doris Mueller,2 study shows for the first time a bortezomib-induced up- Gabriele Gamerith,1 Thomas Auer,1 regulation of AKR1B10. Small interfering RNA–mediated Sergej Skvortsov,1 Bettina Sarg,3 Ira Skvortsova,4 inhibition of this enzyme with known intracellular detoxi- Klaus J. Schmitz,6 Hans-Jörg Martin,7 fication function sensitized HRT-18 cells to therapy with Jens Krugmann,5 Hakan Alakus,8 Edmund Maser,7 the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors,immunohisto- Jürgen Menzel,2 Wolfgang Hilbe,1 Herbert Lindner,3 6 1 chemical expression was shown in 23.2% of 125 tumor Kurt W. Schmid, and Heinz Zwierzina specimens. These findings indicate that AKR1B10 might Departments of 1Internal Medicine, 2Medical Genetics, be a target for combination therapy with bortezomib. Molecular and Clinical Pharmacology, 3Clinical Biochemistry, [Mol Cancer Ther 2009;8(7):1995–2006] 4Radiotherapy-Radiooncology,and 5Pathology,Innsbruck Medical University,Innsbruck,Austria; 6Department of Pathology and Neuropathology,Essen University Hospital,Duisburg-Essen Introduction 7 University,Essen,Germany; Institute of Toxicology and Colorectal cancer is a leading cause of cancer death through- Pharmacology for Natural Scientists,University Medical School Schleswig-Holstein,Kiel,Germany; and 8Department of Visceral out the world (1). Although chemotherapy has improved and Vascular Surgery,Cologne University,Cologne,Germany the outcome of patients with metastatic disease, new thera- pies with novel mechanisms of action are warranted. Major molecular pathways involved in the pathogenesis of colo- Abstract rectal carcinoma such as β-catenin, Smad4, p53, and nuclear Targeting the ubiquitin-proteasome pathway with the pro- factor κB (NF-κB) downstream of Ras are regulated by the teasome inhibitor bortezomib has emerged as a promising ubiquitin-proteasome system providing a rationale for pro- approach for the treatment of several malignancies. The teasome inhibition (2). The 26S proteasome, a large multica- cellular and molecular effects of this agent on colorectal talytic protease complex, degrades several regulatory cancer cells are poorly characterized. This study investi- proteins and inhibitors after their ligation with the protein gated the antiproliferative effect of bortezomib on colorec- ubiquitin and mediates various cellular functions such as tal cancer cell lines (Caco-2 and HRT-18). In order to transcription, stress response, cell cycle regulation, apopto- define the proteins potentially involved in the mechanisms sis, ribosome biogenesis, cellular differentiation, DNA re- of action,proteome profiling was applied to detect the pair, and oncogenesis. Its involvement in malignancy and proteins altered by bortezomib. The in vitro efficacy of the finding that it regulates the key antiapoptotic transcrip- bortezomib as a single agent in colorectal cancer cell lines tion factor NF-κB has formed the basis for the exploration of was confirmed. Proteome profiling with two-dimensional proteasome inhibition as an antineoplastic strategy (3). PAGE followed by mass spectrometry revealed the up- Bortezomib (Velcade, formerly known as PS-341) is a nov- regulation of the major inducible isoform of heat shock el dipeptide boronic acid that reversibly inhibits the chymo- protein 70 (hsp72) and lactate dehydrogenase B in both tryptic activity of the proteasome (4). Preclinical studies cell lines,as well as the induction of aldo-keto reductase have shown that this compound decreases proliferation, in- family 1 member B10 (AKR1B10) in HRT-18 cells. Both duces G2-M arrest and apoptosis, enhances the activity of AKR1B10 and hsp72 exert cell-protective functions. This chemotherapy or radiation, and reverses chemoresistance in a variety of hematologic and solid malignancy models. Bortezomib is already approved for the treatment of pa- Received 10/16/08; revised 4/15/09; accepted 5/4/09; published OnlineFirst tients with relapsed or refractory multiple myeloma and 6/30/09. has shown activity in solid tumors (5). In colorectal cancer, Grant support: Austrian National Bank (project no. 11681), the German preclinical testing with bortezomib has shown synergy with Research Council (DFG-MA 1704/5-1), and the Hans and Blanca Moser-Stiftung (J. Loeffler-Ragg). irinotecan or ionizing radiation and resulted in significant – The costs of publication of this article were defrayed in part by the tumor regression in xenograft models (6 8). However, the payment of page charges. This article must therefore be hereby marked results of clinical efficacy are very limited and biomarkers advertisement in accordance with 18 U.S.C. Section 1734 solely to related to response or resistance are lacking (9). indicate this fact. Recently, proteomic technologies evolved as a useful Requests for reprints: Judith Loeffler-Ragg, Department of Internal Medicine I, – Anichstrasse 35, 6020 Innsbruck, Austria. Phone: 43-512-504-23255; strategy to track biological responses to therapy (10 12). Fax: 43-512-504-24209. E-mail: [email protected] With this method, hundreds to thousands of proteins are Copyright © 2009 American Association for Cancer Research. displayed at the same time and dynamic changes in re- doi:10.1158/1535-7163.MCT-08-0987 sponse to drug therapy can be detected. To define proteins

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that are up-regulated or down-regulated after therapy with the manufacturer (Annexin V FITC Apoptosis Detection bortezomib, we created proteome profiles of the colorectal Kit; BD PharMingen). Corresponding cell culture assays cancer cell lines, HRT-18 and Caco-2, using two-dimensional were prepared in a six-well plate format (150,000 cells/well). PAGE (13). The highly differentiated Caco-2 cell line (dou- Two-dimensional PAGE bling time, 80 hours) and the poorly differentiated HRT-18 HRT-18 and Caco-2 cells (5 × 106) were seeded in T75 cell line (doubling time, 20 hours) are characterized by high flasks and analyzed with two-dimensional PAGE 6, 12, 24, epidermal growth factor receptor expression and a mutation and 48 h after treatment with 0.03 μmol/L of bortezomib. status with mutant APC/mutant p53/wild-type KRAS/ Controls were taken at times 0 and 24h. For sample prep- wild-type BRAF for Caco-2 and mutant APC/mutant p53/ aration, cells were scraped, harvested, and lysed as previ- mutant KRAS/wild-type BRAF for HRT-18 (11, 14–16). Dif- ously described (11). ferential analysis before and after therapy revealed three For first-dimension isoelectric focusing, 100 μg of protein proteins specifically up-regulated. Our data suggest that per sample diluted to 450 μL with rehydration buffer proteome-based technologies can reveal proteins influ- (7 mol/L urea, 2 mol/L thiourea, 1% ASB-14, 0.5% IPG enced by proteasome inhibition and help to further eluci- buffer, and 60 mmol/L DTT) were loaded on immobilized date the complex mode of action. 24cm (pH 3 –10) NL gradient strips (Amersham Biosciences). Active rehydration (50 V) was carried out at 20°C for 12 h. Isoelectric focusing was done at 250 V for 30 min, 500 V for Materials and Methods 1 h, 2,000 V for 1 h, and finally,at 8,000 Vuntil 55,000 V/h were Cell Cultures reached in total. The colorectal cancer cell lines Caco-2 and HRT-18 were For the second dimension, samples were separated on purchased from American Type Culture Collection. Caco-2 12.5% polyacrylamide gels with the Ettan DALTtwelve Sys- was grown in MEM with Earle's salts (PAA Laboratories) tem following the standard procedures recommended by supplemented with 2 mmol/L of L-glutamine, 1.5 g/L of the manufacturer (Amersham Biosciences). After electro- sodium bicarbonate, 0.1 mmol/L of nonessential amino phoresis, gels were silver-stained, scanned with ImageScan- acids, 50 units/mL of penicillin, 50 μg/mL of streptomycin, ner, and analyzed with ImageMaster 2D Platinum software and 10% FCS (v/v; Sigma-Aldrich). HRT-18 cells were (Amersham Biosciences). To obtain a standard gel for each maintained in RPMI 1640 (PAA Laboratories) containing individual cell line, spot detection and matching were done 2 mmol/L of L-glutamine, 50 units/mL of penicillin, using gels from three runs. These standard gels were then 50 μg/mL of streptomycin, and 10% FCS (v/v). Cultures matched to yield information about differentially expressed were incubated in a 5% CO2-humidified atmosphere. proteins. We selected spots overexpressed or underex- Drug Sensitivity Assays pressed by more than 4-fold to exclude artifacts caused by Bortezomib resolved in 0.9% NaCl was obtained from the gel-to-gel variation observed with silver staining, and then institutional pharmacy. Drug sensitivity testing was done checked spots by eye in order to avoid possible software er- using WST-1 assay and direct cell count for Caco-2 and rors (13, 18). HRT-18 cells, at different times (24, 48, 72, and 96 h) and Protein analysis was done as previously published (11). doses (range, 0.01–0.1 μmol/L) of bortezomib. These con- Three protein digests were separated using capillary high- centrations are within those achieved clinically in the performance liquid chromatography connected online to plasma of patients treated with bortezomib (17). To screen an LCQ ion trap instrument (ThermoFinnigan) equipped for IC50 values, an indirect measure of cell viability and with a nanospray interface. Tandem mass spectra were proliferation was done by determination of the metabolic searched against a human database using SEQUEST (LCQ activity of cells with a colorimetric assay based on cleav- BioWorks; ThermoFinnigan). age of the tetrazolium salt WST-1 by mitochondrial dehy- Verification of hsp72 and AKR1B10 Expression Profile drogenases (cell proliferation reagent WST1; Roche by Western Blot Diagnostics). For this assay, cells were seeded in 96-well HRT-18 and Caco-2 cells were lysed and processed as de- plates (5,000 cells/well) and allowed to adhere for 24h. scribed previously (11). For each lane, 40 μg of protein was On day 2, the medium was removed and replaced with separated by 12% SDS-PAGE and electroblotted onto nitro- medium containing bortezomib at the doses and times cellulose membranes (Schleicher & Schuell), incubated for mentioned above. 1 h at room temperature in blocking buffer containing rabbit Direct cell count and viability of bortezomib-treated cells anti-hsp70 (hsp72) polyclonal antibody (Stressgen Bio- was assessed 48 h after exposure to bortezomib (0.01–0.08 technologies), or AKR1B10 polyclonal antibody (A01; Abno- μmol/L) using hemocytometer and trypan blue dye exclu- va Corporation). Immunodetection was done with an sion test (Sigma-Aldrich) according to the protocol of the enhanced chemoluminescence system (Amersham Bio- manufacturer in a six-well plate format (150,000 cells/well). sciences) after incubation with horseradish peroxidase– To determine the percentage of cells that are actively un- conjugated antimouse or antirabbit secondary antibody dergoing apoptosis 48 h after incubation with bortezomib (Amersham Biosciences). (0.02–0.05 μmol/L), Annexin V FITC and propidium NF-κB Western Blot Analysis iodide–stainedcellsweresubjectedtoflowcytometry The predominant form of NF-κB exists as a heterodimeric (Becton Dickinson) according to the recommendations of complex of 50 kDa and 65 kDa protein subunits. In its

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inactive form, NF-κB is sequestered in the cytoplasm. Acti- or negative siRNA control (30 nmol/L) and incubated for vated NF-κB is free to translocate to the nucleus and induces 24h to trigger AKR1B10 silencing. Subsequently, cells were the expression of proinflammatory and antiapoptotic genes. treated with 0.03 μmol/L of bortezomib. After 24h of treat- Isolation of nuclear and cytoplasmic fractions was done ac- ment, cells were harvested, washed twice with PBS, stained in cording to the procedure described by Wang et al. (19). 500 μL of DNA staining solution (50 μg/mL propidium Western blotting for NF-κB was done as described above us- iodide, 0.1% sodium citrate, and 0.1% Triton X-100), incubat- ing p65-specific antibodies (Cell Signaling Technology, Inc.). ed in the dark for 30 min and kept at 4°C until FACS analysis. For a positive control, tumor necrosis factor-α, known to in- Nuclear DNA content was determined by analysis of propi- duce NF-κB, was applied (10 ng/mL, 2 h). Other samples dium iodide–stained nuclei, reflecting the fraction of cells at were treated with bortezomib (0.03 μmol/L), and transloca- each point in the cell cycle (G0-G1, S, and G2-M, FACSCAli- tion of p65 was assessed 3 and 6 h after treatment. bur; Becton Dickinson). Results were represented as DNA AKR1B10 Silencing by Small Interfering RNA Transfection histograms using CellQuest software (Becton Dickinson). Two specific small interfering RNAs (siRNA) targeted to AKR1B10 Enzymatic Activity Assay 3′-untranslational [siRNA1, 5′CGAGAAUCGAGGUGCU- The carbonyl reduction activity of AKR1B10 was moni- GUUtt; according to Yan et al. (20)] and to encoding tored spectrophotometrically by measuring the decrease in (siRNA2, exon 2, 5′GUGCCUAUGUCUAUCAGAAtt) the absorbance of the cofactor NADPH at 340 nm. Prepara- regions of AKR1B10 were chemically synthesized (Ambion). tion of the recombinant enzyme and assay conditions were For negative control, the AllStars Negative Control siRNA done as previously described by coauthor Martin and col- and for a FITC-conjugated transfection control, the Negative leagues (22). Bortezomib was added in concentrations up Control siRNA AF488 was used (Qiagen). For siRNA to 1 mmol/L. delivery, 5,000 HRT-18 cells in complete RPMI medium were Patient Samples seeded in 96-well plates on top of the siRNA-HiPerFect This study was comprised of 125 human consecutive co- Reagent transfection complexes for reverse transfection, lorectal cancer patients, who underwent surgery in the according to the instructions of the manufacturer (Qiagen). years 1996 to 1998 according to the recommendations of Transfected cells were incubated under their normal growth the German Society of Surgery. Complete clinical records conditions (37°C, 5% CO2). Gene silencing was monitored and follow-up information were available in all cases. The after 24to 96 h of incubation with real-time reverse transcrip- minimum follow-up period for patients still alive was tase PCR (RT-PCR) analysis. The effects of combination 60 months. Surgical material was fixed in formalin and rou- therapy with bortezomib were studied with drug sensitivity tinely processed. The tumors were classified according to assays (see above) and cell cycle analysis. Morphologic the Tumor-Node-Metastasis System (6th edition) and histo- changes were characterized by phase contrast microscopy pathologic diagnosis was made according to the WHO clas- of cultured cells (Olympus IX70 inverted tissue culture sification of tumors of the rectum and colon (23). Patients microscope, Olympus SC35 camera). with International Union Against Cancer stages III and IV RT-PCR Analysis colorectal carcinoma received standardized chemotherapy cDNA synthesis was carried out directly with cell lysates in the adjuvant setting. According to the recommendations (5 μL) prepared from cultured HRT-18 cells plated in 96-well of the German Surgical Oncology Working Group (CAO), plates at a density of 5 × 103 cells per well (control, negative patients with stages I and II colorectal carcinoma were not siRNA, siRNA1, and siRNA2) using the FastLane Cell Mul- advised to undergo any adjuvant treatment. tiplex kit (Qiagen). Subsequently, RT-PCR assays were done Tissue Microarray Construction on an ABI Prism 7000 Sequence Detection System using the Vital tumor areas were selected and marked on the re- QuantiTect Probe RT-PCR kit (Qiagen). For AKR1B10 ex- spective H&E-stained slides. The most representative tumor pression determination, we used the FAM-labeled expres- area was carefully selected and marked on each H&E slide. sion assay (Applied Biosystems) spanning exons 3 and 4. In the case of tumor heterogeneity, areas with the lowest de- The control housekeeping gene was human β-actin and gree of differentiation were selected. The tissue microarrays measured with a VIC-labeled expression assay (Applied were constructed using a manual tissue-array instrument Biosystems). Both were analyzed in the same setup. The (Beecher Instruments). Three 0.6-mm-thick tissue cores were variables for PCR amplification were 95°C for 10 min for taken from each colorectal carcinoma specimen. One section activation and 40 cycles at 95°C for 15 s, and 60°C for from each tissue microarray was stained with H&E. Each 1 min. Relative expression of mRNA was determined from block contained normal colon tissue as controls. the point at which each amplification curve crossed the AKR1B10, Cyclooxygenase-2, and Phosphorylated threshold (CT) line. AKR1B10 expression was normalized AKT Immunohistochemistry β to CT values of the housekeeping gene -actin using the To confirm the specificity of the AKR1B10 antibody (mono- −ΔΔ comparative threshold cycle method 2 CT and expressed clonal rabbit anti-human AKR1B10 antibody, clone 1A6; Ab- as a percentage of expression in cells transfected with nega- nova Corporation), and to obtain positive controls, cell tive siRNA (21). pellets of the colorectal cell lines Caco-2 and HRT-18 were Cell Cycle Analysis made. Caco-2 cells and HRT-18 cells (5 × 106) were seeded HRT-18 cells (300,000) were seeded on six-well plates, in T75 flasks, untreated control cells and bortezomib-treated transfected with siRNA1 (30 nmol/L), siRNA2 (10 nmol/L), HRT-18 cells (0.03 μmol/L) were removed from the flasks by

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trypsinization and pelleted in 1× PBS 48 h after incubation. obtained by WST-1 colorimetric assay ranged from 0.03 to The pellets were fixed with 4% formaldehyde, placed in filter 0.04 μmol/L in Caco-2, and from 0.04to 0.06 μmol/L in paper and in a cassette, and routinely processed for paraffin HRT-18 cells (Fig. 1A). For proteome studies, we intended embedding. Immunohistochemistry was done on 5-μm-thick to define a bortezomib concentration with antiprolifera- paraffin sections of cell pellets, or tissue microarray and an- tive but low apoptotic effect. Analysis of cell viability tigen retrieval was carried out with 0.01 mol/L of citrate buff- with trypan blue dye exclusion method 48 hours after er (pH 6.1) for 20 min in a hot water bath (95°C). The primary therapy showed a dose-dependent increase of dead cells, ∼ μ antibody was incubated for 30 min at 1:300 dilution. Detec- reaching LC50 earlier in Caco-2 ( 0.05 mol/L) than in tion was achieved with the Zyto-Chem-Plus HRP kit (Zy- HRT-18 cells (∼0.08 μmol/L; Fig. 1B). At this time point, tomed). Replacement of the primary antibodies with mouse Annexin V FITC/propidium iodide FACS analysis re- immunoglobin served as negative control. Cyclooxygenase-2 vealed apoptosis induction with a dose-dependent in- (COX-2) and phosphorylated Akt immunohistochemistry crease of Annexin V FITC–positive cells (Fig. 1C). Based were done as previously described (24, 25). on these results, proteome studies were done with a bor- Ki67 Immunohistochemistry and Terminal Deoxyri‐ tezomib concentration of 0.03 μmol/L. bonucleotide Transferase–Mediated dUTP Nick End Identification of Proteins Using Two-dimensional PAGE Labeling In order to identify proteins with altered expression le- Ki67 immunohistochemistry was done as reported recent- vels after therapy of HRT-18 and Caco-2 cell lines, the ly (24). The growth fraction was defined as the percentage protein profiles of cell extracts were examined by means of Ki67-positive randomly chosen nuclei per 600 tumor of two-dimensional PAGE before and 6 to 48 hours after cells. In situ DNA fragmentation was established using the treatment with 0.03 μmol/L of bortezomib. As mentioned terminal deoxyribonucleotide transferase–mediated dUTP above, this concentration has a predominant antiprolifera- nick end labeling technique in paraffin-embedded sections. tive effect (G2-M arrest, but low apoptotic fraction; Figs. 1 We used the ApopTag Plus Peroxidase In situ Apoptosis De- and 5D). Silver-stained gels were analyzed with Image tection kit (Intergen Company) according to the recommen- Master 2D Platinum and differentially expressed spots dations of the manufacturer. with a ratio of >4were identified using mass spectrome- Statistical Analysis try in conjunction with the SWISS 2D-PAGE protein data- For statistical evaluation of cell culture experiments, bases to assign identities and accession number. Two mean values and SD were calculated from three indepen- protein spots differed more than 4-fold in expression dent experiments and compared with Student's indepen- in both cell lines: heat shock protein 70 kDa 1 (also dent samples t test using SPSS 15.0 software (SPSS, Inc.). known as hsp70.1, hsp70i, and hsp72) and lactate Significance was defined as P < 0.05. dehydrogenase B (LDHB). In addition, qualitative analysis For patient studies, we analyzed a possible relation be- showed one protein spot exclusively present in HRT- tween AKR1B10 expression with clinicopathologic vari- 18 cells with marked up-regulation after therapy (>4-fold). ables, the Ki-67 labeling index, apoptotic rate, COX-2, and Mass spectrometric analysis identified this protein pAKT expression, as recently reported by coauthor Schmitz as aldo-keto reductase family 1 member B10 (AKR1B10; and colleagues for this patient collective (24). All data were Figs. 2 and 3). statistically analyzed using the SPSS 15.0 software (SPSS). Confirmation of Bortezomib-Induced hsp72 and Relationships between ordinal variables were investigated AKR1B10 Expression using a two-tailed analysis (or Fisher's exact test if the num- In both colorectal cancer cells, treatment with 0.03 μmol/L ber of patients was small). The relationship between cate- of bortezomib increased the level of hsp72 as detected by gorical data and numerical data was determined using the two-dimensional PAGE (Fig. 3A). Western blot analysis con- Kruskal-Wallis test or the Mann-Whitney test, depending on firmed the up-regulation of the stress-inducible form of the number of groups. For survival analysis, only patients hsp70 (hsp72). This effect appeared after 6 hours of treat- who died from colorectal cancer were included in statistical ment, increased progressively with time, and declined after analysis. Disease-specific overall survival curves were esti- 24hours (Fig. 3B). mated using the Kaplan-Meier method, and any differences According to two-dimensional PAGE profiles, up- in the survival curves were compared using the log-rank regulation of AKR1B10 expression occurred in HRT-18 test. Cox regression analysis was done for multivariate sur- cells after treatment with bortezomib (0.03 μmol/L). This vival analysis. Ninety-five percent confidence intervals were cell line constitutively expressed AKR1B10 (Fig. 3A). West- used throughout. ern blot analysis verified an increase in the amount of this enzyme after 6 hours to maximum levels obtained 48 hours after treatment. In the Caco-2 cell line, AKR1B10 Results was not expressed (Fig. 3B). Sensitivity ofCaco-2 and HRT-18 Cells to Bortezomib Involvement ofNF- κB in Bortezomib-Induced The colorectal cancer cell lines Caco-2 and HRT-18 were AKR1B10 Expression exposed for 24to 96 hours to various concentrations of Because of an association observed between NF-κB acti- bortezomib (0.01–0.1 μmol/L), revealing a dose- vation and up-regulation of aldose reductase, another mem- dependent and time-dependent response. The IC50 values ber of the aldo-keto reductase family, we examined a

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Figure 1. Effects of continuous treatment with bortezomib on cell viability and cell death of Caco-2 and HRT-18 cell lines. A, dose-response curves were established by a WST-1–based colorimetric assay 24 to 96 h after drug incubation (0.01–0.1 μmol/L) and cell viability is expressed as a percentage of metabolic activity relative to control (points, mean of three experiments; bars, SD). B, viable and nonviable cell densities at the 48-h time point were determined by trypan blue dye exclusion. The amount of unstained (viable) and trypan blue–positive cells (dead) shown as a percentage of total cell count for bortezomib (0.01–0.08 μmol/L). C, analysis for apoptosis was done 48 h after treatment with bortezomib (0.02–0.05 μmol/L) using Annexin V FITC/ propidium iodide FACS analysis. B and C, columns, mean of three experiments; bars, SD; PI, propidium iodide; pos, positive. potential involvement of NF-κB induction in up-regulation to the nucleus. In the positive control, after treatment with the of AKR1B10 (26). HRT-18 cells were separated into cytosolic known NF-κB–inducer tumor necrosis factor-α (10 ng/mL, and nuclear fractions and translocation of NF-κB(p65) 2 hours), translocation of p65 to the nucleus occurred (Fig. 4). protein was analyzed by Western blot analysis using Influence of Anti-AKR1B10 siRNA on the Sensitivity of p65-specific antibodies. The untreated sample as well as HRT-18 Cells to Bortezomib bortezomib-treated samples (0.03 μmol/L) showed only the siRNA Transfection and Silencing Effects. Two siRNAs, presence of inactive, cytosolic NF-κB without translocation targeted to the 3′ untranslational (siRNA1) and encoding

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(siRNA2) regions of AKR1B10 mRNA were used to confirm Effects of Combined Treatment the specificity of the AKR1B10 knockdown. Transfection After successful transfection, we investigated whether with a nontargeting fluorescent control siRNA indicated the silencing of AKR1B10 mRNA affected susceptibility to that 86.5% (±3.5%) of HRT-18 cells had been transfected (ra- bortezomib in the HRT-18 cell line. In pilot experiments, tio of transfection reagent to culture medium, 1:266.7). As siRNA-transfected and siRNA-untransfected cells were monitored by RT-PCR analysis, both siRNA1 and siRNA2 exposed to bortezomib (0.03 μmol/L; the concentration induced specific AKR1B10 down-regulation in HRT-18 used for proteome and Western blot studies), and effects cells as compared to transfection with a nontargeting have been studied with WST-1 assay, phase-contrast light siRNA-negative control and to cells incubated with a microscopy, and cell cycle analysis. Drug treatment was un- transfection reagent without siRNA (Fig. 5A). Expression dertaken 24hours after siRNA transfection, so that maxi- of AKR1B10 was normalized to the housekeeping gene mum silencing of AKR1B10 occurred 48 hours after drug −ΔΔ β-actin using the 2 CT comparative method, and data delivery. After therapy with 0.03 μmol/L of bortezomib, were expressed relative to cells transfected with negative metabolic activity of specifically transfected cells was re- control siRNA. In the presence of 30 nmol/L of siRNA1 duced to 23.45 ± 4.37% (siRNA1) or 32.67 ± 11.11% (siRNA2) or 10 nmol/L of siRNA2, the relative expression of after 48 hours of incubation, and to 40.85 ± 3.84% (siRNA1) AKR1B10 was reduced and maximum silencing occurred or 17.64± 8.71% (siRNA2) after 72 hours of incubation, as 72 hours after siRNA delivery (74.7 ± 9.3% for siRNA1 compared with control cells (incubated only with transfec- and 78.2 ± 4.5% for siRNA2). tion reagent; Fig. 5B). Phase-contrast light microscopy

Figure 2. Localization and identification number of proteins altered after therapy with bortezomib (0.03 μmol/L) on a representative silver-stained two-dimensional PAGE gel of the HRT-18 cell line. Accession number, molecular weight, and sequence coverage are listed (for differential expression, see Fig. 3A).

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Figure 3. Induction of hsp72 and AKR1B10 after treatment with bortezomib (0.03 μmol/L) in colorectal cancer cell lines. A, proteome analysis with two-dimensional PAGE revealed the up-regulation of hsp72 6 h after treatment in HRT-18 and Caco-2 cell lines and up-regulation of AKR1B10 12 h after treatment in HRT-18 cells. B, Western blot analysis confirmed time-dependent induction of the stress-inducible isoform of hsp72 in HRT-18 and Caco-2 cells and time-dependent induction of AKR1B10 in HRT-18 cells and also in the studied colorectal cancer cell line WiDr (C).

images of these cells showed profound morphologic changes of dose-response curves revealed a significantly lower 72 hours after therapy. Whereas drug-naïve cells reached IC50 with the presence of anti-AKR1B10 siRNA [IC50 with confluency, bortezomib-treated cells showed reduced densi- siRNA1 and siRNA2, 0.015 μmol/L; P =0.02(siRNA1), ty and an increase in cell size. Cells with combined treatment P = 0.01 (siRNA2); Fig. 5E]. Both siRNAs sensitized HRT- presented cell islets with large single cells (Fig. 5C). An ex- 18 cells to low doses of bortezomib (0.01–0.03 μmol/L) with emplary analysis to study effects at the 96-hour time point displayed a decrease of viable cells (trypan blue negative) of 65.5% with combined therapy (siRNA1/bortezomib, 0.03 μmol/L) compared with monotherapy (bortezomib, 0.03 μmol/L + transfection reagent; mean value of viable cells ± SD siRNA1/bortezomib, 455,000 ± 45,000; bortezomib, 0.03 μmol/L + transfection reagent, 1,096,666.7 ± 73,181.6; P = 0.005). Cell cycle analysis revealed an increase in cells in G2- M phase 24hours after therapy with 0.03 μmol/L of bortezomib in transfected and untransfected HRT-18 cells (Fig. 5D). At this time point, the amount of dead cells detectable by means of sub-G1 assay accounted μ for <5%. Figure 4. Effects of treatment with bortezomib (0.03 mol/L) on the activation of NF-κB. After separation of cells into cytosolic To extend these observations, dose-response curves of and nuclear fractions, translocation of NF-κB (p65) protein was an- anti-AKR1B10 siRNA transfected and untransfected borte- alyzed by Western blot analysis using p65-specific antibodies. Translocation of p65 to the nucleus was visible only in the positive zomib-treated HRT-18 cells were done with direct cell control using the NF-κB–inducer tumor necrosis factor-α (10 ng/ counting of viable cells (trypan blue negative). Comparison mL; 2 h).

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2002 Induction of AKR1B10 by Bortezomib

significant earlier and stronger decrease of viable cells. An- AKR1B10 Enzymatic Activity Assay nexin V/propidium iodide FACS analysis confirmed the in- Spectrophotometric measurements of NAPDH oxidation duction of apoptosis, as shown for monotherapy with with recombinant AKR1B10 and bortezomib revealed bortezomib (Fig. 1C). a depletion of the cofactor at high doses of the drug. The

Figure 5. Effects of AKR1B10 silencing on bortezomib-treated HRT-18 cells. Cells were transfected with specific siRNAs directed against AKR1B10 (siRNA1, 30 nmol/L; siRNA2, 10 nmol/L) or a nontargeting siRNA (neg siRNA, 30 nmol/L) and cultured 24 h before being exposed to the indicated concentrations of bortezomib. A, time-dependent AKR1B10 expression in HRT-18 cells transfected with siRNA1 or siRNA2. RT-PCR results are normalized to expression of the housekeeping gene β-actin using the formula 2−ΔΔCT, and data are presented as the ratioofcells transfected with negative controlsiRNA. B, cell viability based on metabolic activity of HRT-18 cells under various experimental conditions determined by means of WST-1 assay 48 and 72 h after therapy with bortezomib (0.03 μmol/L). Columns, means expressed as a percentage of control cells exposed to transfection reagent only (TFR). C, morphologic changes 72 h after 0.03 μmol/L of bortezomib treatment of untransfected and transfected HRT-18 cells visualized by phase contrast imaging (original magnification, ×10). D, cell cycle distribution 24 h after 0.03 μmol/L of bortezomib treatment of untransfected and transfected HRT-18 cells. E, dose-response curves of siRNA transfected (siRNA1 and siRNA2) and untransfected HRT-18 cells (+TFR) 48 h after treatment with bortezomib (0.01–0.03 μmol/L) obtained with trypan blue dye exclusion test. The percentage of viable cells (excluding trypan blue) is expressed relative to control cells (untransfected + TFR, siRNA1 and siRNA2 transfected) without bortezomib treatment. For bortezomib 0.01 and 0.02 μmol/L corresponding data of the AKR1B10-negative cell line Caco-2 were added to the graphic. A and E, points, mean of three experiments; bars, SD; *, P < 0.05; **, P ≤ 0.001 as compared with corresponding controls (Student's t test).

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Molecular Cancer Therapeutics 2003

Figure 6. AKR1B10 immunohistochemical analysis in colorectal cancer. Confirmation of AKR1B10 antibody specificity in cell pellets of untreated (A) or bortezomib-treated (0.03 μmol/L; B) HRT-18 cell line, and untreated Caco-2 cell line (C). Repre- sentative immunostaining of AKR1B10 in colorectal cancer tissue with nuclear and cytoplasmic staining (D). Original magnification, ×100 (A,B, and C); original magnification, ×200 (D).

reaction rate in the presence of 0.167 mmol/L of bortezomib or strong. In all, 26 tumors were classified as negative, 71 as was 2.6 nmol/(min·mg) and 7.85 nmol/(min·mg) with moderate, and 27 as strong. 1 mmol/L of bortezomib. Correlation between Pathologic Data and Immu‐ AKR1B10, COX-2, and Phosphorylated AKT Immu‐ nodetectable AKR1B10 nohistochemical Analysis in Colorectal Cancer To clarify which factors correlate with AKR1B10 immunos- Immunohistochemical analysis of in vitro cell pellets containing, we carried out a statistical analysis that examined firmed the specificity of AKR1B10 staining with constitutive clinicopathologic variables and the expression of molecules expression and bortezomib-related up-regulation in HRT-18 reported previously in this patient cohort (24). AKR1B10 ex- cells and absent expression in Caco-2 cells (Fig. 6A–C). Most pression was not correlated with tumor-node-metastasis specimens lacked AKR1B10 immunostaining. Although stage of tumors, amount of Ki-67–positive cells, or apoptotic normal mucosa revealed a consistent positive immunostain- rate (data not shown). Overall survival did not significantly ing of the apical mucosal epithelial cells, cells in the basal differ between AKR1B10-positive and AKR1B10-negative crypt remained negative. Tumor cells showed a combined tumors as assessed by univariate Kaplan-Meier survival nuclear and cytoplasmic immunostaining. If any of the tu- analysis. With regard to previously characterized molecular mor cells showed specific AKR1B10 immunostaining re- markers in this patient cohort (24), AKR1B10 expression was gardless of staining intensity, the case was classified as significantly associated with the expression of COX-2 positive. In total, AKR1B10 immunohistochemistry of the (P = 0.022) and pAKT (P = 0.045). tissue microarray containing 125 tumor samples from patients with colorectal cancer revealed positive immunostaining in 29 tissue samples (23.2%; for representative example, Discussion see Fig. 6D). The amount of AKR1B10-positive cells ranged The proteasome inhibitor bortezomib exhibits antiprolifera- from 1% to 45% (mean, 9.81 ± 11.26%). tive, proapoptotic, and antiangiogenic activities in several Regarding COX-2 expression, a total of 123 tumors cancer models (6). Inhibition of NF-κB, stabilization of were analyzed; the two others showed loss of discs. Tu- p21, p27, p53, Bid and Bax, inhibition of caveolin-1 activa- Jun mor cells exhibited a specific cytoplasmic staining pat- tion, activation of c- -NH2-kinase, as well as endoplasmic tern. Classification was done according to the amount of reticulum stress response are the molecular effects of this positively stained tumor cells: negative (0–10%) and pos- proteasome inhibition, but the mechanisms involved in sen- itive (>10%). In all, 93 tumors were classified as positive sitivity or resistance to this agent remain poorly understood. and 30 as negative. Although bortezomib showed significant activity in colorec- Phosphorylated Akt immunostaining was available for tal cell lines and xenograft models, it was inactive as a single 124cases. Disc loss occurred in one case. Immunohisto- agent in a phase II trial in metastatic colorectal cancer (7, 9). chemistry of colorectal tumor tissue revealed a specific cy- To define proteins with altered expression following thera- toplasmic staining, whereas normal mucosal tissue py, we investigated colorectal cancer cell lines using a two- exhibited no staining. According to their cytoplasmic stain- dimensional gel-based proteomic approach. Analysis of the ing intensity, tumors were classified as negative, moderate, proteome profiles before and after treatment revealed three

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2004 Induction of AKR1B10 by Bortezomib

proteins with a significant increase in expression levels after targeting encoding and 3′untranslational regions of the treatment with bortezomib. All these proteins have previ- AKR1B10 gene. Consistent with the findings of Yan et al. ously been shown to be involved in malignant growth. (20), AKR1B10 silencing per se caused a time-dependent Whereas the lactate dehydrogenase subunit LDHB is pri- but mild growth inhibition. Combined treatment of siRNA marily a key mediator of glycolysis, hsp72 and AKR1B10, and bortezomib sensitized HRT-18 cells to low doses of bor- are directly involved in cytoprotective mechanisms and tezomib with significant earlier and stronger reduction of vi- their dynamic expression was confirmed by Western blot able cells. G2-M arrest followed by induction of apoptosis (27, 28). Hsp are stress-inducible proteins with antiapoptotic was shown to be the underlying antiproliferative mecha- properties that act as molecular chaperones for nascent pro- nism, as expected for treatment with bortezomib (3). There teins and assist in protecting and repairing proteins whose are two major possible explanations why additional inhibi- conformation is altered by stress, such as heat or anticancer tion of AKR1B10 increased sensitivity to bortezomib. (a) Bor- drugs. The major stress-inducible isoform of the hsp70 fam- tezomib is known to cause the generation of intracellular ily, hsp72, is expressed in human tumors, correlates with reactive oxygen species (46, 47). Consequently, production poor prognosis, and contributes to resistance to cancer ther- of endogenous reactive carbonyls that lead to cellular dam- apy (29–31). Proteasome inhibitors seem to up-regulate hsp age is expected. Inhibition of AKR1B10 may prevent a cyto- synthesis by increasing the amount of misfolded proteins as protective response to drug-induced oxidative injury (37, 48). b shown in breast tumor, hepatoma HepG2,myeloma,and ( ) Furthermore, AKR1B10 could be directly involved in the B-lymphoma cells (32–36). Similar to these reports, in this detoxification of bortezomib, by carbonyl reduction of the study, hsp72 up-regulation occurred very early after the ex- carbonyl moiety present in this agent [N-(2,3-pyrazine)car- posure of the two colorectal cancer cell lines, Caco-2 and bonyl-L-phenylalanine-L-leucine boronic acid; ref. 49]. Silenc- HRT-18, to bortezomib. In contrast to induction of hsp72 af- ing of this oxidoreductase would prolong the duration of ter proteasome inhibition, up-regulation of AKR1B10, a drug exposure and thereby increase cell damage. Using a member of the aldo-keto reductase (AKR) superfamily has NADPH-regenerating system, we could show that bortezo- not been previously shown. The 13 human AKRs are NAD mib is indeed a substrate of AKR1B10. The reaction rate ob- (P)H-dependent oxidoreductases implicated in intracellular served is comparable to values reported for AKR1B10 and detoxification, cell carcinogenesis, and resistance to cancer daunorubicin (22). However, the drug doses necessary to rec- therapeutics (27). They reduce aldehydes and ketones to ognize in vitro enzyme activity are more than a thousand-fold yield primary and secondary alcohols and are regulated higher than the growth-inhibitory concentrations observed in response to osmotic, electrophilic, and oxidative stress. in tumor cells. The detoxification of endogenous reactive car- In addition to endogenous substrates, AKRs are phase I bonyls may be more relevant than a direct interaction with drug-metabolizing enzymes for a variety of carbonyl- bortezomib (48). Nevertheless, the substrate relevance of bor- containing drugs (37). AKR1B10 involvement has been de- tezomib metabolites remains to be elucidated. scribed for resistance to cyclophosphamide, etacrynic acid, The additional detection of AKR1B10 up-regulation in the Adriamycin, daunorubicin, and mitomycin (38–40). Special WiDr colorectal cancer cell line (Fig. 3C) excludes that this is functions of this isoform include the conversion of retinals a unique HRT-18 cell type–specific phenomenon. To rule out into retinols, resulting in the reduction of intracellular reti- the possibility that this protein is present exclusively in cul- noic acid, a signaling molecule regulating cell proliferation tured cells, the in vivo relevance of this protein was deter- and differentiation (41). Furthermore, this enzyme is a reg- mined by immunohistochemical analysis of patient ulator of fatty acid biosynthesis, an essential cell membrane samples. AKR1B10 expression was found in ∼25% of colo- component in cancer cells (42). Recently, the involvement of rectal carcinomas. This finding is comparable to the fre- AKR1B10 has even been shown for non–small cell lung car- quencies observed for adenocarcinoma of the lung cinoma, esophageal carcinogenesis, hepatocellular carcino- (29.2%), cervical cancer (20.0%), and endometrial cancer ma, and uterine cancer in smokers (18, 43–45). Because of (15.8%; refs. 43, 45). A higher percentage of expression has our observation of AKR1B10 induction following therapy been described in squamous cell carcinoma of the lung with bortezomib and its reported biological functions, this (84.4%); moreover, the expression was highly correlated protein was chosen for further functional studies. with smoking (43). Our finding that AKR1B10 expres- In contrast to AKR1B10-negative Caco-2 cells, the faster sion is associated with COX-2 and pAKT expression can- growing cell line HRT-18, with less sensitivity to low doses not be directly explained. All three molecules have of bortezomib, showed strong AKR1B10 up-regulation after proproliferative properties. COX-2 transcription is sup- therapy. This induction was found to be independent of NF- pressed by retinoids, suggesting a possible link to κB activation, contrasting NF-κB dependence observed for AKR1B10 via inhibition of retinoid acid synthesis (41, the enzyme, aldose reductase, the most investigated mem- 50). Despite reported in vitro evidence for enhanced cell ber of the AKR family (26). Constitutive expression of growth in the presence of AKR1B10, we found no asso- AKR1B10 has already been reported for the same cell line ciation with tumor growth fraction or survival of pa- (HRT-18) and knockdown of AKR1B10 expression resulted tients (20). However, a possible association with response in growth inhibition and cell susceptibility to reactive carbo- to bortezomib remains to be discussed, and patient samples nyls (20). We investigated the effect of AKR1B10 silencing from ongoing clinical studies would be helpful in clarifying on treatment with bortezomib using two different siRNAs, this issue.

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Molecular Cancer Therapeutics 2005

In summary, proteomic screening of colorectal cancer sistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB. Nat Med 1999;5:412–7. cell lines before and after treatment with bortezomib allows 20. Yan R, Zu X, Ma J, Liu Z, Adeyanju M, CaoD. Aldo-ketoreductase the identification of proteins that interfere with the complex family 1 B10 gene silencing results in growth inhibition of colorectal can- network of proteasome inhibition. Further functional cer cells: implication for cancer intervention. Int J Cancer 2007;121: – investigation of these proteins might provide a deeper 2301 6. 21. Pfaffl M. A new mathematical model for relative quantification in real- understanding of sensitivity to bortezomib. Especially, the time RT-PCR. Nucleic Acids Res 2001;29:e45. aldo-keto reductase AKR1B10 may act in a cytoprotective 22. Martin H, Breyer-Pfaff U, Wsol V, Venz S, Block S, Maser E. Purifica- manner in response to bortezomib-mediated proteasome in- tion and characterization of akr1b10 from human liver: role in carbonyl re- hibition, thus providing an interesting target for therapeutic duction of xenobiotics. Drug Metab Dispos 2006;34:464–70. interventions. 23. Hamilton SR, Aaltonen LA. World Health Organization histological classification of tumours of the colon and rectum. In: Hamilton SR, Aaltonen LA, editors. Pathology and genetics of tumours of the digestive system. Lyon: IARC Press; 2000. p. 104. Disclosure ofPotential ConflictsofInterest 24. Schmitz K, Wohlschlaeger J, Alakus H, et al. Activation of extracellu- No potential conflicts of interest were disclosed. lar regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k-ras mutations. Virchows Arch 2007;450:151–9. References 25. Schmitz K, Lang H, Wohlschlaeger J, et al. Elevated expression of cyclooxygenase-2 is a negative prognostic factor for overall survival in intra- 1. Faivre J, Bouvier A, Bonithon-Kopp C. Epidemiology and screening of hepatic cholangiocarcinoma. Virchows Arch 2007;450:135–41. colorectal cancer. Best Pract Res Clin Gastroenterol 2002;16:187–99. 26. Lee Y, Paek K, Kang E, et al. Involvement of nuclear factor κB in up- 2. Voutsadakis I. Pathogenesis of colorectal carcinoma and therapeutic regulation of aldose reductase gene expression by 12-O-tetradecanoyl- implications: the roles of the ubiquitin-proteasome system and Cox-2. phorbol-13-acetate in HeLa cells. Int J Biochem Cell Biol 2005;37: – J Cell Mol Med 2007;11:252 85. 2297–309. 3. Mani A, Gelmann E. The ubiquitin-proteasome pathway and its role in 27. Penning T, Drury J. Human aldo-keto reductases: function, gene reg- – cancer. J Clin Oncol 2005;23:4776 89. ulation, and single nucleotide polymorphisms. Arch Biochem Biophys 4. Adams J, Kauffman M. Development of the proteasome inhibitor Vel- 2007;464:241–50. – cade (Bortezomib). Cancer Invest 2004;22:304 11. 28. SõtiC,NagyE,GiriczZ,VíghL,CsermelyP,FerdinandyP.Heat 5. Richardson P, Sonneveld P, Schuster M, et al. Extended follow-up of a shock proteins as emerging therapeutic targets. Br J Pharmacol 2005; phase 3 trial in relapsed multiple myeloma: final time-to-event results of 146:769–80. – the APEX trial. Blood 2007;110:3557 60. 29. Nylandsted J, Brand K, Jäättelä M. Heat shock protein 70 is required 6. Adams J. The development of proteasome inhibitors as anticancer for the survival of cancer cells. Ann N Y Acad Sci 2000;926:122–5. – drugs. Cancer Cell 2004;5:417 21. 30. Wang X, Qiu F, Liu G, Chen R. Correlation between clinicopathology 7. Cusack JJ, Liu R, Houston M, et al. Enhanced chemosensitivity to CPT- and expression of heat shock protein 70 and glucose-regulated protein 11 with proteasome inhibitor PS-341: implications for systemic nuclear 94 in human colonic adenocarcinoma. World J Gastroenterol 2005;11: factor-κB inhibition. Cancer Res 2001;61:3535–40. 1056–9. 8. Russo S, Tepper J, Baldwin AJ, et al. Enhancement of radiosensitivity 31. Ciocca D, Calderwood S. Heat shock proteins in cancer: diagnostic, by proteasome inhibition: implications for a role of NF-κB. Int J Radiat On- prognostic, predictive, and treatment implications. Cell Stress Chaperones col Biol Phys 2001;50:183–93. 2005;10:86–103. 9. Mackay H, Hedley D, Major P, et al. A phase II trial with pharmacody- 32. Bush K, Goldberg A, Nigam S. Proteasome inhibition leads to a heat- namic endpoints of the proteasome inhibitor bortezomib in patients with shock response, induction of endoplasmic reticulum chaperones, and ther- metastatic colorectal cancer. Clin Cancer Res 2005;11:5526–33. motolerance. J Biol Chem 1997;272:9086–92. 10. Cho W. Contribution of oncoproteomics to cancer biomarker discov- 33. Mimnaugh E, Xu W, Vos M, et al. Simultaneous inhibition of hsp 90 ery. Mol Cancer 2007;6:25. and the proteasome promotes protein ubiquitination, causes endoplasmic 11. Loeffler-Ragg J, Skvortsov S, Sarg B, et al. Gefitinib-responsive EGFR- reticulum-derived cytosolic vacuolization, and enhances antitumor activi- positive colorectal cancers have different proteome profiles from non- ty. Mol Cancer Ther 2004;3:551–66. responsive cell lines. Eur J Cancer 2005;41:2338–46. 34. Lauricella M, Emanuele S, D'AnneoA, et al. JNK and AP-1 mediate 12. Loeffler-Ragg J, Sarg B, Mueller D, Auer T, Herbert Lindner H, Zwierzina apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and H. Proteomics, a new tool to monitor cancer therapy? Mag Eur Med Oncol mitochondria-dependent pathways. Apoptosis 2006;11:607–25. 2008;1:129–36. 35. Mitsiades N, Mitsiades C, Poulaki V, et al. Molecular sequelae of pro- 13. Görg A, Weiss W, Dunn M. Current two-dimensional electrophoresis teasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci technology for proteomics. Proteomics 2004;4:3665–85. U S A 2002;99:14374–9. 14. Forbes S, Bhamra G, Bamford S, et al. The Catalogue of Somatic Mu- 36. Shringarpure R, Catley L, Bhole D, et al. Gene expression analysis of tations in Cancer (COSMIC). Curr Protoc Hum Genet 2008;10:unit10.1. B-lymphoma cells resistant and sensitive to bortezomib. Br J Haematol 2006;134:145–56. 15. Tompkins W, Watrach A, Schmale J, Schultz R, Harris J. Cultural and antigenic properties of newly established cell strains derived from adeno- 37. Jin Y, Penning T. Aldo-keto reductases and bioactivation/detoxication. carcinomas of the human colon and rectum. J Natl Cancer Inst 1974;52: Annu Rev Pharmacol Toxicol 2007;47:263–92. – 1101 10. 38. Bacolod M, Lin S, Johnson S, et al. The gene expression profiles of 16. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured medulloblastoma cell lines resistant to preactivated cyclophosphamide. human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst Curr Cancer Drug Targets 2008;8:172–9. – 1977;59:221 6. 39. Ciaccio P, Stuart J, Tew K. Overproduction of a 37.5-kDa cytosolic 17. Ryan D, Appleman L, Lynch T, et al. Phase I clinical trial of bortezomib protein structurally related to prostaglandin F synthase in ethacrynic in combination with gemcitabine in patients with advanced solid tumors. acid-resistant human colon cells. Mol Pharmacol 1993;43:845–53. – Cancer 2006;107:2482 9. 40. Yang X, Tang D, Wang J, CaoD. Screening ofthe drug resistance- 18. Breton J, Gage M, Hay A, et al. Proteomic screening of a cell line mod- associated gene in HepG2 cell line transfected with aldose reductase like el of esophageal carcinogenesis identifies cathepsin D and aldo-keto re- gene-1 (ARL-1). Ai Zheng 2003;22:1289–95. ductase 1C2 and 1B10 dysregulation in Barrett's esophagus and 41. Gallego O, Ruiz F, Ardèvol A, et al. Structural basis for the high all- – esophageal adenocarcinoma. J Proteome Res 2008;7:1953 62. trans-retinaldehyde reductase activity of the tumor marker AKR1B10. Proc 19. Wang C, Cusack JJ, Liu R, Baldwin AJ. Control of inducible chemore- Natl Acad Sci U S A 2007;104:20764–9.

Mol Cancer Ther 2009;8(7). July 2009

2006 Induction of AKR1B10 by Bortezomib

42. Ma J, Yan R, Zu X, et al. Aldo-keto reductase family 1 B10 affects zomib, a novel proteasome inhibitor, in human H460 non-small cell lung fatty acid synthesis by regulating the stability of acetyl-CoA carboxylase-α cancer cells. J Biol Chem 2003;278:33714–23. – in breast cancer cells. J Biol Chem 2008;283:3418 23. 47. Pérez-Galán P, Roué G, Villamor N, Montserrat E, Campo E, Colomer 43. Fukumoto S, Yamauchi N, Moriguchi H, et al. Overexpression of the D. The proteasome inhibitor bortezomib induces apoptosis in mantle-cell aldo-keto reductase family protein AKR1B10 is highly correlated with smo- lymphoma through generation of ROS and Noxa activation independent of kers' non-small cell lung carcinomas. Clin Cancer Res 2005;11:1776–85. p53 status. Blood 2006;107:257–64. 44. Teramoto R, Minagawa H, Honda M, et al. Protein expression profile 48. Martin H, Maser E. Role of human aldo-keto-reductase AKR1B10 in characteristic to hepatocellular carcinoma revealed by 2D-DIGE with su- the protection against toxic aldehydes. Chem Biol Interact 2009;178: pervised learning. Biochim Biophys Acta 2008;1784:764–72. 145–50. 45. Yoshitake H, Takahashi M, Ishikawa H, et al. Aldo-keto reductase fam- 49. Pekol T, Daniels J, Labutti J, et al. Human metabolism of the protea- ily 1, member B10 in uterine carcinomas: a potential risk factor of recur- some inhibitor bortezomib: identification of circulating metabolites. Drug rence after surgical therapy in cervical cancer. Int J Gynecol Cancer 2007; Metab Dispos 2005;33:771–7. – 17:1300 6. 50. Eisinger A, Nadauld L, Shelton D, Prescott S, Stafforini D, Jones D. 46. Ling Y, Liebes L, Zou Y, Perez-Soler R. Reactive oxygen species gen- Retinoic acid inhibits β-catenin through suppression of Cox-2: a role for eration and mitochondrial dysfunction in the apoptotic response to Borte- truncated adenomatous polyposis coli. J Biol Chem 2007;282:29394–400.

Mol Cancer Ther 2009;8(7). July 2009

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Judith Loeffler-Ragg, Doris Mueller, Gabriele Gamerith, et al.

Mol Cancer Ther 2009;8:1995-2006. Published OnlineFirst June 30, 2009.

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