Decreased Selenium-Binding Protein 1 in Esophageal Adenocarcinoma Results from Posttranscriptional and Epigenetic Regulation and Affects Chemosensitivity

Published OnlineFirst March 23, 2010; DOI: 10.1158/1078-0432.CCR-09-2801 Published Online First on March 23, 2010 as 10.1158/1078-0432.CCR-09-2801

Clinical Human Cancer Biology Cancer Research Decreased Selenium-Binding Protein 1 in Esophageal Adenocarcinoma Results from Posttranscriptional and Epigenetic Regulation and Affects Chemosensitivity

Amy L. Silvers1, Lin Lin1, Adam J. Bass3, Guoan Chen1, Zhuwen Wang1, Dafydd G. Thomas2, Jules Lin1, Thomas J. Giordano2, Mark B. Orringer1, David G. Beer1, and Andrew C. Chang1

Abstract Purpose: The chemopreventive effects of selenium have been extensively examined, but its role in cancer development or as a chemotherapeutic agent has only recently been explored. Because selenium- binding protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium. Experimental Design: SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time reverse transcription-PCR, tissue microarrays, immunoblotting, and single-nucleotide polymorphism arrays. Bisulfite sequencing and sequence analysis of reverse transcription-PCR–amplified products explored epigenetic and posttranscriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were done to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells. Results: SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and posttranscriptional mechanisms seemed to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid–supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorgan- ic sodium selenite similarly enhanced cisplatin cytotoxicity, these two forms of selenium elicited different cellular responses. Conclusions: SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues. Clin Cancer Res; 16(7); 2009–21. ©2010 AACR.

Esophageal adenocarcinoma (EAC) is increasing in inci- well as its precursor lesion, dysplastic Barrett's esophagus dence in Western countries and remains a highly lethal (2). Organic selenium compounds, such as methylseleni- malignancy. Despite advances in endoscopic surveillance nic acid (MSA), seem to enhance response to chemother- programs, surgical therapy, and multimodality treatment, apeutic agents, such as cisplatin and paclitaxel, possibly by the prognosis of patients with EAC remains poor, with an downregulation of antiapoptotic signals (3, 4). overall 5-year survival of 5% to 12% (1). Serum selenium Selenium-binding protein 1 (Chr:1q21; SBP1, SELENBP1, deficiency, in particular, has been associated with EAC as hSP56) has been shown to bind selenium covalently (5, 6) and is expressed in a variety of tissues and cell lines (7, 8). Its expression is reduced markedly in multiple tumor types Authors' Affiliations: 1Section of Thoracic Surgery, Department of compared with their corresponding normal tissues, and Surgery and 2Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan and 3Department of Medical Oncology, its reduction has been associated with poor outcome in Dana-Farber Cancer Institute, Harvard Medical School and The Broad lung cancer (9), ovarian cancer (10), colorectal cancer Institute of Massachusetts Institute of Technology and Harvard, (11, 12), and pleural mesothelioma (13). Previous studies Cambridge, Massachusetts have reported a correlation between decreased expression of Note: Supplementary data for this article are available at Clinical Cancer a chromosome 1q21 gene cluster and resistance to preoper- Research Online (http://clincancerres.aacrjournals.org/). ative EAC chemoradiotherapy (14). Corresponding Author: Andrew C. Chang, Section of Thoracic Surgery, Department of Surgery, University of Michigan Medical School, TC2120G/ Higher levels of SELENBP1 expression in nongrowing 5344, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734- versus rapidly growing cells have been reported in both 763-7418; Fax: 734-615-2656; E-mail: [email protected]. mouse and human cells (8, 15). Whereas higher SELENBP1 doi: 10.1158/1078-0432.CCR-09-2801 expression is associated with normal colonic epithelial dif- ©2010 American Association for Cancer Research. ferentiation (12), lower expression was associated with

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Cell lines and treatment. Flo-1 was derived from a pa- Translational Relevance tient with EAC and has been described previously (17). OE33 was derived from an EAC and is maintained by The overall long-term survival for patients with the European Collection of Cell Cultures (Sigma-Aldrich esophageal adenocarcinoma remains poor. Despite Corp.). Het-1A (derived from SV40 large T antigen– improved understanding about the progression from transfected esophageal epithelial cells; ref. 18), SW480 Barrett's esophagus to dysplasia and then adenocarci- (established from a primary colon adenocarcinoma), noma, patients present commonly in advanced stage. and H460 (derived from the pleural fluid of a patient Population-based studies have found that lower serum with large cell lung carcinoma) are all maintained by concentration of selenium is associated with greater in- the American Type Culture Collection. Cell lines were cidence of esophageal cancer, including adenocarcinoma, grown in DMEM or RPMI 1640 supplemented with suggesting a possible basis for selenium chemopreven- 10% fetal bovine serum (Atlanta Biologicals) and 1% tion. Selenium-binding protein 1 (SELENBP1), which penicillin/streptomycin/fungizone (Invitrogen) at 37°C binds selenium covalently, is reduced in the progression in 5% carbon dioxide/95% air. from dysplastic Barrett's esophagus to adenocarcinoma, Cells were seeded at either 4,000 per well in a 96-well but its function has not been well characterized. The plate format, 50,000 per well in a 12-well format, or present study shows that SELENBP1 expression is regu- 325,000 per well in 60-mm plates for 24 h (T0 time lated at both the epigenetic and the posttranscriptional point). All experiments done included a 72-h treatment in vitro levels. Gene overexpression increased esoph- with either 2.5 μmol/L MSA or 10 μmol/L sodium selenite ageal adenocarcinoma cell death in response to seleni- (NaS) and/or a 12- to 24-h treatment with 20 μg/mL cis- um supplementation and cisplatin. These studies platin [cis-diammineplatinum(II) dichloride (CDDP)]. suggest that differential expression of SELENBP1 may The demethylating agent 5-aza-2-deoxycytidine (5-Aza) affect cellular response to selenium supplementation and the histone deacetylase inhibitors trichostatin A both in the chemoprevention and in the treatment of (TSA) and valproic acid (VPA) were used at 5 μmol/L, esophageal cancer. 300 nmol/L, and 5 mmol/L, respectively. RNA extraction and oligonucleotide microarray. Total RNA was isolated from EACs (37 cases) and Barrett's esophageal samples (9 metaplasia, 15 low-grade dysplasia, poor tumor differentiation in lung adenocarcinomas (9). and 7 high-grade dysplasia) as previously described (19) Although clear discrepancies were observed between the le- using Trizol (Invitrogen) followed by RNeasy column pu- vels of mRNA and protein expression, mouse selenium- rification (Qiagen) per the manufacturers' instructions. binding proteins were found to be associated with the ag- cRNA was generated and hybridized to GeneChip HG- ing process in senescence-accelerated mouse models (16). U133A oligonucleotide microarrays (Affymetrix). Image Based on such findings, we evaluated the role of analysis was done by the University of Michigan DNA Mi- SELENBP1 in the tumorigenesis of EAC as well as the ef- croarray Core Facility. A filtering algorithm was used to se- fect of differential SELENBP1 expression and selenium lect genes with either increased or decreased expression in supplementation on cell viability and chemosensitivity. adenocarcinomas or dysplastic Barrett's mucosa when We observed that SELENBP1 expression was decreased in compared with Barrett's metaplasia samples. To normalize primary EAC tumor samples. Decreased expression seemed the microarray data, a summary statistic was calculated us- to be regulated both by methylation of the SELENBP1 pro- ing the robust multichip average method (20) as imple- moter and by alternative splicing of SELENBP1 mRNA. mented in the Affymetrix library of Bioconductor version When SELENBP1 expression was reconstituted in vitro, 1.3,4 which provides background adjustment, quantile tumor cells responded to selenium treatment with normalization, and summarization. Expression values for increased apoptosis, increased cellular senescence, and each sample were then compared with the mean expres- increased sensitivity to cisplatin. sion value for the seven Barrett's metaplasia samples. Fold reduction of 50% was considered significant (21). Quantitative reverse transcription-PCR and DNA sequencing. Materials and Methods Total RNA from treated cells was isolated and column purified using the RNeasy Mini kit (Qiagen) per the manufac- Patients and tissues. Written patient consent and appro- turer's instructions. RNA was eluted from the spin column val of the Institutional Review Board were obtained to col- using RNase-free dH2O and reverse transcribed with lect specimens from patients undergoing esophagectomy 5 units/μL SuperScript II reverse transcriptase (Invitrogen). at the University of Michigan Medical Center (Ann Arbor, Real-time PCR amplification using 20 ng of total RNA, MI). Specimens were transported to the laboratory in Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), DMEM (Invitrogen) on ice. A portion of each sample and 0.2 μmol/L of both forward and reverse primers was frozen in ornithine carbamoyltransferase compound (Miles, Inc.) for cryostat sectioning. The remainder was frozen in liquid nitrogen and stored at −80°C. 4 www.bioconductor.org

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(Supplementary Table S1) was done on the Corbett Rotor- antibodies were used at 1:10,000 to 1:25,000 dilutions. Gene 6000 (Qiagen). Significant differences of relative Antigen-antibody complexes were detected using the −ΔΔC quantification were determined using the 2 T method SuperSignal West Pico Chemiluminescent substrate (22), with normalization to glyceraldehyde-3-phosphate (Pierce Biotechnology, Inc.). dehydrogenase or β-actin. PCR amplification of Construction of SELENBP1 stable cell line. A SELENBP1 SELENBP1 was done using 20 ng of total RNA or genomic mammalian expression construct was created and stably DNA, 0.025 unit/μL of GoTaq DNA polymerase (Promega tranfected into Flo-1 cells. The SELENBP1 clone MGC- Corp.), 0.2 mmol/L deoxynucleotide triphosphates, and 9270 (American Type Culture Collection) was PCR 0.36 μmol/L of both forward and reverse primers (Supple- amplified using primers containing EcoRI and BamHI re- mentary Table S1). Agarose gel (1%)–purified PCR pro- striction sites for directional cloning into the pEGFP-C2 ducts were submitted to the University of Michigan DNA vector containing enhanced green fluorescent protein Sequencing Core. Sequencing results were analyzed with (EGFP; Clontech). The pEGFP-SELENBP1 or empty vec- FinchTV software and the National Center for Biotechnol- tor construct was transfected into Flo-1 cells using ogy Information BLAST software. Agarose gel image densi- FuGENE 6 transfection reagent (Roche). Selected clones were tometry of full-length and alternative splice products was maintained in growth medium containing 200 μg/mL done using ImageJ software.5 geneticin. Immunohistochemistry and tissue microarray. Tissue mi- Proliferation assay. The cell proliferation reagent WST-1 croarrays (TMA) were constructed, as previously described (Roche) was used for spectrophotometric quantification (19, 23), with formalin-fixed, paraffin-embedded tissues of cell proliferation, viability, and chemosensitivity in ac- from 73 patients, including 64 tumor, 8 lymph node me- cordance with the manufacturer's directions. Relative pro- tastases, 8 dysplastic Barrett's mucosa, and 11 nondysplas- liferation rates were calculated as a percentage of the tic Barrett's metaplasia samples. Multiple samples from initial T0 reading within each cell line. Chemosensitivity representative areas of EAC, metaplasia, or dysplasia were analysis of a 3-d treatment with either MSA or NaS and/ included for 33 patients. Normal esophagus was also in- or 12- to 24-h treatment with CDDP was similarly done cluded from three patients who had undergone esopha- using WST-1 reagent. Treatments were initiated following gectomy for benign indications. T0 absorbance readings. Viability was calculated as a Immunohistochemical staining was done on the DAKO percentage of mock-treated cells at 72 h within each Autostainer using DAKO LSAB+ and 3,3′-diaminobenzi- cell line. dine as the chromogen. Dewaxed and rehydrated sections Wound-healing assay. Cells were densely plated and al- of the TMA at 4-mm thickness were labeled with lowed to grow to 100% confluence. Following serum star- SELENBP1 antibody (mouse monoclonal antibody, clone vation for 24 h, cell monolayers were wounded with sterile 4D4, 1:200 dilution; MBL International). We did micro- p200 tips. Digital images of predetermined locations in wave citric acid epitope retrieval for 20 min. Slides were each well were taken at both ×4 and ×10 optical magnifi- lightly counterstained with hematoxylin. Each sample cation on an Olympus CK2 inverted microscope using a was scored independently by two readers using a scale of SPOT Idea 1.3-megapixel camera and analyzed with SPOT 0 (no staining), 1+ (<10% staining), 2+ (10-50%) stain- Basic software (Diagnostic Instruments) immediately after ing, or 3+ (≥50%). wounding and, subsequently, every 24 h until the wounds Protein extraction and immunoblot analysis. Total cellu- were closed. The percentage of initial wound width was lar protein was extracted in lysis buffer [150 mmol/L determined by averaging measurements per well at each NaCl, 20 mmol/L Tris (pH 7.5), 1 mmol/L EDTA, time point. 1 mmol/L EGTA, 2.5 mmol/L Na4P2O7, 1 mmol/L glycerol Single-nucleotide polymorphism analysis. Single-nucleotide 2-phosphate disodium salt hydrate, 1 mmol/L Na3VO4,1% polymorphism (SNP) arrays were done at the Genetic Triton X-100] supplemented with Protease Inhibitor Analysis Platform of the Broad Institute of Massachusetts Cocktail (20 mL/1 mL lysis buffer; Sigma-Aldrich). Total Institute of Technology and Harvard in accordance with cell lysates (20-40 μg) were boiled in 6× sample buffer, re- the manufacturer's instructions as previously described solved on 8% to 16% Tris-glycine gels (Invitrogen), and (24, 25). Briefly, 73 EAC DNAs were processed for Affyme- transferred to Immobilon-P membranes (Millipore). trix genome-wide 250K StyI SNP arrays. The data were Mouse monoclonal SELENBP1 (MBL International) and annotated and visualized using Integrative Genomics rabbit polyclonal poly(ADP-ribose) polymerase (PARP; Viewer software.6 Cell Signaling) antibodies were used at 1:2,000 dilution. Bisulfite sequencing of genomic DNA. Genomic DNA was Mouse monoclonal β-actin antibody (Abcam) was used at isolated using the DNeasy kit (Qiagen), and 1 μg of each 1:10,000 dilution as a loading control. Both horseradish sample DNA was treated with bisulfite using the EZ DNA peroxidase–conjugated mouse (Southern Biotechnology Methylation-Gold kit (Zymo Research Corp.) according to Associates) and rabbit (Vector Laboratories) secondary the manufacturers' instructions. The 5′-untranslated region

5 W.S. Rasband, ImageJ, NIH, Bethesda, Maryland, 1997-2008. Available from: http://rsb.info.nih.gov/ij/. 6 http://www.broad.mit.edu/cancer/software/genepattern/

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Fig. 1. SELENBP1 expression was decreased in the progression from Barrett's metaplasia to adenocarcinoma. A, normalized relative SELENBP1 (probe set: 214433_s_at) expression as determined by HG-U133A oligonucleotide microarray analysis of primary tissue samples from patients with Barrett's metaplasia (B, n = 9), Barrett's metaplasia with indeterminate or low-grade dysplasia (BL, n = 7), low-grade dysplasia (L, n = 8), high-grade dysplasia (H, n = 7), and EAC (T, n = 15). B, mean fold change for each group compared with Barrett's metaplasia. *, P < 0.05; †, P < 0.005 versus Barrett's metaplasia. C, quantitative real-time PCR analysis of SELENBP1 expression in a panel of primary tissue samples from the oligonucleotide microarray analysis compared with normal intestinal tissue. Relative quantitative differences were determined using the 2−ΔΔCT method. LGD, low-grade dysplasia; HGD, high-grade dysplasia. D, comparison of SELENBP1 expression in samples of nondysplastic Barrett's metaplasia and EAC from two independent HG-U133A oligonucleotide microarray analyses.

of SELENBP1 was analyzed using the Transcriptional Reg- extracted using the Quick Plasmid Miniprep kit (Invitrogen) ulatory Element Database7 and promoter prediction soft- and eluted in 30 μL buffer. A minimum of 10 clones sel- ware from the Berkeley Drosophila Genome Project.8 One ected at random was sequenced by the University of of the highest-scored sequences (0.98) spanned −39 to Michigan DNA Sequencing Core. +11. A 186-bp fragment (−95 to +90), which covered Flow cytometry. Following treatment, DNA from whole- 158 bp upstream and 25 bp downstream (inside the first cell nuclei was harvested and stained using modified intron) of the ATG start codon of SELENBP1, was PCR am- Krishan buffer (0.1% citric acid trisodium salt dihydrate, plified using bisulfite-treated DNA and primers designed 0.02 mg/mL RNase A, 0.3% NP40, 0.05 mg/mL propi- by MethPrimer software9 (Supplementary Table S1). PCR dium iodide) at the indicated time points. Apoptosis products were purified by electrophoresis on 1% agarose and cell cycle analyses were done by the University of Mi- gels, ligated into pCR2.1pTOPO-TA vectors (Invitrogen), chigan Flow Cytometry Core and measured on the FACS- and introduced into TOP10 One Shot (Invitrogen) com- Calibur Cytometer (BD Biosciences) using CellQuest and petent bacteria. Insert-containing plasmid DNA was ModFit Light softwares, respectively. Senescence assay. Senescence-associated β-galactosidase activity was determined with the Senescence Detection kit (BioVison Research Products) per the manufacturer's instructions. Digital images were taken at ×20 optical mag- 7 http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=searchPromForm 8 http://www.fruitfly.org/seq_tools/promoter.html nification, and stained cells were counted and expressed as 9 http://www.urogene.org/methprimer/index1.html a percentage of total cell number in three independent

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fields per well per treatment group to obtain an average Fig. 1C). Reduced SELENBP1 expression in EAC was re- value for β-galactosidase staining activity. affirmed using a second set of patient tissues in oligo- Experimental statistical analysis. Two-way ANOVA and nucleotide microarrays (Fig. 1D). Consistent with many Student's t test were done using SPSS for Windows 15 primary EACs, Flo-1 cells showed minimal expression of (SPSS, Inc.) and Microsoft Excel 2002 (Microsoft Corp.). SELENBP1 (Fig. 1C). SELENBP1 expression was decreased significantly in adenocarcinomas compared with meta- Results plastic or dysplastic Barrett's esophagus using immunohistochemistry of TMAs (Fig. 2A-C). Minimal-to-low Patient demographics. Clinical and pathologic features SELENBP1 expression was observed more frequently in for the patients whose tissue samples were used for this adenocarcinoma (P < 0.001, two-way ANOVA) and dys- study are provided in Supplementary Table S2. plastic Barrett's esophagus (P < 0.05, two-way ANOVA) Downregulation of SELENBP1 expression was observed compared with SELENBP1 expression in samples of Barrett's during the progression from metaplastic Barrett's esophagus metaplasia (Supplementary Table S3). Of note, two sam- to EAC. Affymetrix HG-U133A oligonucleotide microarray ples on the oligonucleotide microarray with increased indicated diminished SELENBP1 expression in EAC rela- SELENBP1 expression (36T and 39T) had coordinately tive to both metaplastic and dysplastic Barrett's esophagus increased SELENBP1 protein expression by immuno- (Fig. 1A). SELENBP1 expression in EAC decreased by 25% histochemistry in TMA cores. in samples of high-grade dysplasia (P =0.022)andby Immunoblot analysis of paired EAC and Barrett's meta- ∼50% in EAC (P = 0.005) compared with metaplastic Bar- plasia protein extracts indicated expression of a single rett's esophagus (Fig. 1B). The array results were validated SELENBP1 protein band (Fig. 2D), with reduced expres- using quantitative reverse transcription-PCR (RT-PCR; sion in tumor samples relative to Barrett's metaplasia in

Fig. 2. SELENBP1 expression was decreased in samples of high-grade dysplasia (*) and adenocarcinoma (T) when compared with areas of nondysplastic Barrett's esophagus (arrows) as indicated by immunohistochemical analysis using TMA of esophageal surgical specimens from 73 patients. Representative sections from TMA for SELENBP1 expression at ×4 (A) and ×10 (B and C) magnification. D, Western immunoblotting showed specific binding to SELENBP1 in patient specimens of Barrett's metaplasia (B) and EAC (T). β-Actin was used as a loading control.

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Fig. 3. A, bisulfite sequencing of Flo-1 (EAC), SW480 (colon adenocarcinoma), and Het-1A (immortalized esophageal squamous epithelium) suggested that hypermethylation of the 5′ upstream promoter region of SELENBP1 occurs in esophageal tissues. B, treatment of Flo-1 cells with 5 μmol/L 5-Aza, 300 nmol/L TSA, and 5 mmol/L VPA induced SELENBP1 expression compared with vehicle-treated controls, as determined by quantitative RT-PCR. C, treatment of Flo-1 cells with 2.5 μmol/L MSA, 5 μmol/L 5-Aza, and/or 300 nmol/L TSA did not lead to detectable levels of SELENBP1 protein. Stably transfected SELENBP1.8 cells were used as a positive control. β-Actin was used as a loading control. D, treatment of Flo-1 cells with combinations of 2.5 μmol/L MSA, 5 μmol/L 5-Aza, 300 nmol/L TSA, 10 μmol/L NaS, and/or 20 μg/mL cisplatin (CDDP) induced apoptosis, as determined by PARP cleavage.

four of the five cases analyzed. In the fifth case (36T), SE- SELENBP1 in these samples could not be explained by LENBP1 expression was higher in the tumor compared changes in DNA copy number, suggesting transcriptional with an adjacent noncarcinoma section, corresponding or posttranscriptional modulation. to the gene expression findings observed by both oligonu- DNA methylation and chromatin remodeling of cleotide microarray and quantitative RT-PCR. SELENBP1 may downregulate gene expression. Sequence Decreased DNA copy number changes indicating loss of analysis of SELENBP1 revealed several CpG sites near the heterozygosity were not detected by SNP array analysis. Be- predicted promoter region of this gene, suggesting that cause frequent loss of heterozygosity has been recently DNA methylation may be another mechanism for sup- reported in regions near the SELENBP1 gene (14, 26), pressed SELENBP1 expression in EAC. Bisulfite sequencing we analyzed SNP arrays of 73 EAC samples to deter- of genomic DNA prepared from the EAC cell line Flo-1 mine if the downregulated SELENBP1 expression was and the immortalized esophageal squamous cell line due to decreased DNA copy number at 1q21-q22. No Het-1A indicated that methylation occurred in CpG sites samples were found to have DNA copy number changes at −95 to +90 in the 5′-untranslated region of SELENBP1. less than log2 ratio of −0.737, representing one copy if In contrast, very little if any methylation was detected in ∼30% of the sample DNA contained contaminating nor- the colon cancer cell line SW480 (Fig. 3A). In addition, mal esophagus. Nine patient samples exhibited allelic treatment of Flo-1 cells with the demethylating agent gain (log2 ratio > 0.7) or amplification (log2 ratio > 5-Aza and/or the histone deacetylase inhibitors TSA or 0.848) at the SELENBP1 locus (Supplementary Fig. S1). VPA increased SELENBP1 expression as determined by Eight tumors were examined by both SNP array and oligo- quantitative RT-PCR analysis (Fig. 3B). Combination treat- nucleotide microarray analyses (36T, 40T, 41T, 43T, 44T, ments that resulted in the greatest increase in SELENBP1 45T, 46T, and 50T). The differential expression of mRNA expression levels were not able to produce detectable

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SELENBP1 protein (Fig. 3C). Despite this, treatment of ternative exon and intron retention splicing (27), no bio- Flo-1 cells with demethylating and acetylase-enhancing logical correlation to the different levels of alternatively agents increased sensitivity to apoptosis with cisplatin as spliced fragments in the tumors was found. determined by PARP cleavage (Fig. 3D). These results in- Overexpression of SELENBP1 potentiated the antiproli- dicate that SELENBP1 expression may be regulated at the ferative effect of MSA in vitro and increased chemosensitivity epigenetic level but that the level of inducible expression to cisplatin. As noted earlier, a subset of tumor samples was still too low to detect by protein immunoblotting. (36T and 39T) had higher expression of SELENBP1.To Alternative splicing of SELENBP1 mRNA may diminish examine the biological significance of SELENBP1 expres- gene expression. To determine whether the loss of sion in EAC, Flo-1 cells were stably transfected with ei- SELENBP1 expression was due to internal exon deletion ther an EGFP-SELENBP1 or a corresponding empty or mutation in the coding region of the gene, RT-PCR– vector plasmid, and levels of SELENBP1 expression were amplified cDNA products from patient tumor samples determined by immunoblot analysis. Clone SELENBP1.8 and cell lines were sequenced and compared with the expressed moderate levels, whereas SELENBP1.1 ex- National Center for Biotechnology Information Genbank pressed high levels of EGFP-SELENBP1 compared with database for SELENBP1 using the BLAST program. Five empty vector and untransfected Flo-1 cells (Fig. 5A). Cel- overlapping primer pairs were designed to cover the entire lular proliferation and migration did not change appre- coding sequence (Fig. 4A). PCR amplification of two re- ciably with varying levels of SELENBP1 overexpression gions denoted as the L and mid2 fragments (Fig. 4B) indic- compared with empty vector controls as determined by ated several variants (Fig. 4C), including an exon 4 WST assay (Supplementary Fig. S2) and by wound- − deletion (L, exon 4 ), deletions of both exons 3 and 4 healing assay (data not shown), respectively. Because it − − (L, exon 3 /4 ), and an exon 7 deletion (mid2, exon has been shown that SELENBP1 covalently binds seleni- − 7 ), suggesting the possibility of alternative splicing of um and that a selenium-replete form of SELENBP1 was the SELENBP1 transcript. Whereas deletion of exon 4 required for protein-protein interactions (6), we eval- was in frame, deletion of exons 3 and 4 or of exon 7 in- uated whether selenium supplementation could alter troduced early termination codons that might translate the cellular response to SELENBP1 overexpression in truncated protein products. EAC. Overexpression of SELENBP1 seemed to potentiate Although truncated PCR products were detected in all tu- an antiproliferative response to selenium supplementa- − − mor specimens, tumors with higher levels of exon 3 /4 and tion with MSA but not with NaS, particularly in the higher- − exon 7 deleted PCR products (Fig. 4C) had lower levels of expressing clone SELENBP1.1 (Fig. 5B). full-length PCR products (Fig. 4B). These tumors also To determine if the decrease in cell viability detected af- had reduced expression by both oligonucleotide micro- ter MSA treatment of SELENBP1-overexpressing cells could array and quantitative RT-PCR analyses. Conversely, the enhance the cytotoxicity of CDDP, we examined the sub- two tumors that expressed the highest levels of G1 fraction of treated cells by flow cytometry. Although SELENBP1 (36T and 39T) had lower levels of both exon SELENBP1 overexpression itself did not have an effect, − − − 3 /4 and exon 7 deleted PCR products but higher le- treatment of Flo-1 cells with MSA, CDDP, and the combi- vels of their respective full-length products (Fig. 4D). Al- nation of MSA and CDDP resulted in slight but statistical- though at seemingly reduced levels, alternatively spliced ly significant increases in apoptosis in SELENBP1.1 cells fragments were also detected in normal esophageal and when compared with empty vector–transfected controls gastric tissues (data not shown), suggesting that (Fig. 5C). The proportion of apoptotic cells increased with SELENBP1 is transcriptionally unstable. Interestingly, di- combination treatment, but there was no apparent synergy rect comparison of tumor sample 45T with its matching between MSA and CDDP. The increase in apoptosis fol- normal gastric tissue showed a marked increase in the lowing NaS/CDDP treatment seemed to be SELENBP1 − − − levels of both exon 3 /4 and exon 7 deleted PCR products independent. in tumor cDNA (data not shown). The correlative presence Both MSA and CDDP treatments culminated in PARP − − or absence of the alternatively spliced L (exon 3 /4 ) cleavage, but this measure of apoptosis was not sensitive − and mid2 (exon 7 ) fragments in tumors remains un- enough to detect differences between SELENBP1- and explained. The oligonucleotide microarray probes for control-transfected Flo-1 cells (Fig. 5D). WST-1 analysis SELENBP1 that were used in this study were located 3′ of revealed that overexpression of SELENBP1 enhanced the mid2 fragment (Fig. 4A), and their hybridization cisplatin-mediated cytotoxicity. Additionally, overexpres- should not have been affected by these spliced regions. It sion of SELENBP1 increased cancer cell CDDP sensitivity is possible that alternative splicing affects the stability of when treated with either MSA or NaS in comparison with the resulting transcript. Further investigation of this gene si- the empty vector–transfected control (Fig. 5B). lencing mechanism is warranted. SELENBP1 overexpression potentiated selenium-induced The intronic regions surrounding exons 3, 4, and 7 of senescence. Expression of selenium-binding proteins is as- SELENBP1 were sequenced to examine potential modifica- sociated with accelerated senescence in mice (16). To de- tions near splice junctions. Although a base pair insertion termine if SELENBP1 overexpression activated cellular in intron 3 and several polymorphic sites were identified senescence in human cells, we stained stably transfected (Supplementary Table S4), representing both cassette al- Flo-1 cells for senescence-associated β-galactosidase.

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Whereas baseline senescence was not consistently different sues. The expression levels of the full-length PCR products between empty vector–transfected and SELENBP1- correlated with the SELENBP1 expression levels deter- transfected Flo-1 cells, SELENBP1-transfected cells had in- mined by oligonucleotide microarray and quantitative creased levels of senescence following treatment with MSA RT-PCR. Resulting cDNA sequences from these two alter- but not NaS (Fig. 6A and B). These data suggest that MSA natively spliced variants predicted altered amino acid and SELENBP1 work together to activate this growth- sequences and truncated protein products. Similar NH2- inhibitory pathway. terminally truncated variants have been reported in the closely related mouse homologue Selenbp2, suggesting Discussion that this particular gene is susceptible to alternative splicing (31). These truncated variants may serve to reduce di- In this study, we have shown that expression of rectly the number of full-length transcripts that would be SELENBP1 is decreased in the progression from nondys- translated into functional protein, or they may serve as plastic Barrett's esophagus to Barrett's esophagus with dominant-negative inhibitors of wild-type SELENBP1. high-grade dysplasia and EAC at both mRNA and protein The full significance and functional relevance of these levels. Downregulation of SELENBP1 does not seem to be splicevariantsinEACtumorigenesisisyettobedeter- related to loss of heterozygosity at the 1q21 SELENBP1 mined. Based on these data, strategies to inhibit specific locus as shown by SNP array analysis, suggesting that reg- splicing events might serve as another mechanism for ulation is more likely a transcriptional or posttranscrip- SELENBP1 reexpression in EAC. tional event. Population-based studies suggest a correlation be- Regulation of SELENBP1 may be affected by epigenetic tween higher serum selenium concentration and lower mechanisms. Gene hypermethylation in the “epidermal incidence for a variety of epithelial malignancies (32) differentiation complex,” which is located within 700 kb as well as premalignant diseases such as Barrett's esoph- of the SELENBP1 locus, has been previously reported agus with high-grade dysplasia (2). Comparison of the (28, 29). Bisulfite sequencing of the EAC cell line Flo-1, cancer chemoprotective effects of different forms of sele- which showed very low endogenous levels of SELENBP1, nium in vitro has revealed marked differences between revealed the presence of methylated CpG sites in the 5′ up- methylselenol precursors, such as MSA, and those com- stream sequence of SELENBP1, suggesting that diminished pounds metabolized predominantly to hydrogen sele- SELENBP1 expression might be due, in part, to its prom- nide, such as NaS (33). Although both MSA and NaS oter methylation. In support of this, significantly increased have been shown to inhibit tumor invasion (34, 35), SELENBP1 mRNA expression was observed after 5-Aza– induce apoptosis (36), and cause cell cycle arrest (33, induced demethylation and TSA- or VPA-induced histone 37), methylselenol precursors have been reported to be acetylation. Increased levels of corresponding protein pro- more effective tumor inhibitors than selenite or seleno- ducts were not detected, suggesting that this degree of methionine (38). Whereas MSA treatment induced cell SELENBP1 reexpression was not sufficient to be detected retraction and detachment followed by p53-independent by Western blotting. caspase-mediated apoptosis, NaS treatment resulted in Alternative splicing of primary transcripts is a common cytoplasmic vacuole formation, reactive oxygen species mechanism in higher eukaryotic organisms to increase cell production leading to single-strand DNA breaks, and type–specific and developmental stage–specific protein di- caspase-independent cell death (36). Additionally, MSA versity (27). Unbalanced alternative splicing can lead to the but not NaS increased the potency of SN38 (topoisome- overexpression of antagonistic splice variants of genes in- rase I inhibitor), etoposide (topoisomerase II inhibitor), volved in differentiation, apoptosis, invasion, and metasta- and the microtubule inhibitor paclitaxel in prostate can- sis, often correlating with poor prognosis in cancers (30). cer cells (39). We observed an inverse correlation between the levels of We observed many of these selenium compound– − − two alternatively spliced PCR products (exon 3 /4 or exon specific differences following treatment of Flo-1 EAC cells. − 7 ) and their respective full-length sequences in EAC tis- MSA alone caused cell detachment and subsequent PARP

Fig. 4. PCR amplification and sequence analysis of the coding region of SELENBP1 suggested that full-length mRNA transcripts were alternatively spliced in patient tissue samples and cell lines. A, schematic diagram of the SELENBP1 gene. Boxes and intervening lines represent exons and introns, respectively. Solid black lines both above and below the diagram represent locations of various overlapping PCR products that were generated using the designated primer set. Darker shaded boxes represent exons that were deleted by alternative splicing. Asterisks represent the location of one set of SELENBP1 quantitative RT-PCR primer pairs. Dotted line represents the location of the Affymetrix oligonucleotide array probe sets for SELENBP1. B, agarose gel electrophoreses of PCR-amplified full-length L (21 cycles) and mid2 (25 cycles) products in patient tissues confirmed SELENBP1 expression levels detected by microarray and quantitative RT-PCR analyses. C, extended PCR amplification (35 cycles) and sequencing analyses revealed truncated products for both L (exon 4− and/or exons 3−/4−) and mid2 (exon 7−) primer pairs predominantly in patient tissues with lower levels of full-length products. Flo-1 (EAC), OE33 (EAC), and H460 (lung carcinoma) cell lines were included for comparison. B, nondysplastic Barrett's esophageal tissue; BL, Barrett's with low-grade dysplasia; T, EAC. PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 22 cycles) was used as a loading control. D, integrated densities of electrophoresed PCR products were determined by ImageJ software and plotted as the density ratio of full-length to alternative splice product.

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cleavage, whereas NaS-treated cells showed characteristic ment did not have this differential effect. Although vacuole formation with no apparent PARP cleavage. Al- nontoxic doses of both selenium compounds enhanced though reconstitution of SELENBP1 alone did not signifi- cisplatin cytotoxicity to some extent, the effects were more cantly alter cellular proliferation in Flo-1 cells, addition of pronounced with MSA treatment and were further en- MSA to these cells resulted in significant inhibition com- hanced by SELENBP1 overexpression. In addition, MSA pared with non–SELENBP1-expressing cells. NaS treat- but not NaS treatment of SELENBP1-transfected Flo-1 cells

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Fig. 5. Flo-1 cells were stably transfected with an EGFP-tagged empty vector or SELENBP1-containing plasmid. The transfection efficiency was estimated to be 60% to 70%. A, immunoblot analysis of SELENBP1 protein expression in geneticin-selected clones in the presence or absence of 2.5 μmol/L MSA. β-Actin served as a loading control. B, overexpression of SELENBP1 potentiated the antiproliferative effect of MSA (2.5 μmol/L) and NaS (10 μmol/L) particularly during treatment with CDDP (20 μg/mL) as determined by WST-1 assays. Cells were treated with either MSA or NaS for 72 h and CDDP for 24 h. WST-1 data end points represent four independent wells. Small but statistically significant increases in apoptosis were observed by flow cytometry in SELENBP1-expressing cells treated with 2.5 μmol/L MSA, 20 μg/mL CDDP, or both agents (C) but were not discernible by immunoblot analysis of PARP cleavage (D). β-Actin was used as a loading control. Each flow cytometry data point represents three independently treated wells. All data were confirmed in repeated experiments. *, P < 0.05; †, P < 0.01; ‡, P < 0.005, Student's t test, versus empty vector control.

enhanced senescence as assayed by β-galactosidase stain- SELENBP1 is required for the interaction between the ing. These differences emphasize the importance of the von Hippel-Lindau protein-interacting deubiquitinating chemical form of selenium in cancer chemoprevention enzyme VDU1 and SELENBP1 (6). Many of the tumor- and chemotherapy strategies. suppressive mechanisms analyzed in our studies either The relationship between selenium levels and were enhanced by or were observed only in the presence SELENBP1 expression in cancer progression is still unclear. of MSA. We have also observed increased SELENBP1 ex- Although levels of selenite did not affect the levels of sele- pression following MSA treatment. Our findings suggest nium-binding proteins in rodent cells (40), the level of ex- that selenium affects both SELENBP1 activity and expres- pression of a 58-kDa selenoprotein correlated with the sion in EAC. level of selenite supplementation and with inhibition of Recently, SELENBP1 was shown to contain hypoxia DNA synthesis in mouse mammary epithelial cells (41). response elements in its promoter region and was found Huang et al. (10) have also shown that SELENBP1 expres- to be a target gene for the transcription factor hypoxia- sion is increased with methylselenocysteine treatment in inducible factor-1α (HIF-1α; ref. 45). SELENBP1 mRNA human ovarian cancer cells. It has been reported that sele- and protein expression was increased over 10-fold in the nium compounds promote DNA and histone hypomethy- keratinocytes of transgenic mice expressing constitutive lation (42) and may act as histone deacetylase inhibitors HIF-1α compared with nontransgenic littermates. These in human prostate cancer cells (43, 44), possibly potenti- transgenic mice showed increased resistance in the trans- ating the reexpression of epigenetically silenced genes such formation from papilloma to carcinoma (45). Because as SELENBP1. Selenium may also exert cell growth inhibi- HIF-1α seems to be redox sensitive and the metabolic tion by modifying the function of preexisting proteins. reduction of some forms of selenium resulted in reactive Supporting this is a recent report that selenium-replete oxygen species production (45–47), it is plausible that

2018 Clin Cancer Res; 16(7) April 1, 2010 Clinical Cancer Research

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selenium may also indirectly modify SELENBP1 expres- particularly when cells were also treated with MSA. In sion through HIF-1α. conclusion, diminished SELENBP1 expression in EAC We have found that SELENBP1 is downregulated in the seemed to blunt the cellular response to some forms of se- progression from Barrett's metaplasia to EAC. Although lenium supplementation, and loss of SELENBP1 might be decreased expression of SELENBP1 seemed to be related a marker for limited response to ongoing attempts to use to epigenetic modification, we also observed increased selenium as a chemopreventive or chemosensitization alternative splice products with predicted premature agent. Alternatively, the presence of full-length SELENBP1 truncation in a significant number of tumors with di- in cancer tissues might predict better response to chemo- minished SELENBP1 expression. In vitro overexpression therapy treatment and would support selenium-mediated of SELENBP1 enhanced cisplatin-mediated cell death, secondary chemoprevention strategies.

Fig. 6. SELENBP1 enhanced MSA-induced cell senescence in Flo-1 cells. Stably transfected Flo-1 cells were treated with 2.5 μmol/L MSA, 10 μmol/L NaS, or vehicle for 3 d followed by fixation and staining for senescence-associated β-galactosidase expression. A, representative digital images of treatment groups. Magnification, ×20. B, the number of blue-stained cells versus total cell count per image in three nonoverlapping areas per well was recorded for each treatment group. Although variability was high, these observations were confirmed in triplicate experiments. *, P < 0.05, Student's t test, versus empty vector control.

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Disclosure of Potential Conflicts of Interest Grant Support

No potential conflicts of interest were disclosed. NIH grants 5R01CA071606 (D.G. Beer), 5P30CA046592 (T.J. Giordano), 5K08CA134931(A.J. Bass), and 5K08CA127212 (A.C. Chang) and Thoracic Surgery Foundation for Research and Education (A.C. Chang). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement Acknowledgments in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Laura Lynch and Amrit Misra for their technical assistance in Received 10/20/2009; revised 01/11/2010; accepted 01/25/2010; the experiments presented in this manuscript. published OnlineFirst 03/23/2010.

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Decreased Selenium-Binding Protein 1 in Esophageal Adenocarcinoma Results from Posttranscriptional and Epigenetic Regulation and Affects Chemosensitivity

Amy L. Silvers, Lin Lin, Adam J. Bass, et al.

Clin Cancer Res Published OnlineFirst March 23, 2010.

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