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The Tumor Suppressor Gene WWOX at FRA16D Is Involved in Pancreatic Carcinogenesis

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The Tumor Suppressor Gene WWOX at FRA16D Is Involved in Pancreatic Carcinogenesis Vol. 10, 2459–2465, April 1, 2004 Clinical Cancer Research 2459 The Tumor Suppressor Gene WWOX at FRA16D Is Involved in Pancreatic Carcinogenesis Tamotsu Kuroki,1 Sai Yendamuri,1 Furthermore, transfection with WWOX inhibited colony for- Francesco Trapasso,1 Ayumi Matsuyama,1 mation of pancreatic cancer cell lines by triggering apoptosis. Rami I. Aqeilan,1 Hansjuerg Alder,1 Conclusion: These results indicate that the WWOX gene 1 1 2 may play an important role in pancreatic tumor devel- Shashi Rattan, Rossano Cesari, Maria L. Nolli, opment. Noel N. Williams,3 Masaki Mori,4 5 1 Takashi Kanematsu, and Carlo M. Croce INTRODUCTION 1 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pancreatic cancer is one of the most common human can- Pennsylvania; 2Areta International srl, Gerenzano, Italy; 3Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania; cers and carries a poor prognosis (1). Although complete sur- 4Department of Surgery, Medical Institute of Bioregulation, Kyushu gical resection is the only therapeutic option for a long-term University, Beppu, Japan; and 5Department of Surgery, Nagasaki survival, the 5-year survival rate with curative resection is University, Graduate School of Biomedical Sciences, Nagasaki, Japan Ͻ20% (2). The precise molecular mechanisms of development and/or progression of pancreatic cancer remain unknown, al- ABSTRACT though multiple genetic and epigenetic changes have been de- tected in pancreatic cancer. Therefore, additional elucidation of Purpose: WWOX (WW domain containing oxidoreduc- the molecular mechanisms involved in pancreatic cancer is tase) is a tumor suppressor gene that maps to the common urgently needed for more effective treatment. fragile site FRA16D. We showed previously that WWOX is The tumor suppressor gene FHIT spans the FRA3B fragile frequently altered in human lung and esophageal cancers. site, the most active common chromosome fragile site of the The purpose of this study was to delineate more precisely the human genome, and abnormal FHIT transcripts have been de- role of WWOX in pancreatic carcinogenesis. tected in many human cancers, including pancreatic cancer Experimental Design: We analyzed 15 paired pancreatic (3–6). Additionally, our previous study demonstrated that FHIT adenocarcinoma samples and 9 pancreatic cancer cell lines overexpression enhanced the susceptibility of pancreatic cancer for WWOX alterations. Colony assay and cell cycle analysis cells to exogenous inducers of apoptosis (7). Recently, in the were also performed to evaluate the role of the WWOX as a WWOX (WW domain containing oxidoreductase), a candidate tumor suppressor gene. tumor suppressor gene, chromosome 16q23.3–24.1 was iso- Results: Loss of heterozygosity at the WWOX locus was lated. This gene maps at the location of FRA16D, one of the observed in 4 primary tumors (27%). Methylation analysis most active common chromosomal fragile sites (8–11). The showed that site-specific promoter hypermethylation was WWOX gene is similar to the FHIT gene in that both genes are detected in 2 cell lines (22%) and treatment with the de- Ͼ1 Mb and encompass very active common fragile sites, both methylating agent 5-aza-2؅-deoxycytidine demonstrated an genes show frequent allelic loss and/or loss of homozygous increase in the expression of WWOX. In addition, 2 primary deletion region in several human cancers, and both frequently tumor samples (13%) showed promoter hypermethylation show aberrant transcripts (5, 10, 11). Because increased risk of including the position of site-specific methylation. Tran- pancreatic cancer is associated with cigarette smoking and be- scripts missing WWOX exons were detected in 4 cell lines cause the carcinogens present in tobacco smoke cause deletions (44%) and in 2 tumor samples (13%). Real-time reverse at the FHIT/FRA3B loci (5, 6), we hypothesized that the WWOX transcription PCR revealed a significant reduction of gene may play a role in pancreatic cancer. In this report, we WWOX expression in all of the cell lines and in 6 primary describe that WWOX is altered frequently in pancreatic cancer tumors (40%). Western blot analysis showed a significant by genetic and/or epigenetic changes and is involved in pancre- reduction of the WWOX protein in all of the cell lines. atic carcinogenesis. MATERIALS AND METHODS Received 8/26/03; revised 12/16/03; accepted 1/2/04. Cell Lines and Tissues. Human pancreatic cancer cell Grant support: USPHS Grant CA56036 from the National Cancer lines Panc1, AsPc1, CFPAC1, MiaPaca2, Capan2, BxPc3, Institute. Hs766T, and Su.86.86 were obtained from the American Type The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Culture Collection. PSN1 was obtained from the European advertisement in accordance with 18 U.S.C. Section 1734 solely to Collection of Cell Cultures. All of the cell lines were maintained indicate this fact. in RPMI 1640 with 10% fetal bovine serum at 37°C and 5% Note: T. Kuroki and S. Yendamuri contributed equally to this study. CO . Tumors and corresponding noncancerous tissues were Requests for reprints: Carlo M. Croce, Kimmel Cancer Institute, 2 Jefferson Medical College, Thomas Jefferson University, BLSB Room obtained from 15 patients who underwent surgery for pancreatic 1050, 233S 10th Street, Philadelphia, PA 19107-5799. Phone: (215) 503- adenocarcinoma. The tissue samples were excised and were 4645, Fax: (215) 923-3528; E-mail: [email protected]. immediately stored at Ϫ80°C. DNA was extracted from each of Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. 2460 WWOX Alterations in Pancreatic Carcinoma the 9 cell lines and from the 15 paired pancreatic tissues ac- Biosystems Prism 377 DNA sequencing system. Two primer cording to methods described previously (12). RNA was ex- sets were designed to amplify the entire open reading frame. tracted with the Trizol reagent (Invitrogen Life Technologies, Methylation Analysis. Genomic DNA was treated with Inc., Carlsbad, CA) per the manufacturer’s recommendations. sodium bisulfite according to methods described previously Loss of Heterozygosity (LOH) Analysis. Allelic losses (15). Briefly, 1 ␮g of the genomic DNA was denatured by 2 M were analyzed using a PCR approach, with primers amplifying NaOH, and then incubated in 3 M sodium bisulfite and 10 mM polymorphic microsatellites internal to WWOX at loci D16S3029, hydroquinone for 17 h at 55°C. Bisulfite-treated DNA was D16S3096, D16S504, and D16S518, as described elsewhere (13). extracted using a genomic DNA clean-up kit (Promega, Madi- Briefly, the primer sequences were obtained from the Genome son, WI). Modified DNA was amplified using nested PCR that Database and primers were labeled using 5Ј-fluorescein phosphora- amplify the putative promoter region, including a CpG-rich midite or 5Ј-tetrachlorofluorescein phosphoramidite for microsat- area surrounding the translation start codon (11). Primers for Ј ellite loci, as described by Ishii et al. (14). PCR was performed on the nested PCR amplification were 5 -TAGTTTTTATTAT- Ј Ј 50 ng of DNA for each sample using the conditions described for TATTAGTTTTTATTATT-3 (forward primer) and 5 -ACT- Ј the mutation search. PCR products were denatured in formamide CATCCTCACTATCCATATCAT-3 (reverse primer) for the Ј for 5 min at 95°C and then loaded on a 6% denaturing gel on the first PCR, and 5 -AGTTTTTATTATTATGAGTTTTTATTA- Ј Ј Applied Biosystems 373 DNA sequencer. LOH was analyzed AAT-3 (forward primer) and 5 -CCATATCATCCAAC- Ј using the Applied Biosystems Prism Genescan and the Applied CCCACATA-3 (reverse primer) for the second PCR. PCR was ␮ ϫ Biosystems PRISM GENETYPER ANALYSIS software (Perkin- performed in 50- l reaction volumes containing 1 PCR buffer II (Perkin-Elmer, Branchburg, NJ), 2 mM MgCl , 0.2 mM of Elmer/Applied Biosystems). Cases were defined as having LOH 2 each deoxynucleoside triphosphate, 10 pmol of each primer, 1 when an allele peak signal from tumor DNA was reduced by 50% unit of AmpliTaq Gold polymerase (Perkin-Elmer), and 50 ng compared with the normal counterpart. bisulfite-modified DNA. PCR cycling conditions were 10 min at Mutation Screening of the WWOX Gene. Nine pairs of 95°C, 35 cycles of a 30-s denaturation at 94°C, a 30-s annealing primers for individual exon amplification and screening were at 55°C, a 60-s extension at 72°C, and a final extension step of used for PCR screening with genomic DNA. The primers for 5 min at 72°C. PCR products were subcloned into the pGEM-T each exon are specified in GenBank (accession numbers Easy vector (Promega), and six clones were sequenced. AF325423–325432). The PCRs were performed with the same Real-Time Reverse Transcription PCR. Real-time re- conditions as those described for the LOH analysis. The PCR verse transcription PCR was performed using primers designed products were purified using the QIAquick PCR purification kit to span intron 6. The forward primer on exon 6 was 5Ј-CGTG- (Qiagen, Inc., Valencia, CA), and sequencing reactions and CAGCATTTTGCTGAAG-3Ј and the reverse primer on exon 7 analysis were performed using the Applied Biosystems Prism was 5Ј-AGTTGCTGCGTTGCACACA-3Ј. The probe sequence BigDye terminator reaction chemistry on a Perkin-Elmer Gene 5Ј-AGGCCAAGAATGTGCCTCTTCATGTGC-3Ј was labeled Amp PCR system 9600 and the Applied Biosystems Prism 377 with the reporter dye VIC. 6-Carboxytetramethylrhodamine was DNA sequencing system (Applied Biosystems, Inc., Foster used as the quencher. ␤-Actin was used as an internal control. City, CA). Thirty-five cycles were run with an annealing temperature of Nested Real-Time Reverse Transcription PCR Analysis 62°C and an elongation time of 30 s. All of the reactions were of the WWOX Transcripts. cDNA was synthesized from 2 performed in an ABI PRISM 7000 Sequence Detection System ␮g of total RNA and real-time reverse transcription PCR was (Applied Biosystems). Each sample was performed in triplicate. performed as described previously (13).
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