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Loss of WWOX Expression in Gastric Carcinoma

¹ Kimmel Cancer Institute and ² Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, and ³ Department of Medicine and Therapeutics, University of Aberdeen Medical School, Aberdeen, United Kingdom

	ABSTRACT

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Purpose: WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3–24.1. To investigate the role of the WWOX gene in the development of gastric carcinoma, we examined a large series of primary adenocarcinomas and nine gastric cancer cell lines for the expression of Wwox.

Experimental Design: Loss of heterozygosity, reverse-transcription-PCR, and immunohistochemistry were used to assess the role of WWOX in stomach cancer. A total of 81 primary gastric adenocarcinoma were analyzed.

Results: Loss of heterozygosity was observed in 31% of the cases and loss of Wwox protein expression was found in 65% of gastric adenocarcinoma primary specimens and 33% of gastric cancer cell lines. In addition, we found a high correlation between Wwox and Fhit protein expression.

Conclusions: Our results indicate that alterations of the WWOX gene may be involved quite frequently in gastric tumorigenesis. Our data could be used in future studies to develop diagnostic and targeted therapy of stomach cancer.

	INTRODUCTION

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Gastric cancer was considered the leading cause of cancer-related death in the United States in 1930. Currently, stomach cancer is about one-fourth as common as it was then. Despite this, gastric cancer accounts for approximately 12% of all cancer deaths and remains the world’s second leading cause of cancer mortality behind lung cancer (1) . Higher rates are observed in Japan, China, Eastern Europe, and South America. Ninety percent of stomach cancers are classified as adenocarcinomas. Gastric adenocarcinoma is classified into the following two distinct histological types: a well-differentiated or intestinal type adenocarcinoma and a poorly differentiated or diffuse type adenocarcinoma (2) . Adenocarcinoma of the stomach is associated with specific risk factors, particularly with Helicobacter pylori infection and also with alcohol consumption, tobacco smoking, increased salt and nitrate intake, and decreased antioxidant vitamin intake (1) .

Allelotyping studies of solid tumors, determined by studying of loss of heterozygosity (LOH), and cytogenetic techniques, such as fluorescence in situ hybridization and comparative genomic hybridization, have shown genomic alterations in gastric cancer at specific regions, including 3p, 3q, 5q, 8p, 20pq, 15q, 16p, 16q, 18q and others (3, 4, 5) . Losses on these regions often involve tumor suppressor genes such as FHIT (3p14), APC, MCC (5q21), or DCC (18q21; Refs. 6, 7, 8 ). Allelic losses and homozygous deletions at 16q23 were reported in several tumor types, including adenocarcinomas of stomach (9) . Interestingly, this region also contains FRA16D common fragile site, one of the most active fragile sites in humans (10) . In addition, FRA16D is bracketed by four translocation breakpoints involved in multiple myeloma (11) . Taken together, these results suggest that 16q may contain a tumor suppressor gene associated with cancer initiation or progression including gastric cancer. Indeed, it has been shown by microcell-mediated chromosome transfer that chromosomal region 16q23–24 is a strong suppressor of the metastatic activity of a prostatic cancer cell line (12) .

Recently, the WWOX gene, WW domain-containing oxido-reductase, was mapped to 16q23.3–24.1 (13) . WWOX, also known as FORII (14) , encompasses the active chromosome common fragile site, FRA16D, and is deleted in a number of cancer types including breast, ovarian, prostate, esophageal, lung, and pancreatic carcinomas (13 , 15, 16, 17, 18) . Low, undetectable expression or aberrant transcripts of WWOX were reported in a number of tumor cell lines of different origins (16 , 19) . In addition, Bednarek et al. (20) reported that ectopic Wwox expression strongly inhibits anchorage-independent growth in soft agar of breast cancer cell lines and that Wwox dramatically inhibits tumorigenicity of breast cancer cells in vivo. Altogether, these findings suggest that WWOX is a candidate tumor suppressor gene. The location of WWOX in a fragile site and its inactivation pattern are similar to that of the FHIT gene. The FHIT gene located at 3p14 encompasses the most common human fragile site, FRA3B (21) . FHIT was shown to be inactivated in a significant number of gastric cancers (6 , 22) , and the Fhit protein has lost its expression in large fraction of gastric adenocarcinomas (23) . To identify additional genetic changes involved in gastric cancer pathogenesis, we studied the expression of Wwox in gastric adenocarcinoma.

	MATERIALS AND METHODS

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Cell Lines and Tissues.
Human gastric cancer cell lines Sun1, Sun5, Sun16, RF1, RF48, AgS, Kato3, NCI-N87, and HS746T were obtained from the American Type Culture Collection. All cell lines were maintained in RPMI 1640 with 10% fetal bovine serum at 37°C and 5% CO₂. Fresh frozen tumor and corresponding noncancerous tissue was obtained from 26 pairs of normal and tumor tissues resected from stomach cancer patients at Istituto Regina Elena (Rome, Italy). Samples were taken immediately after surgery and stored at –80°C. DNA was extracted from each of the nine cell lines and from the 26-paired gastric tissues, according to methods described previously (22) . RNA was extracted with the TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsland, CA) according to the manufacturer’s recommendations. For the immunohistochemistry study, 55 cancer cases of fixed gastric tumors and normal tissues were obtained from paraffin archives of the Division of Surgical Pathology at Thomas Jefferson University Hospital, Philadelphia.

LOH Analysis.
Allelic losses were analyzed by PCR amplification with primers amplifying polymorphic microsatellites internal to WWOX at loci D16S3029, D16S3096, D16S504, and D16S518 as described elsewhere (15) . The primer sequences were obtained from the Genome Database, and primers were labeled using 5'-fluorescein phosphoramidite or 5'-tetrachlorofluorescein phosphoramidite for microsatellite loci, as described by Kuroki et al. (15) . PCR was performed on 50 ng of DNA for each sample using conditions described for the mutation search (described below). PCR products were denatured in formamide for 5 min at 95°C and then loaded on a 6% denaturing gel on the Applied Biosystems, Inc. (Foster City, CA) 373 DNA sequencer. LOH was detected by using the Applied Biosystems Prism Genescan and the Applied Biosystems PRISM GENETYPER ANALYSIS software (Perkin-Elmer/Applied Biosystems). Cases were defined as LOH when an allele peak signal from tumor DNA was reduced by 50% compared with the normal counterpart.

Mutation Screening of the WWOX Gene.
Nine pairs of primers, a pair flanking each WWOX exon, were used for PCR amplification with genomic DNA. The primers for each exon are specified in GenBank (accession numbers AF325423–325432). The PCRs were performed under the same conditions as those described above for the LOH analysis. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA), and sequencing reactions and analysis were done using the Applied Biosystems Prism BigDye terminator reaction chemistry on a Perkin-Elmer Gene Amp PCR system 9600 and the Applied Biosystems Prism 377 DNA sequencing system.

Nested Reverse-Transcription (RT)-PCR Analysis of WWOX Transcripts.
cDNA was synthesized from 2 µg of total RNA, and RT-PCR was performed as described previously (15) . Actin amplification served as a control for cDNA quality. One µl of cDNA was used for the first PCR amplification with WWOX-specific primers (forward primer, 5'-AGTTCCTGAGCGAGTGGACC-3'; reverse primer, 5'-TTACTTTCA-AACAGGCCACCAC-3') in a volume of 50 µl containing 20 pmol of each primer, 2.5 mM MgCl₂, 1.5 mM deoxynucleoside triphosphate mix, 1 x PCR buffer, and 2 units of AmpliTaq Gold (Perkin-Elmer). PCR cycles included one cycle of 95°C for 8 min followed by 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min, and a final extension step of 72°C for 5 min in a Perkin-Elmer Gene Amp PCR system 9600. One µl of the first PCR amplification product was used in a second PCR amplification with WWOX-nested primers (forward primer, 5'-AGGTGCCTCCACAGTC-3'; reverse primer, 5'-GTGTGTGCCCATCCGCTCT-3') under the same conditions as the first PCR. The amplified products were analyzed by electrophoresis on a 1.5% agarose gel. DNA bands corresponding to the normal and abnormal size WWOX transcripts were excised from the gel, purified using the QIAquick gel extraction kit (Qiagen), and sequenced on the Applied Biosystems Prism 377 DNA sequencing system. Two primer sets were designed to amplify the entire open-reading frame.

Immunoblotting.
All nine gastric cancer cell lines were lysed using NP40 lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 0.5% NP40, and protease inhibitors. Lysates were resolved by SDS-PAGE and immunodetected using mouse monoclonal anti-Wwox antibody. Mouse monoclonal anti-Tubulin antibody (Oncogene, Cambridge, MA) was used as a control for protein loading. Normal stomach protein lysates were purchased from Clontech (Palo Alto, CA). Monoclonal antibody against the human Wwox protein was raised using a glutathione S-transferase-Wwox fusion protein using standard protocols (18) .

Immunohistochemistry.
Polyclonal glutathione S-transferase-Wwox fusion protein antiserum was prepared in a rabbit using glutathione S-transferase-Wwox protein purified from bacterial cultures. The antiserum was tittered for detection of Wwox in human cells with intact or deleted Wwox genes (S-Y. Han and K. Huebner). The antiserum detects recombinant and endogenous human Wwox, as well as murine Wwox, at 1:5000–1:12,000 dilutions on immunoblots. Specificity for detection of Wwox by immunohistochemistry was determined by blocking detection of the cytoplasmic Wwox protein by preincubation or concurrent incubation with 0.5–1 µg of purified Wwox (data not shown). Immunohistochemical studies were performed using an automated immunostainer (DAKO). Immunohistochemical staining of Wwox (dilution, 1:12,000) was done after antigen retrieval with 10 mM citrate buffer (pH 6.0) in a microwave oven. Detection was done with streptavidin-biotin complex using the LSAB2 system (DAKO) with diaminobenzidine as chromogen. Slides were evaluated and scored by a specialized pathologist (P. Edmonds).

Statistical Analyses.
We used Spearman (nonparametric) correlation to correlate Wwox expression with that of Fhit or with tumor histological grade and tumor stage. The Ps for the analyses with all 55 tumors were two-tailed.

	RESULTS

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LOH and Mutation Analysis.
LOH analysis was performed to assess a correlation between WWOX expression and allelic losses at 16q. We studied LOH using four microsatellite markers within the WWOX gene. The loci D16S3029, D16S3096, and D16S504 are located within intron 8, and D16S518 is located in exon 1 (15) . All 26 cases were informative for at least one of the loci examined, and in 8 cases (31%), LOH was detected (Table 1) . Representative results of LOH analysis are shown in Fig. 1A . To determine whether the WWOX gene was the target of functional inactivation in gastric cancer, we screened for somatic mutations of the WWOX gene by direct sequencing of PCR products. We amplified all nine WWOX exons in tumors and their normal counterpart samples for each patient and from nine human gastric cancer cell lines. We did not detect somatic point mutations in cell lines and clinical samples. However, we found a number of polymorphisms that were reported previously (16) such as C121T in exon 1, C418T in exon 4, A660G in exon 6, C969G in exon 8, and G1403C and T1497G in exon 9. In addition, we found a silent mutation in exon 4 of case 3 where A531 was replaced by G.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, LOH analysis of WWOX gene locus in gastric cancer. Representative loss of heterozygosity analysis of case 20 at the D16S3096 locus. T, tumor; N, corresponding normal tissue. Arrow indicates loss of heterozygosity at D16S3096. B, analysis of the WWOX expression by nested reverse transcription-PCR. A representative experiment showing expression of WWOX in gastric cancer in normal (N) and their tumor (T) counterpart sample for each patient. ACTIN expression was used as a loading control. C, analysis of WW domain-containing oxidoreductase (Wwox) protein expression in stomach cancer. Cells were lysed and immunoblotted using mouse monoclonal anti-Wwox. Anti-Tubulin was used as a control for protein loading. St. Cont, stomach control.

Wwox Protein Expression in Stomach Carcinoma.
To examine Wwox protein expression in stomach cancer, nine gastric carcinoma cell lines were analyzed by immunoblotting using monoclonal anti-Wwox antibody. Normal stomach tissue lysate was used as a positive control. Three cell lines (RF-1, RF-48, and Hs746T) showed absence of Wwox protein expression (Fig. 1C) . The remaining cell lines expressed reduced levels of full-length Wwox when compared with the normal tissue (Fig. 1C) .

To further assess the status of Wwox protein expression in stomach cancer, we performed immunohistochemical staining of Wwox using polyclonal anti-Wwox antibody. We analyzed a second set of 55 patient samples with gastric adenocarcinoma by immunostaining. The same paraffin-embedded tissues had been studied previously for Fhit expression (23) . Tumors were evaluated for histological grade, nuclear grade, disease stage, Lauran classification, presence of signet ring cells, and Tumor-Node-Metastasis classification as well as for survival length after diagnosis. All histological and clinical features and classifications were published previously (23) . Normal gastric tissue was frequently included in sections, serving as an internal control for Wwox expression. Positive Wwox staining was observed in normal gastric glands (Fig. 2A) . The intensity of Wwox staining in normal tissues ranged between moderately (2+) or weakly (1+) positive (Table 2) . Diffuse cytoplasmic staining was characteristically observed in these normal glands (Fig. 2) . Intestinal metaplasia in the stomach, which is a recognized precursor of gastric carcinoma, showed a loss of Wwox protein expression, although adjacent normal glands were still positive for Wwox expression (Fig. 2B) . Thirty-six adenocarcinomas (65%) were completely negative for Wwox protein (Table 2) . Among these, 78% of diffuse gastric adenocarcinomas were negative whereas 60% of the intestinal type showed loss of Wwox expression (Fig. 2, C and D) . Twelve tumors (22%) showed weak staining of Wwox (Fig. 2E) . Seven tumors (15%) exhibited moderate expression of Wwox (Fig. 2F) , comparable with normal gland staining.

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. WW domain-containing oxidoreductase (Wwox) immunostaining in gastric adenocarcinoma. A, uniform Wwox staining in normal gastric glands (x200). B, early gastric cancer showing loss of Wwox protein expression (x200). Wwox is detected in normal glands (arrow). C and D, diffuse; signet ring (C), and intestinal (D) gastric carcinoma showing absence of Wwox expression (x200). E and F, intestinal carcinoma showing weakly and moderately positive Wwox staining (x400).

	DISCUSSION

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Several reports have suggested that 16q23 contains a tumor suppressor gene involved in multiple tumor types (10 , 12) . The WWOX gene was mapped to this region and a loss of WWOX RNA expression has been observed in several carcinomas (13 , 15, 16, 17, 18) . Here, we provide the first evidence that loss of Wwox protein expression may play a role in WWOX function in tumorigenesis. All previous reports that described Wwox alterations based their conclusions on PCR and nested-PCR experiments. Although, the status of DNA and RNA is important, the actual determinant of a gene function is its protein expression. Using immunoblotting and immunohistochemistry experiments, we showed that Wwox protein is absent or greatly reduced in 33% of gastric cancer cell lines and 65% of gastric adenocarcinoma primary specimens (Fig. 1C ; Fig. 2 ). We showed that in 81 primary adenocarcinomas of the stomach, the WWOX gene might play a role in gastric cancer development. Our findings of a high incidence (31%) of LOH at 16q23.3 in gastric cancer (Fig. 1A ; Table 1 ) and loss of Wwox protein expression (Fig. 1C ; Fig. 2 ) contributed to the fact that WWOX encompasses fragile region FRA16D, one of the most active common fragile sites. Almost 80% of diffuse gastric cancers lost Wwox protein expression whereas >60% of intestinal type lost Wwox expression. Our RT-PCR data on additional gastric adenocarcinoma samples showed only 33% of loss and/or alteration of WWOX expression. One possible explanation of this difference between immunohistochemistry and RT-PCR results is that RNAs of tumor samples often contains traces of RNA of normal tissues.

Furthermore, we found a high correlation between loss of Fhit and Wwox expression in gastric carcinomas (Table 3A) . Our findings support the idea that alterations of Wwox and Fhit are likely attributable to their locations in active fragile sites. FHIT is a tumor suppressor gene that encompasses the most active fragile site, FRA3B (21) . Interestingly, WWOX exhibits many features similar to FHIT. It is a large gene that spans a common fragile site and lies in a region of loss of heterozygosity. Both genes also span a region of homozygous deletions in a number of cancers and exhibit a high frequency of aberrant RT-PCR products in tumors. Functional analysis of the FHIT gene showed that Fhit causes apoptosis and suppresses tumorigenicity in vivo (reviewed in Ref. 21 ). Similarly, WWOX suppresses tumor growth (20) , and it was identified as a proapoptotic partner that interacts with p53 (24) .

Stomach cancer is strongly associated with exposure to environmental carcinogens. It has been suggested that carcinogen-induced alterations are more frequent at fragile sites and that genes located at these fragile sites are frequently altered because of such exposures. Indeed, Yakicier et al. (25) reported that genetic alterations at FRA16D are associated with exposure to environmental carcinogens such as aflatoxin B1. In addition, we recently showed that WWOX is deleted or altered in esophageal and lung carcinomas (15 , 17) . Generally, carcinogens can cause DNA damage that may lead to inactivation of tumor suppressor genes such as FHIT and WWOX. The ability of cells to repair this damage may therefore be linked to the loss of function of these genes. Indeed, it was shown that there is a significant correlation between the losses of mismatch-repair proteins, such as Msh2 and Brca2, and Fhit in colorectal and breast cancer (reviewed in 21). Therefore, it would be interesting to determine whether deletions in the WWOX gene may also occur with increased frequency in gene-repair-deficient cancers.

Our results show that the WWOX gene is inactivated in the majority of gastric tumors and suggest that carcinogens could exert their carcinogenic potential on the gastric mucosa by affecting, directly or indirectly, the fragile region contained in the WWOX gene, thus causing breakage and/or deletion within this gene. The observation that the Wwox protein is absent in 65% of primary gastric adenocarcinomas indicates that alterations of the WWOX gene may contribute to gastric tumorigenesis.

	ACKNOWLEDGMENTS

 
We thank Roberto Santoro and Eugenio Santoro for providing tumor samples (Rome, Italy). We also thank Jean Letofsky for technical assistance.

	FOOTNOTES

 
Grant support: National Cancer Institute Grant P01 CA77738.

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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: R. Aqeilan and T. Kuroki contributed equally to this study.

Requests for reprints: Rami I. Aqeilan, Kimmel Cancer Institute, 233 South 10th Street, BLSB room 1050, Philadelphia, PA 19107. E-mail: Carlo.Croce{at}mail.tju.edu

Received 11/17/03; revised 1/14/04; accepted 1/27/04.

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