1546 Vol. 7, 1546–1552, June 2001 Clinical Cancer Research Advances in Brief Fez1/Lzts1 Alterations in Gastric Carcinoma1 Andrea Vecchione,2 Hideshi Ishii,2 tected in one case that did not express Fez1/Lzts1. Hyper- Yih-Horng Shiao, Francesco Trapasso, methylation of the CpG island flanking the Fez1/Lzts1 pro- Massimo Rugge, Joseph F. Tamburrino, moter was evident in six of the eight cell lines examined as well as in the normal control. Yoshiki Murakumo, Hansjuerg Alder, 3 Conclusions: Our findings support FEZ1/LZTS1 as a Carlo M. Croce, and Raffaele Baffa candidate tumor suppressor gene at 8p in a subtype of Kimmel Cancer Center, Jefferson Medical College of Thomas gastric cancer and suggest that its inactivation is attributa- Jefferson University, Philadelphia, Pennsylvania 19107 [A. V., H. I., F. T., J. F. T., Y. M., H. A., C. M. C., R. B.]; Laboratory of ble to several factors including genomic deletion and meth- Comparative Carcinogenesis, National Cancer Institute, Frederick ylation. Cancer Research and Development Center, NIH, Frederick, Maryland 21702 [Y-H. S.]; and Department of Pathology, University of Padova, Padova 35126, Italy [M. R.]. Introduction Gastric cancer is the second most common malignant tu- Abstract mor worldwide, with a much higher incidence in Asian than in 4 Purpose: Loss of heterozygosity (LOH) involving the Western countries (1). The international TNM classification (2) short arm of chromosome 8 (8p) is a common feature of the and Lauren’s system (3) classify gastric cancer into two distinct malignant progression of human tumors, including gastric histological types: intestinal (well differentiated) and diffuse cancer. We have cloned and mapped a candidate tumor (poorly differentiated). Genetic differences have been observed suppressor gene, FEZ1/LZTS1, to 8p22. Here we have ana- in intestinal and diffuse gastric cancer, suggesting two different lyzed whether FEZ1/LZTS1 alterations play a role in the pathways of carcinogenesis (4). Allelotyping studies of solid development and progression of gastric carcinoma. tumors, determined by LOH, have shown genomic alterations in Experimental Design: We examined Fez1/Lzts1 expres- gastric cancer at specific chromosomal regions, including sion in 8 gastric carcinoma cell lines by Western blot, and in 3p14–21, 8p21–23, 11p11–21, and 17p11–21 (5–9). Recently, 88 primary gastric carcinomas by immunohistochemistry. we have cloned a novel candidate tumor suppressor gene, FEZ1/ Twenty-six of these 88 primary gastric carcinomas were also LZTS1 (hereafter called FEZ1), on chromosome 8p22 (10). The microdissected and tested for LOH at the FEZ1/LZTS1 locus FEZ1 gene encodes a putative Mr 67,000 leucine-zipper protein and for mutation of the FEZ1/LZTS1 gene. Furthermore, we with similarities to the cAMP-responsive Atf-5 DNA binding studied the FEZ1/LZTS1 gene regulation and transcrip- protein (11). Immunohistochemistry has been a valuable tool in ؅ tional control and the methylation status of the 5 region of evaluating the expression of different tumor suppressor genes, the gene in all 8 gastric carcinoma cell lines. such as p53 and FHIT, in gastric carcinoma (12, 13). In this Results: Fez1/Lzts1 protein was barely detectable in all report, we raise anti-Fez1 polyclonal antibody and analyze 88 of the gastric cancer cell lines tested and was absent or cases of gastric adenocarcinoma to assess Fez1 expression. significantly reduced in 39 of the 88 (44.3%) gastric carci- Results indicate that loss of Fez1 protein is a frequent event in nomas analyzed by immunohistochemistry, with a signifi- gastric cancer, and reduction of its expression significantly cant correlation (P < 0.001) to diffuse histotype. DNA al- lelotyping analysis showed allelic loss in 3 of 17 (18%) and correlates with diffuse gastric carcinoma. A somatic missense microsatellite instability in 4 of 17 (23.5%) cases informative mutation in a FEZ1 allele has been found in a case negative for for D8S261 at the FEZ1/LZTS1 locus. When we compared Fez1 expression. To gain insight into FEZ1 gene regulation and the presence of LOH with Fez1/Lzts1 expression, we found transcriptional control, we examined the genomic organization, loss of protein expression in all three of the tumors with identified a transcriptional start site, and analyzed a positive allelic imbalance at D8S261. A missense mutation was de- regulatory region of FEZ1 transcription. In addition, we ana- lyzed the methylation status of the region flanking the FEZ1 promoter in gastric cancer cells. Our data suggest that altered Fez1 expression plays a role in the progression of a subset of carcinomas of the stomach, and that many factors, including Received 11/28/00; revised 2/23/01; accepted 2/28/01. The costs of publication of this article were defrayed in part by the DNA deletion, point mutations, and methylation seem to be payment of page charges. This article must therefore be hereby marked involved in switching off Fez1 expression. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported partially by USPHS Grant CA83698-01A1 from the National Cancer Institute. 2 A. V. and H. I. contributed equally to this study. 3 To whom requests for reprints should be addressed, at Kimmel Cancer 4 The abbreviations used are: TNM, Tumor-Node-Metastasis; LOH, loss Center, 1015 Walnut Street, Suite 1102A, Philadelphia, PA 19107. of heterozygosity; LZTS1, leucine zipper putative tumor suppressor gene Phone: (215) 955-9072; Fax: (215) 923-1884; E-mail: R_Baffa@lac. 1; MI, microsatellite instability; BAC, bacterial artificial chromosome; jci.tju.edu. TSS, transcriptional start site. Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 1547 Materials and Methods controls. Sections were reacted with biotinylated antirabbit an- Cell Lines and Tissue Samples. Eight human gastric tibody and streptavidin-biotin-peroxidase (Histostain-SP kit; carcinoma derived cell lines (Ags, KATO III, NCI-N87, RF1, Zymed Laboratories, San Francisco, CA). Diaminobenzidine RF48, SNU1, SNU5, and SNU16), transformed human kidney 293 was used as a chromogen substrate. Finally, sections were cells, and cervical cancer HeLaS3 cells were obtained from the washed in distilled water and weakly counterstained with Har- American Type Culture Collection and maintained in the recom- ry’s modified hematoxylin. All sections were examined inde- mended medium. FEZ1 cDNA was ligated in a pcDNA vector pendently by three investigators (A. V., R. B., and M. R.), and (InVitrogen, Carlsbad, CA), and after confirming the DNA se- complete agreement was reached for Fez1 positivity and nega- quence, DNA transfection was performed with the Gene Porter tivity. Any positive reaction was semiquantified into four ϩ ϩ Ϫ reagent (Gene Therapy, Inc., San Francisco, CA) according to the groups: , 96–100% Fez1-positive cells; / , 51–95% Fez1- positive cells; Ϫ/ϩ, 2–50% Fez1-positive cells; and Ϫ, the manufacturer’s instructions. Eighty-eight formalin-fixed, paraffin- tumors in which Ͼ98% of cells did not express Fez1. Associ- embedded specimens of primary adenocarcinomas of the stomach, ations of Fez1 expression with clinicopathological parameters including 47 diffuse-type, 23 intestinal-type, and 18 unclassified were computed using a two-tailed ␹2 statistic or Fisher’s exact tumors were obtained from patients who underwent radical and test where appropriate. Probability of Ͻ0.05 was considered partial gastrectomy as described previously (14). The tumors were statistically significant. classified histologically and staged according to Lauren’s classifi- Microdissection and DNA Extraction. We were able to cation (3) and to the TNM classification of malignant tumors (2), process 26 of the 88 primary tumors for DNA analysis. Serial respectively. 5-␮m, formalin-fixed, paraffin-embedded sections from these Immunoblot Analysis, Immunoprecipitation, and Re- 26 primary gastric cancers and matched normal tissues were combinant Proteins. Protein extraction and immunoblot subjected to microdissection. This was followed by deparaf- analyses were performed as described (15). Briefly, the protein finization and DNA extraction as described previously (14). concentration was measured with the Protein Assay reagent DNA was quantified in a fluorometer using Pico green dsDNA (Bio-Rad Laboratories, Melville, NY). For immunoblot analy- quantitation reagent (Molecular Probes, Eugene, OR). ␮ sis, 50–100 g of proteins were subjected to SDS-PAGE and LOH Study and Mutation Analysis. PCR amplifica- transferred to a nitrocellulose membrane. After blocking with tions using 5Ј fluorescence-labeled primers for microsatellite 5% nonfat dry milk, the membrane was incubated with a 1:1000 loci (Research Genetics, Huntsville, AL) with tumor and normal dilution of rabbit anti-Fez1 polyclonal antibody. The anti-Fez1 template DNAs were performed as reported (10). MI was inter- antibody was raised in rabbits against glutathione S-transferase- preted when a tumor sample showed a novel abnormal peak fusion Fez1 protein corresponding with nucleotides 1–1128, when compared with the corresponding normal control DNA which was expressed in Escherichia coli and purified with a (8). Mutation was analyzed by direct sequencing of PCR frag- glutathione column (Amersham Pharmacia, Piscataway, NJ). ments with microdissected DNA as a template. Thirteen sets of Mouse monoclonal anti-V5 antibody (InVitrogen) was also primers covering the entire open reading frame (exons 1–3) used. After incubation with horseradish peroxidase-conjugated were used for PCR in a 20-␮l reaction on a 96-well plate with secondary antibodies, membranes were subjected to the chemi- a cycle of 94°C for 2 min; followed by 35 cycles of 94°C for luminescence detection system (Amersham Pharmacia) with 20 s, 62°C for 15 s, and 72°C for 1 min; and a cycle of 72°C for X-ray film. After stripping, the blots were reprobed with anti 1 min. The primer sequences are available upon request. The ␤-actin (Sigma Chemical Co.-Aldrich, St. Louis, MO), followed PCR products were purified with the QIAquick 96 PCR purifi- by incubation with antimouse secondary antibody.