[CANCER RESEARCH 63, 7068–7075, November 1, 2003] Advances in Brief Hypermethylation of XIAP-associated Factor 1, a Putative Tumor Suppressor Gene from the 17p13.2 Locus, in Human Gastric Adenocarcinomas1 Do-Sun Byun, Kyucheol Cho, Byung-Kyu Ryu, Min-Goo Lee, Min-Ju Kang, Hak-Ryul Kim, and Sung-Gil Chi2 Department of Pathology, School of Medicine, Kyung Hee University, Seoul 130-701 [D-S. B., K. C., B-K. R., M-G. L., M-J. K., S-G. C.], and Graduate School of Life Science and Biotechnology, Korea University, Seoul 136-701 [D-S. B., H-R. K.], Korea Abstract gested to play a key role in the aberrantly increased cell viability and resistance to the anticancer therapy in human cancers, whereas over- X-linked inhibitor of apoptosis (XIAP) is the most potent member of the expression seems to suppress apoptosis against a large variety of IAP family that exerts antiapoptotic effects by interfering with the activ- ities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two triggers (4, 5). The human IAP family includes cIAP-1, cIAP-2, mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified XIAP, NAIP, survivin, apollon, ILP2, and livin, and several members to negatively regulate the caspase-inhibiting activity of XIAP. To explore of the human IAP family including XIAP, c-IAP-1, and c-IAP-2 have the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor been shown to be potent inhibitors of caspase-3, -7, and -9 (2, 6, 7). in gastric tumorigenesis, we investigated the expression and mutation Of the eight known human IAP proteins, XIAP is the most potent status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas and versatile inhibitor of caspases and apoptosis. XIAP demonstrates Smac/DIABLO and HtrA2 transcripts were normally expressed in all significant inhibition of apoptosis induced by many triggers, including cancer specimens we examined, XAF1 transcript was not expressed or serum withdrawal and etoposide exposure (8, 9). XIAP possesses present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 three BIR domains and a COOH-terminal RING zinc finger and expression showed a strong correlation with stage and grade of tumors, inhibits both the initiator caspase-9 and the effectors caspase-3 and -7. and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of XIAP mRNA levels are relatively high in the majority of cancer cell 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detect- lines, and high levels of XIAP protein are generally found in renal able alteration in cancers. Whereas loss of heterozygosity within the XAF1 cancer and melanoma cell lines (10, 11). region or somatic mutations of the gene was not detected, expression of The caspase-inhibiting effects of IAPs are antagonized by apoptosis- XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza- promoting proteins. Two mitochondrial proteins, termed Smac/DIABLO 2-deoxycytidine treatment. The 5؅ upstream region of the XAF1 gene and HtrA2, have been identified to promote caspase activation by encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for antagonizing the caspase-inhibitory activity of XIAP (12–14). Smac/ 34 CpG sites in the promoter region revealed a strong association between DIABLO is released from mitochondria together with cytochrome c hypermethylation and gene silencing. Moreover, transcriptional silencing during mitochondria-induced apoptosis. The binding of Smac/DIABLO of XAF1 was tightly associated with hypermethylation of seven CpGs to XIAP is proposed to destabilize the XIAP-caspase interaction by located in the 5؅ proximal region (nucleotides ؊23 to ؊234). Additionally, steric hindrance, resulting in disruption of the XIAP-caspase complex loss or abnormal reduction of XAF1 expression was found to inversely (15, 16). HtrA2 is a mammalian serine protease homologous to the correlate with p53 mutations, suggesting that epigenetic inactivation of bacterial HtrA endoprotease and directly binds to the BIR3 domain of XAF1 and mutational alteration of p53 might be mutually exclusive events XIAP and activates caspases. In a similar manner to Smac/DIABLO, in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to HtrA2 is released from mitochondria into the cytosol and forms the malignant progression of human gastric tumors. complexes with XIAP at high stoichiometry as a result of apoptosis stimulus. These observations demonstrate that Smac/DIABLO and Introduction HtrA2 sensitize cells to apoptosis in response to a number of stimuli by binding to and antagonizing XIAP. Apoptosis is essential for elimination of defective or potentially Very recently, a novel negative regulator of XIAP termed XAF1 dangerous cells and provides a defense against malignant transforma- was isolated based on its ability to bind XIAP (17). Transient over- tion and autoimmunity (1). Several genes critical in the regulation of expression of XAF1 sensitizes tumor cells to the proapototic effects of 3 apoptosis have been identified, including the IAP family (2). The IAP etoposide and reverses the XIAP-mediated inhibition of caspase-3 proteins are a new class of intrinsic cellular regulators of apoptosis activity. In contrast to Smac/DIABLO and HtrA2, XAF1 resides in that are structurally defined by the presence of the evolutionary the nucleus and can effect a marked relocalization of XIAP protein conserved BIR domain (2, 3). Deregulation of IAPs has been sug- from the cytoplasm to the nucleus. The XAF1 gene is located at 17p13.2, approximately 3 cM telomeric to the p53 tumor suppressor Received 4/1/03; revised 8/19/03; accepted 8/27/03. gene, and encodes M 33,100 protein with seven zinc fingers with high The costs of publication of this article were defrayed in part by the payment of page r charges. This article must therefore be hereby marked advertisement in accordance with amino acid similarity with the zinc finger domains of both FLN29 and 18 U.S.C. Section 1734 solely to indicate this fact. TRAF3 (10, 17). XAF1 mRNA is expressed ubiquitously in all normal 1 Supported by a grant (R02-2001-000-00052-0) from the Basic Research Program of the Korea Science and Engineering Foundation and an intramural grant-in-aid from the adult and fetal tissues but is present at very low or undetectable levels Kyung Hee University (2002), Republic of Korea. in various cancer cell lines. Expression analysis using the NCI 60 cell 2 To whom requests for reprints should be addressed, at Department of Pathology, line panel also revealed that cancer cell lines exhibit very low levels College of Medicine, Kyung Hee University, 130-701 Seoul, Republic of Korea. Phone: 82-2-961-0920; Fax: 82-2-961-0277; E-mail: [email protected]. of XAF1 mRNA, whereas the majority of cancer cell lines express 3 The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; relatively high levels of XIAP mRNA, suggesting that deregulation of XIAP, X-linked IAP; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism; obs/exp CpG, observed/expected CpG; XAF1, XIAP-associated factor 1; apoptosis through the loss of XAF1 expression might be important for GAPDH, glyceraldehyde-3-phosphate dehydrogenase. malignant cell survival, and a high level of XIAP to XAF1 expression 7068 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. EPIGENETIC INACTIVATION OF XAF1 IN GASTRIC CARCINOMA in cancer cells might provide a survival advantage through the relative Quantitation was achieved by densitometric scanning of the ethidium bromide- increase of XIAP antiapoptotic function (10). stained gels. Absolute area integrations of the curves representing each spec- Gastric cancer is one of the most commonly diagnosed malignan- imen were then compared after adjustment for GAPDH. Integration and cies worldwide and a leading cause of cancer mortality in certain analysis were performed using the Molecular Analyst software program (Bio- areas, such as Korea, Japan, South America, and Eastern Europe (18). Rad, Hercules, CA). Quantitative PCR was repeated at least three times for each specimen, and the mean was obtained. Although evidence has accumulated indicating the involvement of the Western Blot Assay. Cells were lysed in a lysis buffer containing 20 mM alterations of multiple genes such as p53, K-ras, c-erbB2, K-sam, and Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM E-cadherin, the underlying molecular events that drive the neoplastic ␤ ␮ sodium phosphate, 1 mM -glycerolphosphate, 1 mM Na3VO4,1 g/ml leu- process in gastric cancer are largely undefined (19). In the present peptin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was clarified study, we investigated the expression and mutation status of XAF1, by centrifugation, and 20 ␮g of total protein were supplemented with Laemmli Smac/DIABLO, and HtrA2 in a series of primary gastric adenocarci- buffer and loaded on a 10% SDS-polyacrylamide gel for electrophoresis. XIAP nomas and cell lines to explore the candidacy of these genes as a was detected by immunoblot using an anti-XIAP monoclonal antibody (BD suppressor in gastric carcinogenesis. Our data demonstrate that XAF1 Biosciences, San Diego, CA). Antibody binding was detected by enhanced mRNA expression is lost or significantly down-regulated in a consid- chemiluminescence (Amersham Biosciences, Piscataway, NJ) using a second- erable fraction of gastric cell lines and primary tumors by aberrant ary antibody conjugated to horseradish peroxidase. For stripping, the blots were incubated in a stripping buffer [0.2 M glycine (pH 2.2), 0.1% SDS, and promoter CpG hypermethylation, whereas none of tumors show de- 1% Tween 20] at room temperature for 60 min.