Oncogene (2001) 20, 5062 ± 5066 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Identi®cation of AXUD1, a novel human gene induced by AXIN1 and its reduced expression in human carcinomas of the lung, liver, colon and kidney

Hideyuki Ishiguro1,2, Tatsuhiko Tsunoda3, Toshihiro Tanaka3, Yoshitaka Fujii2, Yusuke Nakamura1 and Yoichi Furukawa*,1

1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; 2Second Department of Surgery, Nagoya City University School of Medicine, 1 Kawasumi Mizuho-ku, Nagoya 467-8601, Japan; 3SNP Research Center, Riken (Institute of Physical and Chemical Research), 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Axin, an important regulator of b-catenin, is frequently Phosphorylated b-catenin is quickly degraded via an mutated in human hepatocellular carcinomas (HCCs), ubiquitin-proteosome pathway in the cytoplasm (Beh- and transduction of the wild-type Axin gene (AXIN1) rens et al., 1998; Nakamura, 1997). Upon Wnt induces apoptosis in HCC cells as well as in colon cancer signaling, phosphorylation of b-catenin is suppressed cells. To investigate the detailed biological function of through unde®ned mechanisms, when b-catenin func- Axin, we searched on a cDNA microarray for genes tions as a transcriptional regulator in the nucleus whose expression was altered by transfer of wild-type coupled with molecules of the Tcf/LEF family (He et AXIN1 into colon-cancer cell line LoVo. Among the al., 1998; Morin et al., 1997). Until now a number of genes showing altered expression, we focused on one, downstream genes such as c-Myc (He et al., 1998), termed AXUD1 (AXIN1 up-regulated1), that revealed cyclinD1 (Shtutman et al., 1999; Tetsu and McCor- enhanced expression in response to exogeneously ex- mick, 1999), c-jun, fra-1, uPAR, ZO-1 (Mann et al., pressed AXIN1 but not to LacZ, a control gene. The 1999), MMP7 (Brabletz et al., 1999), NBL4 (Ishiguro AXUD1 gene consists of ®ve exons and encodes a et al., 2000), DRCTNNB1A (Kawasoe et al., 2000), transcript with an open reading frame of 1767 bp. A 3.2- and MCP-3 (Fujita et al., 2000) have been reported. kb transcript of AXUD1 was expressed in all human However, the precise regulatory mechanisms remain to tissues examined, most abundantly in lung, placenta, be resolved. skeletal muscle, pancreas and leukocyte. By radiation- Impaired regulation of the Wnt signaling pathway is hybrid mapping we assigned its chromosomal location at often involved in tumors that arise in human colon, 3p22, a region where frequent loss of heterozygosity has liver, prostate, uterus, stomach, skin and brain (Chan been reported in lung, renal, prostate, breast and cervical et al., 1999; Fukuchi et al., 1998; Iwao et al., 1998; cancers. AXUD1 was frequently down-regulated in lung, Miyoshi et al., 1998; Smits et al., 2000; Voeller et al., kidney, liver and colon cancers compared with their 1998; Zurawel et al., 1998). Deregulation of this corresponding normal tissues, suggesting that AXUD1 pathway can result from genetic changes in the APC, may have a tumor-suppressor function in those organs. CTNNB1, AXIN1 or AXIN2 genes (da Costa et al., Oncogene (2001) 20, 5062 ± 5066. 1999; Korinek et al., 1997; Miyoshi et al., 1998; Morin et al., 1997; Satoh et al., 2000). We previously reported Keywords: AXIN1; chromosome 3p22; colon cancer; that inactivation of Axin by genetic alterations was a lung cancer frequent feature of hepatocellular carcinomas (Satoh et al., 2000). Moreover, adenovirus-mediated gene trans- fer of wild-type AXIN1 suppressed growth and induced apoptosis not only in Axin-de®cient cells but also in ONCOGENOMICS The Wnt signaling pathway plays important roles in cells with mutant b-catenin or in APC-de®cient cells axis formation in early vertebrate development (Satoh et al., 2000). (McMahon and Moon, 1989). Axin, a negative To identify additional genes regulated by the b- regulator of this pathway, promotes phosphorylation catenin/Tcf4 complex and to clarify the detailed of serine/threonine in exon 3 of b-catenin by forming a mechanism of apoptosis induced by AXIN1,we complex with APC and GSK-3b (Ikeda et al., 1998; investigated genes whose expression was altered in Kishida et al., 1998; Yamamoto et al., 1999). response to the transduction of AXIN1 into LoVo cells using in-house cDNA microarray slides representing 9216 independent genes. Preparation, hybridization and detection of microarray slides as well as data *Correspondence: Y Furukawa; analysis were performed as described previously (Ono E-mail: [email protected] Received 21 February 2001; revised 5 April 2001; accepted 5 April et al., 2000). When we compared expression pro®les 2001 between LoVo cells infected with either adenoviruses AXUD1 is up-regulated by AXIN1 H Ishiguro et al 5063 expressing wild-type AXIN1 (Ad-Axin) or LacZ (Ad- SMART, we looked for conserved protein motifs in LacZ), a gene with in-house accession number A5900 the putative AXUD1 product, but failed to identify showed approximately 2.5-fold up-regulation by any matches with archived motifs. AXIN1 in the cancer cells. Subsequent semi-quantita- To examine the expression of AXUD1 in human tive RT ± PCR experiments con®rmed increased expres- tissues, we performed Northern-blot analysis using the sion of the A5900 gene by Ad-Axin, but not by Ad- AXUD1 cDNA clone as a probe. A 3.2-kb transcript APC expressing the 20-amino-acid repeat domain of was expressed in all human tissues examined, with wild-type APC (Figure 1). Similar induction was expression being most abundant in lung, placenta, observed in other colon-cancer cell lines (SNUC2A skeletal muscle, pancreas and leukocyte (Figure 2). and SW480; data not shown). In addition, we performed radiation-hybrid mapping By screening in a human spleen cDNA library analysis using the GeneBridge 4 (GB4) and G3 (Stratagene) using the A5900 PCR product as a probe radiation hybrid mapping panels (Research Genetics) and subsequent 5' rapid ampli®cation of cDNA ends according to the supplier's protocol (Miano et al., (5'RACE) using a Marathon cDNA ampli®cation kit 1997) and de®ned its position to approximately 3.46cR (Clontech), we identi®ed a putative full-length cDNA from D3S 1561, located at chromosomal band 3p22. In sequence corresponding to A5900 and termed it line with our data, an EST clone (Hs.6607) that AXUD1 (Axin1 up-regulated1). Its cDNA sequence corresponded to AXUD1 was mapped between consisted of 3182 nucleotides with an open reading D3S1609 and D3S1260 according to Gene Map 98 frame of 1767 bp, encoding a deduced 589-amino-acid from NCBI (http://www.ncbi.nlm.nih.gov/genemap/). protein (DNA sequence is available from GenBank, By comparing genomic with cDNA sequences, we accession number AB053121). A homology search with determined that AXUD1 consists of ®ve exons and the nucleotide sequence and predicted amino-acid covers a 12 kb genomic region. sequence using BLAST program by accessing data- To investigate the sub-cellular localization of bases in NCBI (National Center for Biotechnology AXUD1 protein in mammalian cells, we transiently Information, http://www.ncbi.nlm.nih.gov/) revealed transfected a plasmid expressing ¯ag-tagged AXUD1 high identity of AXUD1 to a 5' truncated cDNA protein (pFlag/AXUD1) into COS7 cells and observed sequence (GenBank accession number of AL117565), a the tagged protein by immunocytochemical staining. genomic sequence (NT005498) and a number of ESTs. To con®rm expression of the tagged protein, a whole- Using domain-search programs PROSITE and cell extract from transfected cells was separated by SDS ± PAGE, and tagged protein of Mr 65000 was detected by Western blot analysis with an anti-Flag antibody (Figure 3a). The protein was observed mainly in the nuclei of transfected cells (Figure 3b). By means of cDNA microarray analysis we identi®ed and characterized a novel human gene, AXUD1, whose expression was elevated in colon-cancer cells in response to transduction of wild-type AXIN1. How- ever, its expression was not induced by the 20-amino- acid repeat domain of wild-type APC. Since infection with either Ad-Axin or Ad-APC was able to suppress transcriptional activity of the Tcf/LEF complex in LoVo cells (data not shown), the di€erent e€ect of the two genes on AXUD1 expression implies that tran- Figure 1 Elevated expression of AXUD1 in LoVo cells in scription of AXUD1 is independent of the Tcf/LEF response to Ad-Axin. Human colon-cancer LoVo cells were complex. cultured in HAM's F-12, supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma Chemical Recent progress in basic research has revealed that Co) at 378C in humid air with 5% CO2. Construction and several molecules in the Wnt signaling pathway play infection of adenoviruses expressing wild-type AXIN1, LacZ,or pleiotrophic roles; for example, b-catenin mediates Wnt the 20-amino-acid repeat region of APC was carried out as signals and also regulates cell-to-cell adhesion asso- described previously (Satoh et al., 2000). The transfected cells ciated with E-cadherin (Nakamura, 1997; Nusse and were maintained at 378C for 72 h and total RNA was extracted. Semi-quantitative RT ± PCR was performed with the extracted Varmus, 1992; van den Heuvel et al., 1989), while APC RNAs as templates. Expression of the glyceraldehyde-3-phos- functions as a negative regulator of Wnt signals and is phate dehydrogenase (GAPDH) gene served as an internal also involved in nuclear export and cell motility control. The primers for ampli®cation were GAPDHF (5'- (Rosin- Arbesfeld et al., 2000). In line with this notion, ACAACAGCCTCAAGATCATCAG-3') and GAPDHR (5'- GGTCCACCACTGA CACGTTG-3') for GAPDH, and AX- APC induces CDX2, a gene involved in caudal UD1F (5'-GAGACAGGGCTGAAATCAGG-3'), AXUD1R (5'- determination, independent from the Tcf/LEF GAAGTGCAGTGCCCAGAAAG-3') for AXUD1. Each PCR mediated transcription (da Costa et al., 1999). reaction was carried out in a 25 ml volume of 16PCR bu€er Similarly, Axin may exert a variety of physiological (TAKARA) for 5 min at 948C for initial denaturing, followed by functions, including suppression of b-catenin and up- 20 (for GAPDH) or 27 (for AXUD1) cycles of 948C for 30 s, 588C for 30 s and 728C for 30 s, in the GeneAmp PCR system 9700 regulation of AXUD1 via a b-catenin/Tcf-independent (Perkin-Elmer) pathway.

Oncogene AXUD1 is up-regulated by AXIN1 H Ishiguro et al 5064

Figure 2 Northern blot of AXUD1 in various human tissues. Molecular size is indicated at right. Human multiple-tissue blots (Clontech) were hybridized with the AXUD1 PCR product labeled by random-oligonucleotide-priming, using a Megaprime DNA labeling kit (Amersham Pharmacia Biotech). Pre-hybridization, hybridization and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at 7808C for 24 h

Figure 3 (a) Expression of ¯ag-tagged AXUD1 protein in COS7 cells. To achieve a ¯ag-tagged AXUD1 protein, we cloned the entire coding region of AXUD1 into pFlag-CMV vector (Kodak) and transfected the pFlag/AXUD1 into COS7 cells. The expression of ¯ag-tagged AXUD1 protein in transfected cells was detected by Western blotting, in a manner described elsewhere (Ishiguro et al., 2000). (b) Immunocytochemical staining of ¯ag-tagged AXUD1 protein. Cells were stained with an anti-Flag antibody and visualized by anti-mouse secondary antibody conjugated with rhodamine as described previously (Kawasoe et al., 2000)

The chromosomal location of AXUD1 at 3p22, 1997), breast (Deng et al., 1996) and uterine cervix where frequent LOH has been reported in human (Wistuba et al., 1997), led us to speculate that cancers arising from the lung (Wistuba et al., 1999), inactivation of AXUD1 may be involved in carcinogen- kidney (Yokota et al., 1989), prostate (Dahiya et al., esis of those organs. Therefore we compared expression

Oncogene AXUD1 is up-regulated by AXIN1 H Ishiguro et al 5065 was reduced in 14 of 19 colon cancers, 13 of 15 lung cancers, three of six renal cancers and eight of 10 HCCs examined (Figure 4). The increased expression was observed in none of the cases examined. Although we screened 50 lung cancers for mutations of AXUD1, we detected no genetic alterations (data not shown). However, in addition to allelic losses, transcriptional silencing due to methylation in the promoter region could be responsible for down- regulation of AXUD1. This possibility remains to be examined. We reported earlier that transfection of Ad-Axin induced apoptosis in various human liver- and colon- cancer cells (Satoh et al., 2000). It is noteworthy that this e€ect was observed not only in SNU423 or Alexander cells, which express mutant AXIN1, but also in Huh7 or HepG2 cells, which contain wild-type AXIN1 (Satoh et al., 2000). Therefore overexpression of Axin may induce cell death independently of Tcf/ LEF mediated transcription. Although further investi- gations are needed to clarify the physiological role of AXUD1 protein in cells, it may play a crucial role in the apoptotic process induced by Axin. Since AXIN1 is a good candidate for a gene-therapy approach to treatment of human cancers, identi®cation and characterization of genes regulated by Axin will provide a better understanding of the carcinogenetic process and may identify novel therapeutic targets. Figure 4 Expression of AXUD1 in cancer tissues (T) and corresponding non-cancerous tissues (N) from the colon (A), Investigation of the function of AXUD1, for example, lung (B), kidney (C), and liver (D) as examined by semi- may lead to better diagnosis and yield clues for quantitative RT ± PCR according to the methods described in developing novel anti-cancer drugs and preventive the legend of Figure 1. Cancerous and corresponding non- agents. cancerous tissues were resected specimens obtained from patients who underwent surgery at the Medical Institute Hospital of Tokyo University, the Saitama Medical Center, or the Kyoto University Hospital. All patients provided informed consent

Acknowledgments We are grateful to Tae Makino for her technical assistance. of AXUD1 in cancer tissues from human lung, kidney, This work was supported by a `Research for the Future' liver, and colon and their corresponding non-cancerous Program Grant (96L00102) from the Japan Society for the tissues, using semi-quantitative RT ± PCR. Expression Promotion of Science.

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