Oncogene (2004) 23, 9289–9294 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc SHORT REPORT Isolation and characterization of a novel gene CLUAP1 whose expression is frequently upregulated in colon cancer Meiko Takahashi1, Yu-Min Lin2, 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; 2Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei 11160, Taiwan To disclose mechanisms of colorectal carcinogenesis and appear to be upregulated frequently in the cancer tissues identify novel diagnostic markers and drug targets for compared with the corresponding noncancerous cells. treatment of these tumors, we previously analysed the Since carcinogenesis involves activation of oncogenes expression profiles of 11 colorectal cancers using a and/or inactivation of tumor suppressor genes, en- genome-wide cDNA microarray containing 23 040 genes. hanced expression of at least some of these upregulated Among the genes commonly transactivated in the cancers, genes may reflect oncogenic properties. we identified a novel human gene, which we termed Among the genes with frequently elevated expression CLUAP1 (clusterin-associated protein 1). It encodes a in cancer tissues in our microarray data, an EST with an nuclear protein of 413 amino acids containing a coiled-coil in-house accession number of B9223 corresponding to domain. To investigate its function, we searched for KIAA0643, Hs. 155995 in UniGene cluster (http:// CLUAP1-interacting proteins using yeast two-hybrid www.ncbi.nlm.nih.gov/UniGene/), was upregulated in system and identified nuclear Clusterin. Expression of the cancer tissues compared to their corresponding CLUAP1 was gradually increased in the late S to G2/ noncancerous mucosa in a magnification range between M phases of cell cycle and it returned to the basal level in 2.1 and 3.5 in five out of seven cases that passed the the G0/G1 phases. Suppression of this gene by short cutoff filter (data not shown). To clarify the results of the interfering RNAs resulted in growth retardation in the microarray, we carried out semiquantitative RT–PCR transfected cells. These data provide better understanding and revealed that expression of B9223 was increased in of colorectal carcinogenesis, and inactivation of CLUAP1 14 of additional 20 colon cancers (70%) compared with may conceivably serve in the future as a novel therapeutic their corresponding normal mucosae (Figure 1a). intervention for treatment of colon cancer. Homology searches with the sequence of B9223 in Oncogene (2004) 23, 9289–9294. doi:10.1038/sj.onc.1208100 public databases using BLAST program in National Published online 11 October 2004 Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/BLAST/) identified a gene that Keywords: CLUAP1; clusterin; colon cancer had been annotated as KIAA0643 protein, clone MGC:9638 (GenBank Accession number BC017070), and a genomic sequence with GenBank Accession number of NT_010552.13 assigned to chromosomal Colorectal carcinomas are leading causes of cancer band 16p13. To determine the coding sequence of the death worldwide. In spite of recent progress in gene, we predicted candidate–exon sequences in the diagnostic and therapeutic strategies, prognosis of genomic sequence using GENSCAN (http://genes.mit. patients with advanced cancers remains very poor. edu/GENSCAN.html) and Gene Recognition and Although molecular studies have revealed that altera- Assembly Internet Link (GRAIL, http://compbio.ornl. tions of tumor suppressor genes and/or oncogenes are gov/Grail-1.3/) program, and performed exon-connec- involved in colorectal carcinogenesis, the precise me- tion experiments. As a result, we obtained an assembled chanisms remain to be fully elucidated. To disclose sequence of 1681 nucleotides (GenBank Accession mechanisms underlying these tumors from a genome- number AB089691) containing an open-reading frame wide point of view and to discover target molecules for of 1239 nucleotides encoding a putative 413-amino-acid diagnosis and for development of novel therapeutic protein, and termed the gene CLUAP1 (clusterin- drugs, we have been analysing their expression profiles associated protein 1) because of the reason described by cDNA microarray representing 23 040 genes (Kita- below. The first ATG was flanked by a sequence hara et al., 2001; Lin et al., 2002). These efforts have (AGCGTTATGT) that agreed with the consensus pinpointed a number of genes, including ESTs, which sequence for the initiation of translation in eukaryotes, with an in-frame stop codon upstream. Comparison of *Correspondence: Y Furukawa; E-mail: [email protected] CLUAP1 cDNA and the genomic sequence disclosed Received 19 February 2004; revised 29 July 2004; accepted 2 August that this gene consisted of 11 exons. The amino-acid 2004; published online 11 October 2004 sequence of the predicted CLUAP1 protein showed 89, CLUAP1 is upregulated in colon cancer M Takahashi et al 9290 a N T N T N T N T N T N T N T N T N T N T B9223 GAPDH N T N T N T N T N T N T N T N T N T N T B9223 GAPDH b Figure 1 (a) Semiquantitative RT–PCR showing that B9223 expression was increased in 14 of 20 colon cancers compared to their corresponding normal mucosae. Extraction of RNA, cDNA synthesis, and PCR reaction were carried out as reported previously (Shimokawa et al., 2003). Amplification proceeded for 4 min at 941C for denaturing, followed by 21 (for GAPDH) and 32 (for CLUAP1), cycles of 941C for 30 s, 601C for 30 s, and 721C for 60 s, in the GeneAmp PCR system 9700 (Perkin-Elmer, Foster City, CA, USA). Primer sequences were as follows: GAPDH, forward 50-ACAACAGCCTCAAGATCATCAG-30 and reverse 50-GGTCCAC- CACTGACACGTTG-30; CLUAP1, forward 50-GAGTGGAAGTAACGATGACTC-30 and reverse 50-GTCATTGTCACTCT- CATCCAG-30.(b) Alignment of CLUAP1 (GenBank Accession number AB089691), a mouse RIKEN cDNA 2610111M03 (BC024910), a zebra fish unknown protein for MGC: 56115 (AAH45921), A. ganbiae ENSANGP00000008052 (XP_310880), and a C. elegans putative protein for 4E162 (NP_500339). Boxed region corresponds to the coiled-coil motif, and shading indicates residues that are conserved in at least three proteins 62, 38, 38, and 31% identities to a mouse RIKEN expressed abundantly in human testis, thyroid and cDNA 2610111M03 (NP_084014), a zebra fish unknown trachea, and moderately in spinal cord and adrenal protein for MGC:56115 (AAH45921), Anopheles gan- gland (Figure 2a). To examine the expression and biae ENSANGP00000008052 (XP_310880), a Caenor- explore the function of CLUAP1, we prepared poly- habditis elegans hypothetical protein (BAD 23996), and clonal antibody against CLUAP1. Western blot analysis Drosophila melanogaster CGI7599-PA (NP_608470), using whole extracts of colon cancer cells, including respectively. A search for protein motifs with the Simple HCT116, SNU-C4, and SW480, showed a 53 kDa band Modular Architecture Research Tool (SMART, http:// that corresponded to CLUAP1 (Figure 2b). As a smart.embl-heidelberg.de) revealed that the predicted negative control for the Western blot analysis, we used protein contained a single coiled-coil region (codons extracts from TE8 esophageal cancer cells that showed 195–267), which was conserved in the homologues. In low level of CLUAP1 expression by semiquantitative addition to the coiled-coil motif, these homologues also RT–PCR (Figure 2b). shared similarities in amino acids between codons 1 and To reveal its subcellular localization, fluorescent 184 of CLUAP1 (Figure 1b). immunocytochemical staining of endogenous CLUAP1 Multiple-tissue Northern blotting with CLUAP1 was carried out in HCT116 cells. Cells were stained with cDNA as a probe showed that a 1.6-kb transcript was anti-CLUAP1 and visualized by fluorescein-conjugated Oncogene CLUAP1 is upregulated in colon cancer M Takahashi et al 9291 a Ovary Lung Thymus Lymph node Lymph Prostate Thyroid Trachea Spinal cord Spinal Liver Spleen Kidney Pancreas Testis Heart Brain Placenta Peripheral blood leukocytes blood Peripheral Colon Adrenal gland Adrenal Small intestine Bone marrow Bone Stomach Skeletal muscle CLUAP1 1.6 kb ß-actin bcHCT116 d e N T N T N T HCT116 SW480 SNU-C4 TE8 CLUAP1 CLUAP1 ß-actin DAPI Figure 2 (a) Human multiple-tissue blots (Clontech) were hybridized with a 32P-labeled PCR product of CLUAP1. Prehybridization, hybridization, and washing were performed according to the supplier’s recommendations. A 1.6-kb transcript was expressed abundantly in human testis, thyroid, and trachea. Expression of b-actin served as an internal control. (b) Western blot analysis of CLUAP1 in cancer cell lines. HCT116, SW480, and SNU-C4 were established from colon cancer tissues, while TE8 was from esophageal cancer tissue. The band indicated with an arrow corresponded to CLUAP1. Expression of b-actin served as an internal control. (c) Immunocytochemical staining using anti-CLUAP1 antibody in HCT116 cells showing the nuclear localization of endogenous CLUAP1. Recombinant His-tagged CLUAP1 protein was produced in Escherichia coli and inoculated for the immunization of rabbits. The polyclonal antibody to CLUAP1 was purified from the sera as described elsewhere (Shimokawa et al., 2003). Immunohistochemical staining of HCT116 cells with rabbit anti-CLUAP1 antibody was performed as reported previously (Okabe et al., 2003). The reaction was visualized after incubation with fluorescein-conjugated