Oncogene (2004) 23, 3716–3720 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc SHORT REPORTS Expression of CD109 in human cancer Mizuo Hashimoto1,2, Masatoshi Ichihara1, Tsuyoshi Watanabe1, Kumi Kawai3, Katsumi Koshikawa4, Norihiro Yuasa2, Takashi Takahashi4, Yasushi Yatabe5, Yoshiki Murakumo1, Jing-min Zhang1, Yuji Nimura2 and Masahide Takahashi*,1,3 1Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; 2Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; 3Division of Molecular Pathology, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; 4Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan; 5Department of Pathology and Molecular Diagnsotics, Aichi Cancer Center, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan It was recently reportedthat the human CD109 gene phenotype with the association of MTC, pheochromo- encodes a glycosyl-phosphatidylinositol-anchored glyco- cytoma, and developmental abnormalities such as protein that is a member of the a2-macroglobulin/C3, C4, mucosal neuroma, hyperganglionosis of the intestinal C5 family of thioester-containing proteins. In this study, tract, and marfanoid skeletal changes. To elucidate the we foundthat the expression of mouse CD109 gene was mechanisms of the development of MEN2A and upregulatedin NIH3T3 cells expressing RET tyrosine MEN2B phenotypes, we recently performed differential kinase with a multiple endocrine neoplasia 2B mutation. display analysis to identify the genes that are differen- Northern blot analysis showeda high level of expression tially expressed among NIH 3T3 cells and NIH 3T3 of the CD109 gene only in the testis in normal human and transfectants expressing RET with a MEN2A or mouse tissues. In addition, its expression was high in some MEN2B mutation (designated RET-MEN2A or RET- human tumor cell lines, which included squamous cell MEN2B) (Watanabe et al., 2002). We identified 29 carcinoma andglioblastoma cell lines, whereas it was known genes and 13 previously unidentified sequences undetectable in neuroblastoma and small-cell lung carci- that were differentially expressed among these three cell noma cell lines. When CD109 expression was examinedin lines. These included a variety of genes that are involved 33 cases of human lung cell carcinomas by quantitative in cell growth, tumor progression or suppression RT–PCR, a significant high expression of CD109 was (Watanabe et al., 2002). In this study, we further detected in about half of squamous cell carcinomas characterized one of the previously unidentified se- examined, but not in adenocarcinoma, large-cell carcino- quences that was upregulated in NIH-RET(MEN2B) ma andsmall-cell carcinoma. Similarly, upregulation of cells and may become potential a molecular target for CD109 was observedin nine out of 17 esophageal new cancer therapeutics. squamous cell carcinomas. Thus, these results suggested By differential display, we detected a transcript that that CD109 might be a useful molecular target for the was upregulated in NIH-RET(MEN2B) cells (Figure 1a development of new therapeutics for malignant tumors, and b). Using its cDNA fragment, we screened a cDNA such as squamous cell carcinoma. library constructed from RNA of NIH-RET(MEN2B) Oncogene (2004) 23, 3716–3720. doi:10.1038/sj.onc.1207418; cells and obtained a cDNA clone of 4717 base pairs in length that contained a 4329 bp open reading frame. It encoded a protein of 1442 amino acids with an amino- Keywords: CD109; squamous cell carcinoma; lung terminal signal peptide of 22 amino acids and carcinoma; esophageal carcinoma a carboxyl-terminal hydrophobic glycosyl-phospha- tidylinositol (GPI)-anchored cleavage-addition site (Figure 1c). In addition, a sequence for the thioester bond (CGEQ) was found (amino acids 923–926 in Germline mutations of the RET proto-oncogene are Figure 1c) (Hofmann et al., 1999). After completing the responsible for the development of multiple endocrine sequence, it turned out that this cDNA encoded a mouse neoplasia (MEN) type 2A and 2B (Takahashi, 2001). orthologue of the human CD109 gene, a member of the MEN2A is characterized by the development of a2-macroglobulin/C3, C4, C5 family that was recently medullary thyroid carcinoma (MTC) and pheochromo- reported (Lin et al., 2002). cytoma, whereas MEN2B shows a more complex To confirm that mouse CD109 is a GPI-anchored cell surface protein, COS7 cells were transfected with FLAG-tagged mouse CD109 cDNA cloned into an *Correspondence: M Takahashi, Department of Pathology, Nagoya expression vector. Western blot analysis revealed that University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; E-mail: [email protected] the transfectant expressed a protein of 180 kDa detected Received 12 August 2003; accepted 3 December 2003 by an anti-FLAG monoclonal antibody that was the CD109 expression in human cancer M Hashimoto et al 3717 Figure 1 Expression and amino-acid sequence of mouse CD109 gene. (a) Detection of a mouse CD109 cDNA fragment by differential display analysis. Total RNAs from NIH 3T3, NIH-RET(MEN2A) and NIH-RET(MEN2B) cells were analysed by differential display (Watanabe et al., 2002). An arrowhead indicates a cDNA band derived from RNA of NIH-RET(MEN2B) cells that encodes mouse CD109. (b) Upregulation of CD109 mRNA in NIH-RET(MEN2B) cells. Total RNAs (20 mg) from NIH 3T3, NIH-RET(MEN2A) and NIH-RET(MEN2B) cells were analysed by Northern blotting with a CD109 cDNA fragment isolated by differential display. A 5.5 kb CD109 transcript was expressed at a high level in NIH-RET(MEN2B) cells (upper panel). An arrowhead indicates the location of 28S ribosomal RNA. The same membrane was rehybridized with glyceraldehyde-3-phophate dehydrogenase (GAPDH) cDNA as a control (lower panel). (c) Amino-acid sequences of mouse and human CD109. Identical amino acids between human and mouse sequences are shaded. The amino-terminal signal sequence (underlined), the thioester signature sequence (double underlined) and the GPI anchor cleavage-addition site (closed triangle) are shown expected molecular mass of a FLAG-tagged CD109 gene is observed in human tumors, we next investigated fusion protein (Figure 2a). As shown in Figure 2b, its the CD109 expression in 30 human tumor cell lines. As cell surface expression was observed by immunostaining shown in Figure 3c, it was highly expressed in with an anti-FLAG antibody. In addition, when the glioblastoma and squamous cell carcinoma cell lines as transfectant was treated with phosphatidylinositol- well as in some adenocarcinoma and sarcoma (osteo- specific phospholipase C (PI-PLC) that cleaves the sarcoma and melanoma) cell lines. In contrast, its GPI anchor, the CD109 expression on the cell surface expression was very low and undetectable in the became almost undetectable (Figure 2c), confirming that examined neuroblastoma, small-cell lung carcinoma, mouse CD109 is a GPI-anchored cell surface protein. and leukemia/lymphoma cell lines. When CD109 gene expression was examined in CD109 expression was further analysed in human various human and mouse adult tissues by Northern cancer tissues that were surgically resected. In total, 33 blotting, a high level of expression was detected only in of lung carcinomas (14 adenocarcinomas, 11 squamous the testis (Figure 3a and b). A 5.5 kb mRNA was a main cell carcinomas, five large-cell carcinomas and three transcript, although a low level of 10 kb transcript was small-cell carcinomas) were examined by quantitative also detected. To see whether upregulation of CD109 real-time RT–PCR. An average of expression level of Oncogene CD109 expression in human cancer M Hashimoto et al 3718 the CD109 gene was significantly higher in squamous Moreover, we investigated the CD109 gene expression cell carcinoma than in adenocarcinoma, large-cell in esophageal squamous cell carcinomas. The expression carcinoma, and small-cell carcinoma (Figure 4a). Upre- levels were compared between 17 cases of human gulation of CD109 expression (more than 2.5-fold than esophageal squamous cell carcinomas and seven cases the control level) was observed in six out of 11 of normal esophageal mucosae adjacent to carcinomas. squamous cell carcinomas examined. Upregulation of CD109 expression (more than 2.5-fold than the average in normal mucosa) was detected in eight cases of carcinomas. In contrast, when its expression was examined in nine cases of gastric adenocarcinomas, only one case showed more than 2.5-fold upregulation of CD109. The elevated levels of CD109 expression did not correlate with the disease stages of the examined lung and esophageal squamous cell carcinomas (data not shown). In the current study, we cloned the mouse CD109 gene by differential display analysis that was highly expressed in NIH(RET-MEN2B) cells. Lin et al. (2002) recently reported the cloning of human CD109 gene that encodes a novel member of the a2-macroglobulin/C3, C4, C5 family, including a2-macroglobulin-like protease inhibitors and complement proteins. The amino-acid identity between human and mouse genes was approxi- mately 69%. Based on the amino-acid sequence, it turned out that CD109 is closely related to a2-macro- globulin-like protease inhibitors and more distantly related to C3 and C4 proteins (Lin et al., 2002), although its physiological function still remains un- known. Human CD109 is a monomeric glycoprotein of 170 kDa that contains several N-linked endoglycosidase H-sensitive hybrid-type glycans, but no O-linked gly- cans, and is susceptible to cleavage with PI-PLC, indicating that CD109 is a GPI-anchored cell surface protein (Sutherland et al., 1991; Haregewoin et al., 1994; Smith et al., 1995; Solomon et al., 1998). We confirmed by immunofluorescence and FACS analyses that mouse CD109 is expressed as a GPI-anchored cell surface protein that is cleaved by PI-PLC.