Oncogene (2002) 21, 4108 ± 4119 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Restoration of E-cadherin-based cell ± cell adhesion by overexpression of nectin in HSC-39 cells, a human signet ring cell gastric cancer cell line Ying-Feng Peng1, Kenji Mandai1, Hiroyuki Nakanishi1, Wataru Ikeda1, Masanori Asada1, Yumiko Momose1, Sayumi Shibamoto3,6, Kazuyoshi Yanagihara4, Hitoshi Shiozaki2,7, Morito Monden2, Masatoshi Takeichi5 and Yoshimi Takai*,1 1Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan; 2Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan; 3Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan; 4Central Animal Laboratory, National Cancer Center Research Institute, Tokyo 104-0045, Japan; 5Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan Nectin is an immunoglobulin-like adhesion molecule that Introduction comprises a family consisting of four members, nectin-1, -2, -3, and -4. Nectin is associated with the actin In about 50% of carcinomas with highly invasive and cytoskeleton through afadin, a nectin- and actin ®lament- metastatic nature, mutations of the components of the binding protein. The nectin-afadin and cadherin-catenin E-cadherin-catenin system have been reported (Shioza- systems are associated with each other and cooperatively ki et al., 1991). E-Cadherin functions as a key cell ± cell form cell ± cell adherens junctions in intact epithelial adhesion molecule in a Ca2+-dependent manner at cells. HSC-39 cells, a human signet ring cell gastric cell ± cell adherens junctions (AJs) in intact epithelial cancer cell line, express E-cadherin but do not form cells (Takeichi, 1991; Kemler, 1992; Marrs and Nelson, cell ± cell adhesion. The b-catenin gene has been shown to 1996; Yap et al., 1997). The cytoplasmic region of E- be truncated at the N-terminal region including the a- cadherin is directly associated with b-catenin which catenin-binding domain in HSC-39 cells, but overexpres- directly interacts with a-catenin (Ozawa et al., 1989; sion of normal b-catenin failed to form cell ± cell McCrea et al., 1991; Nagafuchi et al., 1991; Oda et al., adhesion. HSC-39 cells expressed nectin-1, -2, and 1993). a-Catenin directly binds to actin ®laments (F- afadin, but not nectin-3. Overexpression of nectin-3 or actin) (Rimm et al., 1995) and two other F-actin- -2 formed cell ± cell adhesion and accumulation of E- binding proteins, vinculin and a-actinin (Knudsen et cadherin, but not actin ®laments, at the cell ± cell al., 1995; Watabe-Uchida et al., 1998; Weiss et al., adhesion sites. Overexpression of a truncated form of 1998). Thus, E-cadherin is indirectly linked to the actin nectin-2 incapable of interacting with afadin failed to cytoskeleton through these peripheral membrane form cell ± cell adhesion. However, the nectin-formed proteins. These peripheral membrane proteins together cell ± cell adhesion was not so strong as that observed in with E-cadherin play important roles in the formation epithelial cells, such as CaCo-2 cells. Co-expression of and maintenance of cell ± cell AJs (Takeichi, 1991). The nectin-2 and normal b-catenin did not form strong cell ± E-cadherin-catenin system is furthermore required for cell adhesion. These results suggest that an unidenti®ed organization of tight junctions (TJs) and desmosomes mechanism, by which nectin and E-cadherin form the in epithelial cells (Gumbiner and Simons, 1986; actin cytoskeleton-associated adherens junctions to form Gumbiner et al., 1988; Watabe et al., 1994). Thus, strong cell ± cell adhesion, is impaired in HSC-39 cells. the mutational inactivation or reduced function of the Oncogene (2002) 21, 4108 ± 4119. doi:10.1038/sj.onc. E-cadherin-catenin system inevitably leads to abnormal 1205517 E-cadherin-mediated cell ± cell adhesion. However, in the carcinomas of which the E-cadherin-catenin system Keywords: nectin; afadin; cadherin; catenin; cell ± cell is intact, the causative gene mutations responsible for adhesion their highly invasive and metastatic nature have not been identi®ed. The nectin-afadin system is another cell ± cell adhe- sion system at AJs (Mandai et al., 1997; Takahashi et *Correspondence: Y Takai; al., 1999; Miyahara et al., 2000; Satoh-Horikawa et al., 2+ E-mail: [email protected] 2000). Nectin is a Ca -independent immunoglobulin- Current addresses: 6Department of Molecular Oncology, Genentech, like cell ± cell adhesion molecule (Aoki et al., 1997; Inc., South San Francisco, California 94080-4990, USA; 7First Lopez et al., 1998; Takahashi et al., 1999). This Department of Surgery, Kinki University School of Medicine, adhesion molecule was originally isolated as the Osaka-Sayama, Osaka 589-8511, Japan Received 21 January 2002; revised 1 March 2002; accepted 22 poliovirus receptor-related protein and has recently March 2002 been shown to serve as an a-herpes virus entry and The nectin-afadin system in cancer cells Y-F Peng et al 4109 a1 a2 a3 b1 b2 b3 Figure 1 Morphology of HSC-39 cells. (a1 ± a3) Phase contrast imaging. (a1) Clustered cells. The cells were cultured under normal culture conditions and the photograph was taken; (a2) Single cells. The clustered cells were scattered by pipetting and cultured in suspension. The photograph was taken immediately after pipetting; and (a3) Reclustered cells. The scattered cells were cultured in suspension for 1 h and the photograph was taken. (b1 ± b3) E-Cadherin staining. (b1) Low magni®cation; and (b2 ± b3) High magni®cation. (b2), a lateral view; and (b3), an apical view. Arrows, bud-like structures. Bars, 100 mm(a1 ± a3); 20 mm(b1); and 10 mm(b2 ± b3). The results shown are representative of three independent experiments cell ± cell spread mediator (Cocchi et al., 1998; 2000; afadins. 1-Afadin, a larger splicing variant, is an F-actin- Geraghty et al., 1998; Warner et al., 1998; Lopez et al., binding protein with one PDZ domain and three 2000; Sakisaka et al., 2001). Nectin comprises a family proline-rich domains and connects nectin to the actin consisting of four members, nectin-1, -2, -3, and -4, cytoskeleton (Mandai et al., 1997; Takahashi et al., and each of nectin-1, -2, and -3 has two or three 1999). s-Afadin, a smaller splicing variant, has one PDZ splicing variants: nectin-1a,-1b,-2a,-2d,-3a,-3b,and domain but lacks the F-actin-binding domain and the -3g (Morrison and Racaniello, 1992; Aoki et al., 1994; third proline-rich domain (Mandai et al., 1997). Human Lopez et al., 1995; Eberle et al., 1995; Cocchi et al., s-afadin is identical to the product of the AF-6 gene that 1998; Satoh-Horikawa et al., 2000; Reymond et al., has been identi®ed as an ALL-1 fusion partner involved 2001). Each member of the nectin family forms a in acute myeloid leukemia (Prasad et al., 1993). For the homo-cis-dimer, followed by formation of a homo- purpose of this study, we will refer to 1-afadin simply as trans-dimer, causing cell ± cell adhesion (Lopez et al., afadin. The nectin-afadin system is ubiquitously ex- 1998; Miyahara et al., 2000; Satoh-Horikawa et al., pressed not only in epithelial cells but also in non- 2000; Sakisaka et al., 2001; Reymond et al., 2001). epithelial cells lacking TJs, such as ®broblasts and Nectin-3 furthermore forms a hetero-trans-dimer with neurons (Mandai et al., 1997; Takahashi et al., 1999; either nectin-1 or -2 and the formation of each hetero- Nishioka et al., 2000; Mizoguchi et al., 2002). trans-dimer is stronger than that of each homo-trans- We have obtained several lines of evidence that the dimer (Satoh-Horikawa et al., 2000). Nectin-4 also nectin-afadin system plays an important role in the forms a hetero-trans-dimer with nectin-1 and the formation of AJs in cooperation with the E-cadherin- formation of the hetero-trans-dimer is stronger than catenin system: (1) the nectin-afadin system is strictly that of the homo-trans-dimer (Reymond et al., 2001). con®ned to AJs, which are undercoated with F-actin All the members except nectin-1b,-3g and -4 have a C- bundles in polarized epithelial cells, whereas the E- terminal conserved motif of four amino acid (aa) cadherin-catenin system is more widely distributed residues (E/A-X-Y-V) which interacts with the PDZ from AJs to the basal area along the lateral membrane domain of afadin (Takahashi et al., 1999; Satoh- (Mandai et al., 1997; Takahashi et al., 1999); (2) Horikawa et al., 2000). Nectin-4 does not have the studies using afadin (7/7) mice and (7/7) embryoid conserved motif, but interacts with the PDZ domain of bodies indicate that the proper organization of AJs and afadin through its C-terminus (Reymond et al., 2001). TJs are markedly impaired in epithelial cells (Ikeda et Afadin has at least two splicing variants, 1- and s- al., 1999); (3) cadherin-de®cient L ®broblasts stably Oncogene The nectin-afadin system in cancer cells Y-F Peng et al 4110 a c b Figure 2 Expression of the components of the E-cadherin-catenin and nectin-afadin systems in HSC-39 cells. (a,b) Western blotting. The lysates (100 mg of protein for nectin-1a and 30 mg of protein for others) of HSC-39 and the control cells were subjected to SDS-polyacrylamide gel electrophoresis (8% polyacrylamide gel), followed by Western blotting with the indicated Abs. CaCo-2, NS20Y, nectin-1a-L, nectin-2a-L, and nectin-3a-L cells were used as control cells.