Oncogene (2008) 27, 264–273 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Interaction of cancer cells with platelets mediated by Necl-5/poliovirus receptor enhances cancer cell metastasis to the lungs K Morimoto1, K Satoh-Yamaguchi1, A Hamaguchi1, Y Inoue1, M Takeuchi1, M Okada1, W Ikeda2, Y Takai2 and T Imai1 1KAN Research Institute Inc., Kobe MI R&D Center, Kobe, Hyogo, Japan and 2Department of Molecular Biology and Biochemistry, Faculty of Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Necl-5 is an immunoglobulin (Ig)-like molecule that was identified and their modes of action in normal cell originally identified as a poliovirus receptor and is often proliferation and as well as the modes of action of their upregulated in cancer cells. We recently found that it mutations, suppression or augmentation in abnormal colocalizes with integrin avb3 at the leading edges of cell proliferation are fairly well established. In contrast, moving cells and enhances growth factor-induced cell disruption of cell–cell adhesion, enhanced cell move- movement and proliferation. Upon cell–cell contact, Necl- ment and loss of contact inhibition of cell movement 5 is removed from the cell surface by its trans-interaction and proliferation are involved in the invasiveness and with the cell adhesion molecule nectin-3, resulting in metastasis of cancer cells, but the mechanisms for how reduced cell movement and proliferation. Here, we mutations and modifications of oncogenes and tumor investigated the role of Necl-5 in the interaction of cancer suppressor genes cause these pathological events are cells with platelets. Necl-5 was upregulated in CT26 cells, poorly understood. a colon adenocarcinoma cell line. When CT26 cells were We recently found that an immunoglobulin (Ig)-like injected into the tail vein of mice, they were arrested in the molecule, Necl-5/poliovirus receptor (PVR)/CD155/ pulmonary vessels by adhering to platelets and subse- Tage4, colocalizes with integrin avb3 at the leading quently metastasized to the lungs. Overexpression of edges of moving cells and enhances growth factor- Necl-5 in CT26 cells enhanced this metastasis, while induced cell movement and proliferation (Ikeda et al., inhibition of the trans-interaction of Necl-5 with CD226 2004; Kakunaga et al., 2004). Human PVR/CD155 was by an anti-Necl-5 monoclonal antibody reduced the originally identified as a PVR (Mendelsohn et al., 1989; metastasis. Depletion of platelets by treatment with a Koike et al., 1990), while rodent Tage4 was originally rabbit anti-mouse platelet serum reduced the Necl-5- identified as the product of an overexpressed gene in enhanced metastasis in mice. Thus, the trans-interaction rodent colon carcinoma (Chadeneau et al., 1994). PVR/ of upregulated Necl-5 in cancer cells with its counter- CD155 is also overexpressed in many human cancer cells receptor in platelets, probably CD226, is critical for (Gromeier et al., 2000; Masson et al., 2001). This efficient metastasis of cancer cells to the lungs. molecule with four nomenclatures has tentatively been Oncogene (2008) 27, 264–273; doi:10.1038/sj.onc.1210645; designated nectin-like molecule-5, Necl-5 (Takai et al., published online 16 July 2007 2003). Integrin aVb3 is often upregulated in cancer cells and appears to be involved in their invasiveness and Keywords: Necl-5; poliovirus receptor; CD226; cancer metastasis (Guo and Giancotti, 2004). cell; platelet; metastasis The modes of action of Necl-5 in cell movement and proliferation have been elucidated. Specifically, Necl-5 enhances growth factor-induced activation of Cdc42 and Rac small G proteins, causing the formation of Introduction filopodia and lamellipodia, respectively, which even- tually enhances cell movement (Ikeda et al., 2004). The The characteristics of malignant cancer cells are cytoplasmic region of Necl-5 binds to Tctex-1, a subunit abnormal proliferation, invasion into the surrounding of the dynein motor complex, which may also be tissues and metastasis to other organs (Thiery, 2002). involved in regulating cell movement in cooperation Many oncogenes and suppressor genes have been with microtubules (Mueller et al., 2002). Necl-5 en- hances the activation of Ras-Raf-MEK-ERK signaling, and induces upregulation and downregulation of cell Correspondence: Dr T Imai, KAN Research Institute Inc., Kobe MI cycle regulators, including cyclins D2 and E, and p27kip1, R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, thereby shortening the period of the G1 phase of the cell Hyogo 650-0047, Japan. E-mail: [email protected] cycle (Kakunaga et al., 2004). Necl-5 is upregulated in Received 20 February 2007; revised 29 May 2007; accepted 5 June 2007; NIH3T3 cells transformed by the oncogene V12-Ki-Ras published online 16 July 2007 and this upregulation is mediated by transcriptional Necl-5-CD226 interaction in lung metastasis K Morimoto et al 265 activation of the Necl-5 gene through the V12-Ki-Ras- Results Raf-MEK-ERK-AP-1 pathway (Hirota et al., 2005). Necl-5 does not show any homophilic cell–cell Enhancement of metastasis of cancer cells to the lungs by adhesion activity, but does heterophilically trans-inter- Necl-5 act with nectin-3, a member of the Ig-like nectin family Necl-5 has been shown to be upregulated in many types that acts as a Ca2 þ -independent cell–cell adhesion of cancer cells (Chadeneau et al., 1994, 1996; Gromeier molecule and forms adherens junctions cooperatively et al., 2000; Masson et al., 2001; Sloan et al., 2004). We with cadherins (Ikeda et al., 2003; Takai et al., 2003). used fluorescent-activated cell sorting (FACS)analysis When cells come into contact with other cells, Necl-5 is to examine the expression levels of cell surface Necl-5 in downregulated from the cell surface by its trans- various cancer cell lines, namely CT26, Colon 26, interaction with nectin-3 (Fujito et al., 2005). However, B16F1, Meth A and OVHM. Necl-5 was strongly when cells do not come into contact with other cells, expressed in all of these cancer cell lines (Figure 1a). Necl-5 is upregulated at the transcriptional level by To evaluate the metastatic potential of Necl-5 on growth factor-induced signaling (Hirota et al., 2005). cancer cells, CT26 cells, a colon adenocarcinoma cell Upregulation of Necl-5 enhances growth factor-induced line, were infected with a retroviral supernatant gener- cell movement and proliferation, whereas downregula- ated by transfecting pMX-Necl-5 into packaging cells. tion of Necl-5 reduces them (Fujito et al., 2005). Puromycin-resistant CT26 cells were established and Cultured normal cells continue to move until they come designated Necl-5-CT26 cells. FACS analysis revealed into contact with other cells and become confluent. They that the expression level of surface Necl-5 was about 10- then undergo cell–cell adhesion and stop moving. This fold higher in Necl-5-CT26 cells than in parental CT26 phenomenon has been known for more than 50 years as cells and control pMX-CT26 cells infected with a contact inhibition of cell movement and proliferation retroviral supernatant using the pMX vector alone (Abercrombie and Heaysman, 1953; Abercrombie, (Figure 1b). Next, we compared the rates of metastasis 1979). This contact inhibition is lost when normal cells to the lungs among wild-type CT26, Necl-5-CT26 and are transformed. We previously proposed that Necl-5 pMX-CT26 cells. We injected each cell line into the tail plays a role, at least in part, in this contact inhibition of vein of BALB/c mice and counted the numbers of tumor cell movement (Fujito et al., 2005). nodules in the lungs. The sizes of the lungs containing These biological properties of Necl-5 suggest that metastasized Necl-5-CT26 cells were larger than those its upregulation in many cancer cells plays a role in containing metastasized CT26 and pMX-CT26 cells their metastasis. The metastasis of cancer cells consists (Figure 1c). The number of metastatic foci in the lungs of multiple steps: (1)disruption of cell–cell adhesion of mice receiving Necl-5-CT26 cells was 175740 and and enhancement of cell movement, (2)invasion into the four- to sevenfold higher than those in the lungs of mice surrounding tissues, (3)entry into blood/lymph vessels, receiving CT26 cells (2476)or pMX-CT26 cells (46 716; (4)transport to other organs, (5)arrest in the vessels, (6) Figure 1d). Histological examination revealed that exit from the vessels and (7)proliferation there (Thiery, the metastasized Necl-5-CT26 cells in the lungs formed 2002). Among these steps, we examined steps (5)-(7) large foci beneath the veins (Figure 1e). These results by injecting cancer cells into the tail vein of mice suggest that Necl-5 enhances metastasis of CT26 cells to and measuring metastasis to the lungs. We found the lungs. that the upregulated Necl-5 in cancer cells trans- We subsequently used another cancer cell line, the interacts with its counter-receptor in platelets, probably OVHM ovarian cancer cell line, to further examine the CD226, and that this trans-interaction enhances the potential effect of Necl-5 on metastasis. The number of metastasis of the cancer cells. CD226, also called Necl-5-overexpressing OVHM cells (Necl-5-OVHM DNAM-1, is another Ig-like molecule and was originally cells)that metastasized to the lungs was 356 711 and identified as a DNAX accessory molecule (Shibuya about 10-fold higher than the numbers of metastasized et al., 1996). CD226 is expressed on the majority of parental OVHM cells (1177)and control pMX-OVHM peripheral blood T lymphocytes, and also on platelets cells introducing the pMX vector alone (270.5) (Kojima et al., 2003).