Interaction of Cancer Cells with Platelets Mediated by Necl-5/Poliovirus Receptor Enhances Cancer Cell Metastasis to the Lungs

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 enhances 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). On the other hand, platelets are (Supplementary Figures 1a–d). These results demon- involved in the metastasis of cancer cells (Nash et al., strate that Necl-5 augments the metastasis of cancer 2002). When cancer cells enter blood vessels, platelets cells of different organ origins to the lungs. attach to the cells and the resulting aggregates of platelets and cancer cells arrest the cancer cells in a Inhibition of cancer metastasis by an anti-Necl-5 blood vessel and then induce extravasation of cancer monoclonal antibody cells. Although the formation of these aggregates is well Next, we analysed the effects of an anti-Necl-5 mono- accepted, the detailed mechanisms of the interaction clonal antibody (mAb)on cancer metastasis to the lungs between platelets and cancer cells remain unknown. using CT26 cells. This mAb inhibits the trans-interaction Therefore, it is important to clarify the mechanisms of of Necl-5 with nectin-3 (Ikeda et al., 2003). BALB/c the interaction between platelets and cancer cells to mice were injected i.v. with CT26 cells treated with the understand the metastasis of cancer cells and develop anti-Necl-5 mAb, or with phosphate-buffered saline therapies for preventing this metastasis. Here, we (PBS)or rat immunoglobulin G (IgG)as controls. The describe a role of Necl-5 in the metastasis of cancer animals were killed on day 14 and the tumor nodules on cells to the lungs. the surface of their lungs were counted. The apparent

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 266

Figure 1 Enhancement of cancer cell metastasis to the lungs by Necl-5. (a)Cell surface expression levels of Necl-5 on various cancer cells. The cells were analysed by FACS with anti-Necl-5 mAb (open profile)and control rat IgG (gray profile).( b–e)Enhancement of CT26 cell metastasis to the lungs by Necl-5. (b)Cell surface expression levels of exogenous Necl-5 on CT26 cells. CT26, pMX-CT26 and Necl-5-CT26 cells were analysed by FACS with anti-Necl-5 mAb (open profile)and control rat IgG (gray profile).( c)Photographs of metastasized lungs. (d)Numbers of tumor nodules in the lungs. Groups of BALB/c mice ( n ¼ 5)were given CT26, pMX-CT26 and Necl-5-CT26 cells by i.v. injection and the numbers of pulmonary metastases in the different groups were counted on day 14 after the injection. (e)Hematoxylin–eosin staining of the lungs. Bars, 100 mm.

lung size was smaller in mice injected with CT26 cells the mean volumes in cubic millimeters were calculated treated with the anti-Necl-5 mAb than in control mice (Figure 2c). The animals were killed on day 28 and the treated with PBS or IgG (Figure 2a). The number of tumor volumes were measured (Figure 2d). Tumors tumor nodules formed by CT26 cells treated with the appeared at day 6 in all three mouse groups, and anti-Necl-5 mAb was reduced to one-third of the injection of the anti-Necl-5 mAb had no effect on number formed by control CT26 cells treated with the tumor size. These results indicate that the anti-Necl- PBS or IgG (Figure 2b). These results provide another 5 mAb does not affect the proliferation of cancer cells line of evidence that Necl-5 is involved in the metastasis in vivo. of cancer cells to the lungs. Next, we analysed the effect of the anti-Necl-5 mAb Arrest of cancer cells in pulmonary vessels on tumor growth in the s.c. tissue. BALB/c mice were Next, we analysed how cancer cells are metastasized to injected i.v. with the anti-Necl-5 mAb, PBS or rat IgG the lungs. To assess the arrest of cancer cells in the before and after a s.c. injection of CT26 cells. Tumor lungs, we injected carboxyﬂuorescein diacetate-succini- growth was measured two to three times per week, and midyl ester (CFSE)-labeled CT26, pMX-CT26 and

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 267

Figure 2 Inhibition of cancer metastasis by an anti-Necl-5 mAb. (a and b)Effects of the anti-Necl-5 mAb on cancer metastasis. (a)Photographs of metastasized lungs. ( b)Numbers of tumor nodules in the lungs. CT26 cells were pretreated with phosphate-buffered saline (PBS), control rat immunoglobulin G (IgG) or anti-Necl-5 mAb. BALB/c mice (n ¼ 5)injected i.v. with 2 Â 105 of the cells were killed on day 14, and the numbers of tumor nodules in the lungs were counted. (c and d)Lack of effect of the anti-Necl-5 mAb on tumor growth. (c)Tumor growth curve. ( d)Tumor volume on day 28. BALB/c mice ( n ¼ 5)were injected s.c. with 1 Â 105 CT26 cells on day 0. PBS, control rat IgG or anti-Necl-5 mAb was injected i.v. on days À1, 0, 3, 6, 8, 10, 13, 15, 17, 20, 22, 24 and 27 at a dose of 0.5 mg. The tumor dimensions were measured and the mean tumor volume in each group was monitored.

Necl-5-CT26 cells into the tail vein of mice and collected the lung tissue at 24 h after the injection. Clusters of CFSE-labeled CT26 and pMX-CT26 cells were observed inside the capillaries (Figure 3a). It should be noted that Necl-5-CT26 cells were also observed around the capillaries. Quantitative analysis revealed that the number of arrested cells was twofold higher for Necl-5- CT26 cells (86721)than for CT26 cells (43 717)or pMX-CT26 cells (45711; Figure 3b). These results suggest that Necl-5 plays a role in the lodgment of cancer cells in the pulmonary vessels during the early stages of metastasis.

Arrest of cancer cells in pulmonary vessels by Necl-5- dependent attachment to CD226-expressing platelets Necl-5 has been shown to heterophilically trans-interact with CD226 (Bottino et al., 2003)and nectin-3 (Ikeda Figure 3 Arrest of cancer cells in pulmonary vessels. BALB/c mice et al., 2003). CD226 is an Ig-like cell–cell adhesion (n ¼ 5)injected i.v. with 1 Â 106 CFSE-labeled CT26 or Necl-5- molecule with two Ig-like loops in the extracellular CT26 cells were killed after 24 h, and their lungs were isolated. (a) region (Shibuya et al., 1996). First, we examined Immunofluorescence images of the lungs. Samples were stained whether immunofluorescence signals for CD226 and with anti-CD31/PECAM-1 Ab. Red, CD31/PECAM-1; green, CFSE-labeled cells. Bars, 100 mm. (b)Number of arrested CFSE- nectin-3 were detected at sites surrounding CT26 cells labeled cells. The number of CFSE-labeled cells in a determined arrested in the lungs. Immunofluorescence microscopy area of the surface was counted as described in the ‘Materials and revealed that signals for CD226, but not nectin-3, were methods’.

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 268

Figure 4 Arrest of cancer cells in pulmonary vessels via attachment to platelets through the trans-interaction of Necl-5 with CD226. (a)Concentration of CD226, but not nectin-3, at sites surrounding arrested CT26 cells in the lungs. BALB/c mice ( n ¼ 5)injected i.v. with 1 Â 106 carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled CT26 cells were killed after 24 h, and their lungs were isolated. Samples were stained with anti-nectin-3 or anti-CD226 mAbs. Bars, 10 mm. (b)Effects of the anti-Necl-5 mAb on the adhesion of Necl-5 to CD226 or nectin-3. Calcein-AM-labeled CD226-B300 or nectin-3-B300 cells were seeded into control AP or Necl-5-AP-immobilized 96-well enzyme- linked immunosorbent assay plates in the presence of phosphate-buffered saline (PBS), control rat immunoglobulin G (IgG) or anti-Necl-5 mAb. Bound cells were measured using a multilabel counter. (c)Analysis of the cell surface expression of CD226 on platelets. The platelets were analysed by FACS with the indicated mAbs (open profile)and control IgG (gray profile).( d)Expression of CD226 in platelets surrounding CT26 cells in the lungs. BALB/c mice (n ¼ 5)injected i.v. with 1 Â 106 CFSE-labeled CT26 cells were killed after 24 h, and their lungs were isolated. Samples were double stained with various combinations of anti-CD226, anti-CD41 and anti-Necl-5 mAbs. Bars, 10 mm. (e)Depletion of platelets. BALB/c mice were injected i.p. with a rabbit anti-mouse platelet serum and the numbers of platelets and white blood cells were monitored. (f)Role of platelets in Necl-5-induced metastasis. Untreated or anti-platelet serum-pretreated BALB/c mice ( n ¼ 6)were injected i.v. with 1 Â 106 CT26 or Necl-5-CT26 cells. The mice were killed after 24 h, and their lungs were isolated. Samples were double-stained with anti- Necl-5 mAb and 4,6-diamidino-2-phenyl indole. The number of Necl-5-positive cancer cells in a determined area of the surface was counted as described in the ‘Materials and methods’.

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 269

Figure 4 Continued. concentrated at sites surrounding CT26 cells nectin-3 or Necl-5 was not detected (Figure 4c). (Figure 4a). Next, we examined whether the anti-Necl- Immunofluorescence microscopy revealed that signals 5 mAb inhibits the trans-interaction of Necl-5 with for CD226 as well as a platelet marker, CD41, were CD226, nectin-3 or both. We plated B300-19 cells concentrated in the cells, presumably platelets that expressing CD226 or nectin-3 onto dishes, which were surrounded CT26 cells (Figure 4d). Furthermore, the precoated with the extracellular region of Necl-5 fused signals for CD226 roughly colocalized with those of to alkaline phosphatase (Necl-5-AP), in the presence or Necl-5, which was expressed in CT26 cells. These results absence of the anti-Necl-5 mAb, washed the cells and suggest that adhesion of CT26 cells to platelets through then counted the numbers of cells that attached to the the trans-interaction of Necl-5 with CD226 is involved dishes. B300-19 cells expressing either CD226 or nectin- in the lung metastasis of CT26 cells. 3 adhered to the dishes, and their adhesion to Necl-5-AP To further confirm the role of platelets in the Necl-5- was efficiently inhibited by the anti-Necl-5 mAb enhanced metastasis, BALB/c mice were depleted of (Figure 4b). platelets by treatment with a rabbit anti-mouse platelet It has been shown that CD226 is expressed in platelets serum. Peripheral blood platelets were selectively (Kojima et al., 2003), and that platelets play important decreased to o5% of the normal level from 6 to 48 h roles in thrombosis and the induction of tumor after the serum injection without any drastic reduction metastasis. We used FACS analysis to confirm that in the white blood cells (Figure 4e). Next, CFSE-labeled CD226 was expressed in platelets, and that expression of pMX-CT26 and Necl-5-CT26 cells were injected into

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 270

Figure 5 Necl-5 as a major platelet adhesion molecule that trans-interacts with CD226. (a)Effects of the anti-Necl-5 mAb on the adhesion between CT26 cells and platelets. Calcein-AM-labeled CT26 cells were preincubated with PBS, control rat IgG or anti-Necl-5 mAb, and seeded into platelet-coated 96-well enzyme-linked immunosorbent assay plates. Bound cells were measured using a multilabel counter. (b)Analysis of the cell surface expression of nectin-2 in CT26 cells. CT26 cells were analysed by FACS with anti- nectin-2 mAb (open profile)and anti-rat IgG Ab (gray profile).( c)Inhibition of the adhesion of nectin-2 to CD226 by anti-nectin-2 mAb. Gray bar, reaction with anti-nectin-2 mAb; white bar, control experiment with no Ab. (d)Inhibition of the binding of CD226– CT26 cells by the anti-Necl-5 mAb, but not the anti-nectin-2 mAb. CT26 cells were preincubated with anti-Necl-5 and/or nectin-2 mAbs, incubated with control-AP or CD226-AP and analysed by FACS. Open profile, CD226-AP; gray profile, control-AP.

mice at 6 h after the anti-platelet serum treatment. analysis (Figure 5b). We then examined whether Necl-5 Depletion of platelets significantly suppressed the on CT26 cells is the major CD226 counter-receptor. To pulmonary attachment of cancer cells (Figure 4f). The achieve this, we prepared an anti-nectin-2 mAb that mean number of arrested pMX-CT26 and Necl-5-CT26 inhibited the trans-interaction of nectin-2 with CD226. cells decreased from 4178to1475 and from 117714 This mAb inhibited the attachment of CD226-expres- to 2478, respectively, in the platelet-depleted mice. sing B300-19 cells to dishes precoated with the extra- These results suggest that platelets predominantly cellular region of nectin-2 fused to alkaline phosphatase mediate Necl-5-dependent tumor cell attachment to (nectin-2-AP; Figure 5c). The interaction between CT26 pulmonary vessels. cells and CD226-AP was not significantly reduced by the anti-nectin-2 mAb alone, but was markedly inhibited by Necl-5 is a major platelet adhesion molecule that the anti-Necl-5 mAb alone or a mixture of the anti-Necl- trans-interacts with CD226 5 and anti-nectin-2 mAbs (Figure 5d). These results We further confirmed that Necl-5 is a major cell–cell indicate that Necl-5 is a major cell–cell adhesion adhesion molecule of CT26 cells that trans-interacts with molecule of CT26 cells that trans-interacts with CD226. its counter-receptor of platelets. Adhesion between CT26 cells and platelets was inhibited by the anti- Necl-5 mAb, suggesting that Necl-5 is involved in the Discussion adhesion (Figure 5a). CD226 has also been shown to trans-interact with nectin-2 (Bottino et al., 2003). Necl-5 is upregulated in many types of human and Furthermore, nectin-2 and Necl-5 are both expressed rodent cancer cells (Chadeneau et al., 1994, 1996; in various tumor cell lines (Bottino et al., 2003). We Gromeier et al., 2000; Masson et al., 2001; Sloan detected nectin-2 expression in CT26 cells by FACS et al., 2004). We further confirmed upregulation of

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 271 Necl-5 in several cancer cell lines, including CT26, as well as increasing the cell motility and proliferation of Colon 26, B16F1, Meth A and OVHM. The physiolo- cancer cells. CD226 was originally identified as a signal- gical and pathological roles of this upregulated Necl-5 transducing adhesion molecule involved in the cytolytic are unknown, although we found that it enhances cell function mediated by cytolytic T lymphocytes and movement and proliferation. In the present study, we natural killer (NK)cells. Initiation of the cytotoxicity have shown that Necl-5 enhances cancer cell metastasis function by cross-linking of the CD226 receptor depends to the lungs. Among the multiple steps involved in upon its Ser329 phosphorylation by protein kinase C cancer cell metastasis to the lungs, we have demon- (PKC)and physical and functional interactions with strated that upregulated Necl-5 is involved in at least the LFA-1 (Shibuya et al., 1999). CD226 is also known as steps of arrest in the lungs, exit from the vessels and platelet and T-cell antigen 1 and its cross-linking by a proliferation in the lungs, although it still remains mAb stimulates platelet secretion and activation in a unknown whether the upregulated Necl-5 is involved in PKC-dependent manner (Sherrington et al., 1997). On the initial steps of metastasis, such as invasion to the other hand, Necl-5 colocalizes with integrin avb3 at surrounding tissues and entry into blood and lymph the leading edges of moving cells and enhances growth vessels. We initially anticipated that disruption of the factor-induced cell movement and proliferation by trans-interaction of Necl-5 with nectin-3 by an anti- facilitating growth factor-induced activation of Cdc42 Necl-5 mAb would lead to enhanced metastasis of and Rac small G proteins (Ikeda et al., 2004; Kakunaga cancer cells to the lungs, since this trans-interaction et al., 2004). Necl-5 in glioma cells increases motility and reduces cell movement and proliferation. Unexpectedly, substrate-dependent reductions in adhesion and cell however, we found that this mAb reduced cancer cell spreading by modulating an adhesion-induced Src/ metastasis to the lungs. Our subsequent detailed FAK/paxillin/p130Cas signaling pathway to destabilize experiments revealed that the upregulated Necl-5 in focal adhesions at the cell-substrate interface (Sloan cancer cells trans-interacts with a counter-receptor other et al., 2005). Thus, the trans-interaction of Necl-5 and than nectin-3, probably CD226, in platelets and that CD226 may enhance the lung metastasis by inducing this trans-interaction enhances the metastasis of the activation signals in both cancer cells and platelets. cancer cells. In this context, molecules activating both platelets Platelets play an important role in thrombosis and are and cancer cells have been shown to promote metastasis. also involved in the induction of inflammation, tissue Aggrus/podoplanin is a type-I transmembrane sialomu- repair and tumor metastasis. The adhesive interaction cin-like glycoprotein that induces platelet aggregation between cancer cells and platelets has been reported to and its expression is frequently upregulated in colorectal play a critical role in metastasis (Nash et al., 2002). tumors (Kato et al., 2003). A mAb against Aggrus However, the molecules that facilitate the arrest of inhibited tumor-induced platelet aggregation in vitro as cancer cells in blood vessels have not yet been fully well as the pulmonary metastasis of highly metastatic identified. We have shown that Necl-5 on cancer cells clones of a mouse colon adenocarcinoma cell line in vivo. and a counter-receptor other than nectin-3, probably Recently, Aggrus was shown to bind ezrin-radixin- CD226, on platelets are important cell–cell adhesion moesin proteins to activate RhoA small G protein and molecules involved in the adhesion between cancer cells promote the epithelial-mesenchymal transition, leading and platelets in vitro as well as in pulmonary metastasis to increased cell migration and invasiveness (Martin- in vivo. It was previously reported that association of Villar et al., 2006). Im et al. (2004)reported that tumor melanoma cells with adherent thrombi results in stable cells initially make contact with exposed basement arrest under in vitro flow conditions and that inhibition membrane, followed by spreading of the tumor on the of platelet activation, platelet integrin aIIbb3 or melano- vessels in a manner that is morphologically reminiscent ma integrin b3 function abolishes the cell attachment of cell spreading in vitro. Inhibition of coagulation with (Felding-Habermann et al., 1996). In vivo, association of a thrombin inhibitor did not alter the association of platelets with tumor cells attached to the vascular tumor cells with platelets and fibrin or fibrinogen at the endothelium has been reported (Crissman et al., 1988), time of early arrest. However, tumor cell spreading at suggesting that platelets may stabilize and protect 6–24 h and subsequent retention of tumor cells in the attached tumor cells during blood flow. Consistent with lungs was markedly inhibited by anticoagulant treat- this hypothesis, we found that CD226 þ /CD41 þ platelets ment. Thus, tumor cell spreading on the vascular surface surrounded CT26 cells arrested in the pulmonary constitutes an important component of the metastatic vasculature and that signals for CD226 colocalized with cascade mediated by coagulation. It remains to be seen those of Necl-5 on CT26 cells. Furthermore, depletion whether Necl-5 facilitates the platelet-dependent initial of platelets, but not leukocytes, significantly suppressed attachment, the cell spreading leading to stable arrest in the pulmonary attachment of cancer cells. These results the vessel or both. clearly show that platelets predominantly mediate Necl- Under inflammatory conditions, leukocytes are cap- 5-dependent tumor cell attachment to pulmonary vessels tured by selectins, activated by chemokines and firmly probably through the trans-interaction of Necl-5 with adhered by integrins on endothelial cells, before finally CD226. transmigrating from the blood circulation into the Beyond the physical interaction between cancer cells peripheral tissues by passing through the endothelial and platelets, it is tempting to speculate that these cell–cell junctions. The multistep adhesion molecule molecules also enhance metastasis by activating platelets cascade involved in leukocyte trafficking may also

Oncogene Necl-5-CD226 interaction in lung metastasis K Morimoto et al 272 regulate the cell adhesion, migration and tissue tropism Necl-5 on platelets. However, CD96/Tactile was re- of cancer cells during tumor metastasis. In this regard, cently found to interact with Necl-5 and promote NK Reymond et al. (2004)reported that CD226 on cell–target cell adhesion (Fuchs et al., 2004). Thus, we monocytes interacts directly with Necl-5 at endothelial cannot exclude the possibility that Necl-5 also interacts cell–cell junctions and promotes their transendothelial with CD96 on the platelets in addition to CD226. migration by acting speciﬁcally during the diapedesis Further studies by using neutralizing anti-CD226 anti- step. Furthermore, CD226 mediates the adhesion of body or CD226 knockout mice are needed to determine thrombin-activated, but not resting, platelets to vascular whether CD226 is the sole counter-receptor for Necl-5 endothelial cells (Kojima et al., 2003). Considering that on platelets. cancer cells could be covered with platelets, their adhesion to and diapedesis through endothelial cells may be facilitated indirectly by the platelet–endothelial Materials and methods interaction. Further studies are needed to clarify whether Necl-5 enhances the diapedesis of cancer cells See Supplementary Information. arrested in blood vessels. In the present study, we have shown that Necl-5 is the major counter-receptor for CD226 on cancer cells, Abbreviations despite the fact that both Necl-5 and nectin-2 are expressed on these cells. Similarly, Necl-5, but not Necl-5-AP, extracellular region of Necl-5 fused to alkaline nectin-2, was previously found to be the major CD226 phosphatase; nectin-2-AP, extracellular region of nectin-2 counter-receptor on endothelial cells (Reymond et al., fused to alkaline phosphatase; PVR, poliovirus receptor. 2004). These results suggest that the CD226-binding domain of nectin-2 may be in an inactive conforma- Acknowledgements tional state in cancer cells as well as in resting endothelial cells. We have further indicated that We thank Dr H Yamauchi (KAN Research Institute Inc.)for CD226, but not nectin-3, is a counter-receptor for helpful comments and encouragement.

References

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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