Oncogene (2006) 25, 6480–6488 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE MRP-1/CD9 gene transduction regulates the actin through the downregulation of WAVE2

C-L Huang1, M Ueno2, D Liu1, D Masuya1, J Nakano1, H Yokomise1, T Nakagawa3 and M Miyake4

1The Second Department of Surgery, Faculty of Medicine, Kagawa University, Kagawa, Japan; 2Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, Japan; 3Life Science Research Center, Kagawa University, Kagawa, Japan and 4The Fifth Department of Oncology and the Department of Thoracic Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan

Motility-related -1 (MRP-1/CD9) is involved Introduction in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein Motility-related protein-1 (MRP-1/CD9), a member (Arp) 2 and Arp3 and the Wiskott–Aldrich syndrome of the , is implicated in various biological protein (WASP) family according to MRP-1/CD9 gene functions including motility (Miyake et al., 1991; transduction into HT1080 cells. The frequency of cells Ikeyama et al., 1993). Experimental studies have with lamellipodia was significantly lower in MRP-1/CD9- demonstrated that MRP-1/CD9 plays a functional role transfected HT1080 cells than in control HT1080 cells as a tumor metastatic suppressor. Transfection with (Po0.0001). MRP-1/CD9 gene transduction affected the MRP-1/CD9 cDNA revealed that cell motility was subcellular localization of Arp2 and Arp3 . suppressed in MRP-1/CD9-expressing cells (Ikeyama Furthermore, MRP-1/CD9 gene transduction induced a et al., 1993). The adenoviral transfection of MRP-1/ downregulation of WAVE2 expression (Po0.0001). CD9 cDNA also suppressed pulmonary metastasis However, no difference was observed in the expression and prolonged the survival of tumor-bearing immuno- of Arp2, Arp3 or other WASPs. A neutralizing anti- deficient mice (Miyake et al., 2000). Furthermore, MRP-1/CD9 monoclonal antibody inhibited downregula- clinical studies have revealed that a reduced expression tion of WAVE2 in MRP-1/CD9-transfected HT1080 of MRP-1/CD9 is associated with nodal metastases and cells (Po0.0001), and reversed the morphological effects a poor prognosis in cancer patients (Higashiyama et al., of MRP-1/CD9 gene transduction. Furthermore, down- 1995; Huang et al., 1998). However, its biological regulation of WAVE2 by transfection of WAVE2-specific mechanism in regulating cell motility is still not clearly small interfering RNA (siRNA) mimicked the morpho- understood. logical effects of MRP-1/CD9 gene transduction and Intravasation is considered to be a first step in suppressed cell motility. However, transfection of each metastasis (Condeelis et al., 2001). The reorganization siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect of cortical actin filaments plays an important role in cell WAVE2 expression. Transfection of WAVE2-specific motility. The actin-related protein (Arp) 2/3 complex siRNA also did not affect expressions of these Wnts. and the Wiskott–Aldrich syndrome protein (WASP) These results indicate that MRP-1/CD9 regulates the family are involved in the reorganization of cortical actin cytoskeleton by downregulating of the WAVE2, actin filaments (Takenawa and Miki, 2001; Vartiainen through the Wnt-independent signal pathway. and Machesky, 2004). The Arp2/3 complex is composed Oncogene (2006) 25, 6480–6488. doi:10.1038/sj.onc.1209654; of two Arps, Arp2 and Arp3, which act as templates for published online 8 May 2006 new actin filaments, and five additional subunits called ARPC1–5 (Vartiainen and Machesky, 2004). The Arp2/ Keywords: MRP-1/CD9; WAVE2; actin; motility 3 complex-dependent de novo actin nucleation is critical for the rapid formation of actin networks at the leading edge of the cells. The resulting reorganization of the actin skeleton provides the protruve force required for the extension of lamellipodia and filopodia observed during cell movement (Bailly et al., 1999). Further- more, the WASP family proteins, including the WASP subfamily (WASP and N-WASP) and the WAVE Correspondence: Dr C-L Huang, The Second Department of Surgery, subfamily (WAVE1, WAVE2 and WAVE3), regulate Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, the activation of the Arp2/3 complex (Takenawa and Kagawa 761-0793, Japan. E-mail: [email protected] Miki, 2001). Received 8 August 2005; revised 15 March 2006; accepted 30 March To clarify the regulation of motility in tumor cells 2006; published online 8 May 2006 would be helpful in developing new treatment strategies MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6481 for cancer patients. Therefore, we performed an experi- et al., 2000). Seventy-two hours after the transfection mental study on Arps and WASP family proteins in of MRP-1/CD9 into HT1080 using rAd5CAG-MRP-1/ relation to MRP-1/CD9 gene transduction. Conse- CD9, we performed real-time quantitative reverse quently, we found that MRP-1/CD9 gene transduction transciption–PCR (RT—PCR) analyses to deter- downregulated WAVE2 expression, and it affected the mine the MRP-1/CD9 gene expression, Western blot subcellular localization of the Arp2/3 complex. Further- analyses to determine the MRP-1/CD9 protein expres- more, we studied the relationship between the Wnt sion and motility assays using Transwell plates to signal pathways, including Wnt1, Wnt2b1 and Wnt5a, evaluate cell motility. As a result, transfection using and WAVE2 expression because we recently found that rAd5CAG-MRP-1/CD9 was found to effectively MRP-1/CD9 gene transduction downregulates these restore MRP-1/CD9 expression to suppress cell motility Wnt signal pathways (Huang et al., 2004). in HT1080 cells, as reported previously (Huang et al., 2004). Next we performed immunocytochemical analyses of F-actin, Arp2 and Arp3 to investigate the cell shape and Results actin . Lamellipodia was frequently seen in the highly motile control HT1080 cells (Figure 1a). MRP-1/CD9 gene transduction affects the actin Furthermore, Arp2 and Arp3 proteins tended to localize cytoskeleton and the subcellular localization of Arp2 in the lamellipodia in control HT1080 cells. In contrast, and Arp3 in HT1080 cells Arp2 and Arp3 proteins tended to diffusely locate We used a human fibrosarcoma cell line HT1080, which in cells after treatment with rAd5CAG-MRP-1/CD9 is highly motile and has a negative expression of (CD9-HT1080) (Figure 1b). Furthermore, the frequency MRP-1/CD9, and two adenoviral vectors, a vector of cells with lamellipodia was significantly lower in encoding human MRP-1/CD9 (rAd5CAG-MRP-1/ CD9-HT1080 than in the control HT1080 cells (29.77 CD9) and a control vector containing the bacterial lacZ 2.3 vs 77.7712.3%, Po0.0001, Figure 1c). As a result, gene (rAd5CAG-LacZ), as reported previously (Miyake the MRP-1/CD9 gene transduction affected the

Figure 1 Immunocytochemistry with staining of F-actin (green), Arps and WAVE2 (red) in control HT1080 cells (a) and CD9- HT1080 cells (b). Bars, 10 mm. One of the three experiments with similar results is shown. (c) The percentage of cells with lamellipodia according to MRP-1/CD9 gene transduction. Control, untreated HT1080 cells; LacZ, HT1080 cells after the treatment with a control vector rAd5CAG-LacZ; CD9, HT1080 cells after the treatment with rAd5CAG-MRP-1/CD9.

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6482 remodeling of the actin cytoskeletons in accordance with the changes in the subcellular localization of Arp2 and Arp3 proteins.

MRP-1/CD9 gene transduction downregulates the WAVE2 expression To clarify the mechanism of the remodeling of the actin cytoskeletons by MRP-1/CD9 gene transduction, real- time quantitative RT–PCR was performed to evaluate the gene expression of Arps and the WASP family. Consequently, MRP-1/CD9 gene transduction induced a downregulation of the WAVE2 gene expression (Figure 2a). The WAVE2 gene expression was signifi- cantly lower in CD9-HT1080 than in the control HT1080 (0.30670.011-fold, Po0.0001). However, there was no difference in the gene expression of Arp2 (0.87870.086- fold), Arp3 (0.96970.062-fold), WASP (0.84970.043- fold), N-WASP (0.98670.074-fold), WAVE1 (1.2597 0.116-fold) or WAVE3 (0.91270.081-fold). Furthermore, we performed Western blot analyses to evaluate the protein expression of WAVE2, Arp2 and Arp3 according to MRP-1/CD9 gene transduction. MRP-1/CD9 gene transduction downregulated the WAVE2 protein expression (Figure 2b). However, there was no difference in the protein expression of Arp2 or Arp3. We next performed immunocytochemical analyses with WAVE2 staining. WAVE2 protein also tended to localize in the lamellipodia in control HT1080 cells (Figure 1a). In contrast, WAVE2 expression was low in CD9-HT1080 cells (Figure 1b). Furthermore, the frequency of cells with WAVE2 at the lamellipodia was significantly lower in CD9-HT1080 cells than in the control HT1080 cells (6.172.9 vs 28.275.2%, Po0.0001; Figure 2c).

Anti-MRP-1/CD9 monoclonal antibody inhibits downregulation of WAVE2 in MRP-1/CD9- transfected cells To confirm whether the MRP-1/CD9 gene negatively regulates WAVE2 expression, we examined the effect of an anti-MRP-1/CD9 monoclonal antibody (mAb), ALB-6, which neutralizes the function of the MRP-1/ CD9 protein (Park et al., 2000). After transfection of MRP-1/CD9 into HT1080, cells were cultured in media Figure 2 (a) Fold changes in the gene expressions of Arp2, Arp3 and WASP family according to MRP-1/CD9 gene transduction containing 1 mg/ml of ALB-6, or a control mouse IgG. evaluated by real-time quantitative RT–PCR. (b) Western blot Seventy-two hours after transfection of MRP-1/CD9, analysis for the protein expression of WAVE2, Arp2 and Arp3 real-time quantitative RT–PCR to determine the according to MRP-1/CD9 gene transduction and treatment with a WAVE2 gene expression, Western blot analyses to neutralizing anti-MRP-1/CD9 mAb, ALB-6. Equal protein loading determine the WAVE2 protein expression, immuno- was evaluated by blotting against actin. One of the three experiments with similar results is shown. (c) The percentage of cytochemistry and motility assays were performed, on cells with WAVE2 protein at the lamellipodia. Control, untreated each group of cells. HT1080 cells; LacZ, HT1080 cells after the treatment with a Consequently, MRP-1/CD9-transfected HT1080 cells control vector rAd5CAG-LacZ; CD9, HT1080 cells after the maintained in media containing ALB-6did not have a treatment with rAd5CAG-MRP-1/CD9; CD9 þ IgG, MRP-1/ 7 CD9-transfected HT1080 cells maintained in media containing a decreased gene expression of WAVE2 (0.921 0.043- control mouse IgG; CD9 þ anti-CD9, MRP-1/CD9-transfected fold) in comparison to the control HT1080 cells HT1080 cells maintained in media containing a neutralizing anti- (Figure 3a). Furthermore, the WAVE2 gene expression MRP-1/CD9 mAb, ALB-6. was significantly higher in ALB-6-treated CD9-HT1080 than in CD9-HT1080 (Po0.0001). Western blot ana- lyses also revealed that ALB-6-treated CD9-HT1080

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6483

Figure 3 (a) Fold changes in the WAVE2 gene expression evaluated by real-time quantitative RT–PCR. (b) Immunocytochemistry with staining of F-actin (green), Arps and WAVE2 (red) in ALB-6-treated CD9-HT1080. Bars, 10 mm. One of the three experiments with similar results is shown. (c) The percentage of cells with lamellipodia according to MRP-1/CD9 gene transduction and treatment with ALB-6. (d) Motility assays using Transwell plates. *Po0.0001. LacZ, HT1080 cells after the treatment with a control vector rAd5CAG-LacZ; CD9, HT1080 cells after the treatment with rAd5CAG-MRP-1/CD9; CD9 þ IgG, MRP-1/CD9-transfected HT1080 cells maintained in media containing a control mouse IgG; CD9 þ anti-CD9, MRP-1/CD9-transfected HT1080 cells maintained in media containing a neutralizing anti-MRP-1/CD9 mAb, ALB-6. cells had an increased WAVE2 protein expression in chamber in ALB-6-treated CD9-HT1080 was signifi- comparison to CD9-HT1080, and had the same level of cantly higher than that in CD9-HT1080 (133.0711.0 vs WAVE2 protein expression as that of control HT1080 83.178.0 at 6h, Po0.0001; 249.6723.2 vs 171.7712.8 (Figure 2b). These results revealed that the neutralizing at 12 h, Po0.0001) whereas it was of the same level as anti-MRP-1/CD9 mAb treatment inhibited the down- that in the control HT1080 cells (Figure 3d). regulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells. Immunocytochemical analyses revealed that Arps and Decreasing the WAVE2 expression affects the actin WAVE2 protein tended to localize in the lamellipodia in cytoskeleton and suppresses cell motility ALB-6-treated CD9-HT1080 cells (Figure 3b). The To confirm the involvement of WAVE2 in the actin frequency of cells with lamellipodia in ALB-6-treated cytoskeleton and cell motility, the WAVE2 expression CD9-HT1080 was significantly higher than that in CD9- was knocked down by transfection of WAVE2-specific HT1080 (73.178.8 vs 29.772.3%, Po0.0001) and of small interfering RNA (siRNA) (WAVE2 siRNA). the same level as that in the control HT1080 cells Seventy-two hours after transfection of WAVE2 siRNA (Figure 3c). Furthermore, in motility assays, the number or ScraWA siRNA (control) into HT1080 cells, of cells passing through the membrane into the lower Western blot analyses to determine the WAVE2 protein

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6484 expression, immunocytochemistry and motility assays that in ScraWA siRNA-transfected HT1080 cells were performed on each group of cells. (76.076.1 vs 131.2711.9 at 6h, Po0.0001; 160.07 Consequently, the WAVE2 protein expression in cells 14.6vs 244.4 726.8 at 12 h, Po0.0001) (Figure 4d) and after the transfection of WAVE2 siRNA was reduced by of the same level as that observed in CD9-HT1080 cells. 90% of the value in cells after the transfection of ScraWA siRNA (Figure 4a). Immunocytochemical analyses demonstrated that Arp2 and Arp3 proteins Expressions of Wnt1, Wnt2b1 or Wnt5a are not tended to diffusely locate in WAVE2 siRNA-transfected associated with WAVE2 expression HT1080 cells (Figure 4b). The frequency of cells with We recently found that MRP-1/CD9 gene transduction lamellipodia in WAVE2 siRNA-transfected HT1080 downregulates several Wnt signal pathways, including cells was significantly lower than that in ScraWA Wnt1, Wnt2b1 and Wnt5a (Huang et al., 2004), and siRNA-transfected HT1080 cells (24.674.3 vs 74.57 therefore we performed further experiments using the 10.4%, Po0.0001) (Figure 4c) and of the same level as transfection of Wnt1-specific siRNA (Wnt1 siRNA), that in CD9-HT1080 cells. Furthermore, in motility Wnt2b1 siRNA and Wnt5a siRNA to evaluate the assays, the number of cells passing through the relationship between these Wnt signal pathways and membrane into the lower chamber in WAVE2 siRNA- the WAVE2 expression. Control scramble oligonucleo- transfected HT1080 cells was significantly smaller than tides were used for each siRNA. Seventy-two hours

Figure 4 (a) Western blot analysis for the protein expressions of WAVE2 and Wnt family at 72 h after transfection of WAVE2 siRNA and ScraWA siRNA. Equal protein loading was evaluated by blotting against actin. One of the three experiments with similar results is shown. (b) Immunocytochemistry with staining of F-actin (green), Arps and WAVE2 (red) in WAVE2 siRNA-transfected HT1080 cells. Bars, 10 mm. One of the three experiments with similar results is shown. (c) The percentage of cells with lamellipodia at 72 h after siRNA transfection. (d) Motility assays using Transwell plates. *Po0.0001. ScraWA siRNA, a control scramble siRNA.

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6485 after the transfection of each siRNA into HT1080 Discussion cells, real-time quantitative RT–PCR to evaluate each gene expression and Western blot analyses to determine Metastasis is a critical biological behavior of malignant each protein expression were performed for each group tumors, and intravasation is considered to be a first step of cells. for metastasis (Condeelis et al., 2001). The reorganiza- Consequently, the transfection of Wnt1 siRNA, tion of actin filaments at the leading edges of the cells is Wnt2b1 siRNA and Wnt5a siRNA effectively down- a crucial event in cell motility, and it is regulated by a regulated the protein expressions of their target Wnts, highly integrated signaling cascade (Takenawa and respectively (Figure 5). However, no difference was Miki, 2001; Vartiainen and Machesky, 2004). This observed in the WAVE2 gene expression (1.04370.039- signaling cascade is governed by the Rho family, the fold) or the WAVE2 protein expression between Wnt1 WASP family and the Arp2/3 complex. Among the Rho siRNA-transfected HT1080 cells and control HT1080 family, RhoA, Rac and Cdc42 play important roles cells (Figure 5a). In addition, no difference was observed in the reorganization of the cortical actin networks in the WAVE2 gene expression (0.95370.076-fold) (Hall, 1998). Through the activation of these members or the WAVE2 protein expression between Wnt2b1 of the Rho family, the WASP family can activate the siRNA-transfected HT1080 cells and control HT1080 Arp2/3 complex (Yamaguchi et al., 2000; Vartiainen cells (Figure 5b). Furthermore, no difference was and Machesky, 2004). Finally, the Arp2/3 complex- observed in the WAVE2 gene expression (0.8457 dependent de novo actin nucleation is critical for the 0.047-fold) or the WAVE2 protein expression between rapid formation of actin networks and protrusions at Wnt5a siRNA-transfected HT1080 cells and control the leading edge of the cells. Briefly, Rac regulates HT1080 cells (Figure 5c). the formation of lamellipodia through the activation On the other hand, there was also no difference in the of WAVE (Miki et al., 1998; Machesky et al., 1999; gene expression of Wnt1 (0.98070.046-fold), Wnt2b1 Eden et al., 2002). Cdc42 controls the extension of (0.97470.081-fold) or Wnt5a (1.09470.032-fold) be- filopodia and microspikes through the activation of tween the WAVE2 siRNA-transfected HT1080 cells and WASP (Rohatgi et al., 2000; Yamaguchi et al., 2000). the control HT1080 cells. In addition, there was also no RhoA controls the assembly of stress fibers resulting in difference in the protein expression of Wnt1, Wnt2b or contraction at the rear of the cell to translocate the cell Wnt5a between WAVE2 siRNA-transfected HT1080 body (Maekawa et al., 1999). In total, these coordinated cells and ScraWA siRNA-transfected HT1080 cells reorganizations of actin filaments permit cell migration. (Figure 4a). On the other hand, MRP-1/CD9 is a member of the tetraspanins, which are forming multi- molecular complexes on the cell surface associating numerous proteins, the ‘ web’ (Berditchevski and Odintsova, 1999; Le Naour et al., 2004). MRP-1/ CD9 is implicated in various biological functions including cell motility (Miyake et al., 1991; Ikeyama et al., 1993). Many studies have demonstrated that MRP-1/CD9 plays a functional role as a tumor meta- static suppressor (Higashiyama et al., 1995; Huang et al., 1998; Miyake et al., 2000). However, the bio- logical mechanism by which it regulates cell motility is still not clearly understood. Therefore, we carried out the present study to understand the mechanism by which MRP-1/CD9 regulates cell motility, which could help in developing new treatment strategies for cancer patients. The present study has demonstrated that MRP-1/ CD9 gene transduction downregulates the WAVE2 expression in HT1080 cells. Furthermore, the neutraliz- ing anti-MRP-1/CD9 mAb treatment inhibited down- regulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells, and reversed the morphological effects of MRP-1/CD9 in immunocytochemical analyses. These Figure 5 (a) Western blot analysis for the protein expressions of results indicate that the MRP-1/CD9 signal is located Wnt1 and WAVE2 at 72 h after transfection of Wnt1 siRNA and ScraW1 siRNA (a control scramble siRNA). (b) Western blot upstream of the WAVE2-mediated pathway. Although analysis for the protein expressions of Wnt2b and WAVE2 at 72 h our previous study using microarray assays did not after transfection of Wnt2b1 siRNA and ScraW2 siRNA (a control detect this fact (Huang et al., 2004), this oversight might scramble siRNA). (c) Western blot analysis for the protein be partly owing to the fact that the signals of the expressions of Wnt5a and WAVE2 at 72 h after transfection of Wnt5a siRNA and ScraW5 siRNA (a control scramble siRNA). WAVE2 gene spots were weak in the microarray assays Equal protein loading was evaluated by blotting against actin. One we used. A recent study also reported that the difference of the three experiments with similar results is shown. in measurement of low-expression genes was considered

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6486 to contribute to the limited correlation between the including integrins (Berditchevski and Odintsova, 1999; results of microarray assays and the true values Gutierrez-Lopez et al., 2003), proteins with Ig domains, evaluated by real-time RT–PCR (Zhu et al., 2005). such as EWI-2 (Stipp et al., 2001), CD9P-1 (Charrin Furthermore, the present study has demonstrated that et al., 2001) and major histocompatibility complex MRP-1/CD9 gene transduction affects the subcellular molecules (Zilber et al., 2005), protein kinase C (Zhang localization of the Arp2/3 complex in HT1080 cells et al., 2001) and phosphoinositide 4-kinase (Yauch and whereas there was no difference in the protein expres- Hemler, 2000). Therefore, MRP-1/CD9 could have sion of Arp2 or Arp3. Regarding the cell shape, multiple biological functions through various signaling lamellipodia frequently appeared in high motile parental pathways, such as integrin-linked kinase and phospha- HT1080 cells. In contrast, the frequency of cells with tidyl inositol 3-kinase pathways (Chen et al., 1996; King lamellipodia was significantly lower in MRP-1/CD9- et al., 1997). The present study has demonstrated that transfected HT1080 cells than in parental HT1080 cells. MRP-1/CD9 regulates the actin cytoskeleton by the The WAVE, also known as Scar, was identified as a downregulation of WAVE2, thus resulting in the downstream effector for Rac-mediated lamellipodia suppression of cell motility. However, further investiga- formation (Miki et al., 1998). The WAVE family proteins tions are necessary to clarify the molecular mechanism consist of three orthologs, WAVE1, WAVE2 and of WAVE2 regulation by MRP-1/CD9. WAVE3. WAVE2 is widely expressed, whereas WAVE1 and WAVE3 are predominantly expressed in the brain (Soderling et al., 2003; Vartiainen and Machesky, 2004). Materials and methods Furthermore, WAVE2 is involved in various biological functions including cell motility, development and Cell lines and cell culture angiogenesis (Yamazaki et al., 2003; Yan et al., 2003). We used a human fibrosarcoma cell line HT1080 (MRP-1/ WAVE2 is essential for the peripheral ruffles at the CD9-negaitve). It was maintained in RPMI-1640 medium leading edge of cells, and it promotes directional cell supplemented with 10% fetal calf serum. migration (Suetsugu et al., 2003; Yamazaki et al., 2003). WAVE2 silencing by RNA interference on tumor cells Transfection of adenoviral vectors dramatically suppressed cell motility and metastasis An E1-deleted replication-deficient recombinant adenoviral (Kurisu et al., 2005). These results indicate that WAVE2 vector encoding human MRP-1/CD9 under the control of the acts as a primary effecter downstream of Rac to induce human CAG promoter (rAd5CAG-MRP-1/CD9) was con- lamellipodia formation and metastasis (Suetsugu et al., structed using an Adenovirus Expression Vector (Takara 2003; Kurisu et al., 2005). Biomedicals, Otsu, Japan), as reported previously (Miyake et al., 2000). In addition, a control adenoviral vector contain- In general, WAVE2 and N-WASP are widely ing the bacterial lacZ gene under the control of the human expressed among WASP family proteins. WAVE2 plays CAG promoter (rAd5CAG-LacZ) was also constructed. a key role in the formation of lamellipodia downstream HT1080 was maintained in 10 cm culture dishes at 90% of Rac, whereas N-WASP plays a key role in the form- confluency. Infections with rAd5CAG-MRP-1/CD9 or rAd5- ation of filopodia downstream of Cdc42 (Takenawa and CAG-LacZ were performed at an MOI of 30 plaque-forming Miki, 2001). The present study revealed that MRP-1/ units per cell in a volume of 200 ml for 2 h incubation. Seventy- CD9 gene transduction downregulated WAVE2 expres- two hours after treatment with each adenoviral vector, 2 Â 105 sion. The coordinated reorganization of actin filaments cells, one-twentieth of the population maintained in 10 cm is considered to be essential for cells to move. As a culture dishes, were placed into each tube on ice for result, the downregulation of WAVE2 induced by immunofluorescence staining to evaluate MRP-1/CD9 protein expression in each group of cells, as described previously MRP-1/CD9 gene transduction could disrupt the (Huang et al., 2004). Simultaneously, other subsequent coordinated reorganization of actin filaments to sup- experiments, including extraction of total RNA, protein press directional cell migration. In fact, the present sample preparation for Western blot analysis, immunocyto- study revealed that the downregulation of WAVE2 by chemistry and motility assays, were performed using the RNA interference mimicked the functional effects of remaining cells maintained in 10 cm culture dishes. MRP-1/CD9 transduction. However, the molecular mechanism by which Antibodies MRP-1/CD9 downregulates WAVE2 still remains un- We used two mouse anti-human MRP-1/CD9 mAbs, M31-15 clear. We recently found that MRP-1/CD9 gene and ALB-6. M31-15 was initially developed in our laboratory transduction downregulated the Wnt signal pathways, (Miyake et al., 1991). ALB-6, a neutralizing mAb, was including Wnt1, Wnt2b1 and Wnt5a (Huang et al., purchased from Immunotech (Marseille, France) (Park et al., 2004). However, transfection of each siRNA for Wnt1, 2000). A rabbit anti-human WAVE2 polyclonal Ab and a Wnt2b1 or Wnt5a did not affect WAVE2 expression. rabbit anti-human Arp3 polyclonal Ab were obtained from Transfection of WAVE2-specific siRNA also did not Upstate Biotechnology (Lake Placid, NY, USA). A rabbit affect the expressions of these Wnts. As a result, MRP- anti-human Arp2 polyclonal Ab (H-84), a rabbit anti-human Wnt1 polyclonal Ab (H-89), a goat anti-human Wnt5a 1/CD9 was thus considered to regulate cell motility by polyclonal Ab (C-16) and a mouse anti-human actin mAb downregulation of WAVE2, through the Wnt-indepen- (C-2) were obtained from Santa Cruz Biotechnology (Santa dent signal pathway. Cruz, CA, USA). A rabbit anti-human Wnt2b polyclonal To date, the MRP-1/CD9 protein is considered to antibody was obtained from Zymed Laboratories (South San form functional complexes with numerous proteins, Francisco, CA, USA). Fluorescein isothiocyanate-conjugated

Oncogene MRP-1/CD9 downregulates WAVE2 C-L Huang et al 6487 AffiniPure F(ab0)2 fragment anti-mouse IgG antibody was WAVE2 (1:1000), Arp2 (1:500) and Arp3 (1:500). Next, the obtained from Immunotech. Secondary horseradish peroxi- cells were incubated with Texas Red-conjugated anti-rabbit dase (HRP)-labeled anti-mouse IgG Ab and HRP-labeled anti- IgG for 1 h. The cells were then incubated with fluorescein rabbit IgG Ab were purchased from Amersham Pharmacia phalloidin solution (Molecular Probes, Eugene, OR, USA) for Biotech (Buckinghamshire, UK). HRP-labeled anti-goat IgG 20 min to visualize F-actin. After the cells were mounted, the Ab was purchased from ICN Pharmaceuticals (Aurora, OH, fluorescent signals were viewed under confocal microscopy USA). Texas Red-conjugated anti-rabbit IgG was obtained (Radiance 2100, Bio-Rad). A ruffled membrane structure with from Vector Laboratories (Burlingame, CA, USA). dense cortical actin was regarded as the lamellipodia. For each determination, the photographs of nine randomly selected Real-time quantitative RT–PCR fields were analysed. At least three independent experiments Total cellular RNA was extracted using the acid guanidinium were performed for each type of cells. thiocyanate procedure. First-strand cDNA synthesis was performed with 5 mg of total RNA using a cDNA synthesis Motility assay kit (Amersham). To quantify gene expression, TaqMan real- The motility assay was performed using Transwell plates time quantitative PCR was performed with the ABI PRISM (polycarbonate membrane, 8 mm pores; Costar, Cambridge, 7700 Sequence Detection System using TaqMan Gene MA, USA). 2 Â 105 cells were added to the top chamber, and Expression Assays probes and TaqMan Universal PCR assays were performed at 371C, 5% CO2, for 12 h. Cells passing Master Mix (Applied Biosystems, Foster City, CA, USA). through the membrane into the lower chamber were counted. Each sample was run in triplicate, and each PCR experiment Each condition was tested in triplicate per experiment, and each included three nontemplate control wells. In addition, experiment repeated three times with consistent results. triplicate assays were performed with RNA samples isolated from at least two independent experiments. A standard curve siRNA transfection for the serial dilutions of cDNA of control HT1080 was The target sequences for WAVE2, Wnt1, Wnt2b1 and Wnt5a similarly generated. The comparative threshold cycle method siRNA oligonucleotides were designed by siRNA Design (Applied Biosystems) was used to calculate the gene expression Support System (Takara Biomedicals). Scrambled oligonu- in each sample relative to the value observed in control cleotides were also designed for each of the above sequences HT1080 cells, using glyceraldehyde-3-phosphate dehydrogen- as negative controls. The sense strand sequences were as ase as a control for normalization among samples. follows: WAVE2 siRNA, 50-GCAUCAAGCCUCUAAAGA AdTdT-30; ScraWA siRNA (a negative control for WAVE2 Western blot analysis siRNA), 50-ACCAGCGCUAGUACAUAAAdTdT-30; Wnt1 Western blot analysis was performed to evaluate the expres- siRNA, 50-GAAGGUUCCAUCGAAUCCUdTdT-30; ScraW1 sion of various proteins. Briefly, cells were harvested and siRNA (a negative control for Wnt1 siRNA), 50-GCCAUCU resuspended in lysis buffer (62.5 mM Tris-HCl, 2% sodium CGAGUUAGUCAAdTdT-30; Wnt2b1 siRNA, 50-CAACUC dodecyl sulfate (SDS), 10% glycerol, 4 M urea). Protein UCCAGAUUACUGUdTdT-30; ScraW2 siRNA (a negative samples (50 mg) were each diluted into a 20 ml solution of lysis control for Wnt2b1 siRNA), 50-AGAUUCCAACCUCAUUC buffer and 5% 2-mercaptoethanol (Bio-Rad Laboratories, GUdTdT-30; Wnt5a siRNA, 50-GAAGUCCAUUGGAAUA Hercules, CA, USA) and heated in a water bath at 951C UUAdTdT-30; ScraW5 siRNA (a negative control for Wnt5a for 5 min. Then, protein extracts were separated by 10% siRNA), 50-AGAGACUCGAUUAAUGAUUdTdT-30. HT1080 SDS polyacrylamide gel electrophoresis in a vertical-slab gel cells were seeded in 6cm culture dishes 24 h before transfection electrophoresis cell (Bio-Rad). Next, the separated proteins to reach 50–60% confluency. The siRNA transfection was were transferred from gel to nitrocellulose membrane performed in a total volume of 2.5 ml containing each siRNA (Hybond-ECL; Amersham) and then blocked in a blocking (final concentration of 25 nM) and 25 ml of TransIT-TKO solution (5% dry milk and 0.2% Tween 20 in phosphate- Transfection Reagents (Mirus, Madison WI, USA) according buffered saline (PBS)) for 1 h. Then, membranes were to the manufacturer’s instructions. The efficiency of each siRNA incubated overnight with the primary antibodies detecting on protein suppression was ascertained by Western blot analyses, M31-15 (1:100), WAVE2 (1:1000), Arp2 (1:500), Arp3 (1:500), and cell viability was microscopically analysed by a Trypan blue Wnt1 (1:500), Wnt2b (1:100) and Wnt5a (1:100). The exclusion assay. Seventy-two hours after the siRNA transfection, membranes were then incubated with HRP-labeled secondary protein sample preparations for Western blot analyses, immuno- antibodies for 1 h. The proteins were visualized on enhanced cytochemistry and motility assays were performed. chemiluminescence film (Hyperfilm; Amersham) by applica- tion of Western blotting detection system (Amersham). Statistical analyses Finally, the blots were reprobed using antibodies against actin Data were presented as mean7s.d. Because the distributions (1:1000) to ensure equal loading and transfer of proteins. of the normalized WAVE2 gene expression (P ¼ 0.8245), frequencies of cells with lamellipodia (P ¼ 0.8011) and cells Immunocytochemistry with WAVE2 expression at the lamellipodia (P ¼ 0.8695), Cells grown on dishes were fixed for 10 min in 3.7% showed normal distributions (Kolmogorov–Smirnov analysis), paraformaldehyde in PBS. After the cells were incubated with differences in these data were analysed by analysis of variance 1% bovine serum albumin in PBS for 30 min, the cells were with the Bonferroni/Dunn test. A P-value of less than 0.05 was incubated for 1 h with the primary antibodies detecting considered to be statistically significant.

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