Oncogene (1999) 18, 4718 ± 4725 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc RalA requirement for v-Src- and v-Ras-induced tumorigenicity and overproduction of urokinase-type plasminogen activator: involvement of metalloproteases

Julio A Aguirre-Ghiso*,1,4, Paul Frankel2, Eduardo F Farias1, Zhimin Lu2, Hong Jiang2,5, Amanda Olsen2, Larry A Feig3, Elisa Bal de Kier Jo€e1 and David A Foster2

1Cell Biology Department, Research Area, Institute of Oncology, `Angel H Ro€o', University of Buenos Aires, Buenos Aires 1417, Argentina; 2Department of Biological Sciences, Hunter College of The City University of New York, New York, NY 10021, USA; 3Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts, MA 02111, USA

Overproduction of urokinase-type plasminogen activator Introduction (uPA) and metalloproteases (MMPs) is strongly corre- lated with tumorigenicity and with invasive and meta- Metastasis is the last and most lethal stage of tumor static phenotypes of human and experimental tumors. progression. This process follows several steps including We demonstrated previously that overproduction of uPA the exit of tumor cells from the primary tumor, in tumor cells is mediated by a phospholipase D (PLD)- intravasation after degradation of the interstitial and and protein kinase C-dependent mechanism. The onco- the blood vessels extracellular matrix, dissemination genic stimulus of v-Src and v-Ras results in the through the lymphatic or arterial system, and ®nally activation of PLD, which is dependent upon the extravasation, settlement, and growth in a distant organ monomeric GTPase RalA. We have therefore investi- (Hart et al., 1989). In several of these steps, as well as gated whether RalA plays a role in uPA and MMP during primary tumor organization, proteases like overproduction that is observed in response to oncogenic urokinase-type plasminogen activator (uPA) and metal- signals. We report here that NIH3T3 cells transformed loproteases (MMPs) are highly involved (Liotta and by both v-Src and v-Ras, constitutively overproduce uPA Stetler-Stevenson 1991; Ossowski, 1992). Tumorigenicity and that expression of a dominant negative RalA mutant has been described as being dependent on the expression (S28N) blocks overproduction of uPA in both the v-Src- of tumor- or host-derived proteases, since they enable the and v-Ras-transformed cells. v-Src and v-Ras also remodeling of the local tissue and angiogenesis, two induced an upregulation of the activity of MMP-2 and critical steps for the successful development of a primary MMP-9 as detected by zymograms, however only the v- tumor (Liotta et al., 1991; Werb et al., 1996). Strong Src induction correlated with MMP protein levels correlations between uPA and/or MMP production and detected by Western blot analysis. The dominant the invasive and metastatic ability of tumor cells have negative RalA mutant blocked increased MMP-2 and 9 been described (Ossowski 1992; Moller 1993; Boyd overproduction induced by v-Src, but not the increased 1996). uPA is a serine-protease that converts plasmino- activity of MMP-2 and 9 induced by v-Ras. And, gen to active plasmin (Moller 1993; Yu and Schultz, consistent with a role for the RalA/PLD pathway in 1990). Enhanced pericellular proteolysis, primary tumor mitogenesis and tumor development, the dominant organization, and invasiveness are mediated via the uPA/ negative RalA mutant completely blocked tumor forma- uPA receptor/plasmin system. Upregulation of uPA and tion by v-Src- and v-Ras-transformed NIH3T3 cells the uPA receptor enables tumor cells to keep their injected subcutaneously in syngeneic mice. The data surface completely saturated with these enzymes presented here implicate RalA and PLD as signaling (Ossowski and Reich 1983; Yu et al., 1997). MMPs are mediators for tumor formation and protease production a group of matrix proteases, such as the type IV by transformed cells. collagenases (MMP-2/gelatinase B, MMP-9/gelatinase A), which are implicated in tissue remodeling, angiogen- Keywords: GTPase; metalloproteases; Ral; Ras; Src; esis, in¯ammation, invasion and metastasis (Matrisian, tumorigenicity; uPA 1992). Tumor proteases like uPA and MMPs have been reported to be upregulated upon transformation by v- Src and v-Ras (Sato et al., 1993; Bell et al., 1993; Silberman et al., 1997; Gum et al., 1996). Signaling pathways activated by v-Src and v-Ras (Burgering and Bos, 1995) include those involving phosphatidylinositol- 3-kinase (Carpenter and Cantley, 1996), protein kinase C (Song et al., 1991), Raf-1 (Blobe et al., 1994) the extracellular regulated kinases (ERK-1 and 2) (Burger- *Correspondence: JA Aguirre-Ghiso ing and Bos, 1995) and phospholipase D (PLD) (Song Current addresses: 4 Division of Neoplastic Diseases, Department of et al., 1991). It has been reported that Raf-1, MEK-1 Medicine, Mount Sinai School of Medicine, New York, NY 10029, and ERK1 mediate the signal leading to uPA, uPA 5 USA; Department of Microbiology, Columbia University College of receptor, and MMP-9 upregulation (Lengyel et al., Physicians and Surgeons, New York, NY 10032, USA Received 27 August 1998; revised 8 March 1999; accepted 16 March 1996, 1997; Gum et al., 1997). We recently reported 1999 that overexpression of uPA in murine mammary RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4719 adenocarcinoma cells is controlled by a PLD and response to both v-Src and v-Ras (Jiang et al., protein kinase C-dependent pathway (Aguirre Ghiso et 1995b). NIH3T3 cells transformed by either v-Src al., 1997). Additionally, MMP-2 expression induced by (Figure 2a and 2c) or v-Ras (Figure 2b and c) that laminin has been reported to be coupled to PLD expressed the dominant negative S28N-RalA (Figure activation (Reich et al., 1995). PLD converts phospha- 2c) showed signi®cant (P50.01) reductions in secreted tidylcholine to phosphatidic acid, which has been uPA activity relative to the v-Src- and v-Ras- implicated in vesicle budding and transport, as well as transformed cells. Zymographic analysis showed that the transduction of intracellular signals (Bi et al., 1997; the uPA activity reduced by the dominant negative Exton, 1998; Roth and Sternweis, 1997). Phosphatidic mutant of RalA corresponded to a reduction of a acid may also be metabolized further to the biologically 48 kDa band (Figure 2d). uPA protein levels as active diacylglycerol or lysophosphatidic acid (Foster, determined by Western blot analysis were also reduced 1993; Exton, 1994). PLD activity is increased in in cells expressing the dominant negative RalA mutant response to most, if not all, mitogenic signals (Foster, (Figure 2e). As reported previously (Jiang et al., 1993; Exton, 1998). A PLD activity was found to be associated with the Ras-family GTPase RalA, which is required for the activation of PLD by both v-Src and v- Ras (Jiang et al., 1995b) and the active complex includes PLD1, RalA and Arf (Luo et al., 1997, 1998). In the present work, we have investigated the involvement of the RalA pathway in protease produc- tion and tumor formation induced by v-Src and v-Ras.

Results uPA production is upregulated in v-Src- and v-Ras-transformed NIH3T3 cells Several reports have described increases in uPA production in response to transformation by v-Src and v-Ras (Bell et al., 1993; Silberman et al., 1997). To establish whether uPA production is elevated in NIH3T3 cells in response to v-Src and v-Ras, we examined uPA expression levels in parental, and in v- Src-, and v-Ras-transformed NIH3T3 cells. As shown in Figure 1a, NIH3T3 cells secreted a 48 kDa plasminogen activator activity. This activity was completely blocked by 1 mM amiloride or an anti- catalytic anti-uPA antibody, con®rming the identity of uPA. We then compared the uPA activity in conditioned medium (CM) from v-Src- and v-Ras- transformed cells relative to that in the parental NIH3T3 cells. As shown in Figure 1b, both v-Src and v-Ras-transformed cells displayed increased levels of uPA activity relative to the parental NIH3T3 cells (Figure 1b). Consistent with the increased uPA activity in v-Src and v-Ras-transformed cells, we also detected increased levels of uPA protein in these cells relative to the NIH3T3 cells (Figure 1c).

Overproduction of uPA in v-Src- and v-Ras-transformed cells is mediated by RalA We demonstrated previously that uPA production is dependent on PLD (Aguirre Ghiso et al., 1997), and Figure 1 uPA production is upregulated in v-Src- and v-Ras- transformed NIH3T3 cells. (a) Plasminogen activator activity was that the activation of PLD by v-Src and v-Ras is investigated by zymographic analysis on the CM from NIH3T3 dependent upon RalA (Jiang et al., 1995b). We (pZip-neo transfectant) cells (lane 1). The same assay was therefore wished to determine whether the elevated performed in the presence of plasminogen-casein underlays uPA levels observed in v-Src and v-Ras cells were prepared with anticatalytic anti-uPA antibody (10 mg/ml) (lane dependent on RalA. To investigate the role of RalA on 2), or in the presence of the speci®c uPA inhibitor amiloride (1 mM) (lane 3); LM3 cells, which overexpress uPA were used as a uPA overproduction induced by v-Src and v-Ras, we positive control (lane 4). (b) uPA activity in the CM from examined uPA activity in v-Src and v-Ras transformed parental NIH3T3-pZip-neo cells was compared with that present cells stably expressing a mutant RalA protein (S28N), in the v-Src- and v-Ras-transformed NIH3T3-pZip-neo cells. (c) that functions as a dominant negative mutant for RalA uPA protein levels were determined by Western blot analysis of cell lysates from NIH3T3-pZip-neo cells, v-Src-transformed (Jiang et al., 1995b; Urano et al., 1996). This mutant NIH3T3-pZip-neo cells and v-Ras-transformed NIH3T3-pZip- blocked the increased PLD activity observed in neo cells RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4720

Figure 2 Overproduction of uPA in v-Src- and v-Ras-transformed cells is mediated by RalA. (a) Measurement of uPA activity present in the CM of NIH3T3-pZip-neo cells, v-Src-transformed NIH3T3-pZip-neo cells (v-Src) or v-Src cells expressing the S28N dominant negative RalA mutant (v-Src-S28N-Ral) was determined by radial caseinolysis as in Figure 1. (b) uPA activity in the CM from v-Ras-transformed NIH3T3-pZip-neo cells (v-Ras) and v-Ras cells expressing the S28N dominant negative RalA mutant (v- Ras-S28N Ral) was determined as in (a). The data in a and b represent the mean+the standard deviation for six samples from a representative experiment that was repeated three times. *indicates P50.01 for v-Src or v-Ras relative to the parental NIH3T3- pZip-neo cells, **indicates P50.01 for v-Ras-S28N-Ral or v-Src-S28N-Ral cells relative to the v-Ras or v-Src cells respectively as determined by the Students t-test. (c) Western blot analysis of levels of Src protein in the NIH3T3, v-Src and v-Src-S28N-Ral cells (upper left panel), and levels of Ras protein in the NIH3T3, v-Ras and v-Ras-S28N-Ral cells (upper right panel), or levels of RalA protein in the v-Src, v-Src-S28N-Ral, and v-Ras and v-Ras-S28N-Ral cells. (d) Zymograms of the uPA activity present in NIH3T3- pZip-neo cells and v-Src- and v-Ras-transformed NIH3T3-pZip-neo cells and these cells expressing the dominant negative Ral S28N mutant are presented along with the densitometric analysis of the zymogram. (e) Western blot analysis of uPA protein levels was performed on cell lysates from the NIH3T3, v-Src-transformed, v-Src-transformed cells expressing the S28N RalA mutant, as well as the v-Ras-transformed and v-Ras-transformed cells expressing the S28N RalA mutant

1995b), expression of the dominant negative RalA been reported in response to transformation by v-Src mutant had no e€ect upon either Src or Ras protein and v-Ras (Sato et al., 1993; Gum et al., 1996). MMP levels, or on the level of phosphotyrosine in the cells production in tumor cells has been reported to be expressing v-Src (Figure 2c and data not shown). Thus regulated by Ras (Sato et al., 1993) and dependent on the reduced uPA levels in cells expressing the dominant PLD (Reich et al., 1995). We therefore examined the negative RalA observed in Figure 2 are not due to role of RalA in the v-Src and v-Ras induction of reduced levels of the transforming oncoprotein in MMPs. Zymograms of the CM from v-Src- and v-Ras- response to the RalA mutant. transformed NIH3T3 cells showed an enhancement of proenzyme forms of a 72 kDa (Gelatinase B/MMP-2) and a 105 kDa (Gelatinase A/MMP-9) production Increased MMP-2 and MMP-9 production by v-Src, but compared to the control parental NIH3T3 cells not v-Ras is dependent on RalA (Figure 3a and b). MMP-2 and MMP-9 activity was It has been suggested that MMPs play a role in also elevated in cell lysates from v-Src- and v-Ras- tumorigenesis and metastasis formation (Liotta and transformed NIH3T3 cells (data not shown). v-Src- Stetler-Stevenson, 1991; Werb et al., 1996). Consistent transformed cells expressing the dominant negative with this hypothesis, increased MMP production has S28N RalA had substantially reduced levels of both RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4721

Figure 4 v-Src- and v-Ras-induced colony formation is partially dependent upon RalA. 16103 NIH3T3-pZip-neo cells, v-Src- and v-Ras-transformed cells, and v-Src- and v-Ras-transformed cells expressing the S28N RalA mutant or the D49N RalA mutant were suspended in soft agar and the percentage of cells forming colonies was determined. Error bars represent the standard errors for triplicate samples from duplicate experiments

transformed cells (Figure 3a and b). These low molecular weight species may represent the active forms of MMP-2. Interestingly, this form of MMP-2 was reduced in the v-Ras-transformed cells expressing the dominant negative RalA. Figure 3 Dependence of increased MMP-2 and MMP-9 activities in v-Src- and v-Ras-transformed cells upon RalA. (a) Zymograms showing the MMP activities secreted into CM by NIH3T3, v-Src- transformed or v-Src-transformed cells expressing the S28N The dominant negative S28N RalA mutant prevents v- dominant negative RalA mutant. (b) MMP activity from Src and v-Ras induced tumorigenicity NIH3T3, v-Ras-transformed, and v-Ras-transformed cells expres- sing the S28N dominant negative RalA mutant. Duplicate lanes It was reported previously that the dominant negative represent the activities secreted in two independent samples. S28N RalA inhibited v-Ras- and v-Raf-induced focus MMP activity was abolished in gels incubated in the presence of formation in cultured cells (Urano et al., 1996). 40 mM EDTA and una€ected in the presence of aprotinin (not However, the inhibitory e€ect of the dominant shown) indicating their speci®city as metalloproteases. (c) Western negative RalA on focus formation was not as strong blot analysis of MMP-2 protein in cell lysates of NIH3T3, v-Src- and v-Ras-transformed NIH3T3 cells, and these cells expressing as that observed for dominant negative mutants of the dominant negative Ral S28N mutant Ras and Raf on v-Src-induced transformation where colony formation in soft agar was inhibited almost completely (Qureshi et al., 1993; Jiang et al., 1995a,b). We therefore investigated the e€ect of the MMP-2 and MMP-9 in the CM (Figure 3a). dominant negative RalA on the ability of the v-Src- Consistent with these results Western blots of cell and v-Ras-transformed cells to form colonies in soft lysates from NIH, v-Src or v-Src-S28N-Ral cells agar. As shown in Figure 4, the dominant negative showed elevated MMP2 protein in the v-Src-trans- RalA mutant reduced the colony forming eciency of formed cells and a reduction in these cells expressing the v-Src- and v-Ras-transformed cells; however, as the dominant negative S28N RalA (Figure 3c). In reported previously for the focus assay (Urano et al., contrast, the increased MMP-2 and MMP-9 activity 1996), the dominant negative RalA only partially induced by v-Ras was largely una€ected by the S28N inhibited the colony formation. The S28N mutant RalA mutant (Figure 3b). Surprisingly, MMP-2 protein reduced colony formation to about 75% that seen in levels were not elevated in the v-Ras and v-Ras-S28N- the Ras-transformed cells and 54% of that seen in Ral cells relative to the parental NIH3T3 cells (Figure the Src-transformed cells. Another RalA mutant, 3c). This may explain why the dominant negative RalA D49N, which was slightly more e€ective at inhibiting prevents v-Src-induced increases MMP2, but not v- colony formation where the eciency was reduced to Ras-induced increases in MMP2. These data suggest 44 and 51% for the Ras and Src transformed cells two distinct mechanisms for elevating MMP-2 activity, respectively. one involving increased expression that is dependent We next examined the e€ect of the dominant upon RalA and one that involves a change in speci®c negative RalA on the ability to form tumors in activity that is independent of RalA. Also of interest syngeneic mice. Cell suspensions from v-Src- and v- here were the additional low molecular weight bands Ras-transformed NIH3T3 cells and these cells expres- observed for MMP-2 in both v-Ras- and v-Src- sing the S28N dominant negative RalA mutant were RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4722 Table 1 The e€ect of S28N RalA on the lung colony formation ability of v-Ras- and v-Src-transformed cells Mice with colonies/ Incidence Lung tumor nodules Cells total mice (%) median (range) NIH3T3- 0/7 0 0 (0 ± 0) pZip-neo v-Ras 10/11 90 7 (0 ± 31) v-Ras+ 1/7 14 0 (0 ± 2) S28N RalA v-Src 7/7 100 3 (1 ± 15) v-Src+S28N 0/8 0 0 (0 ± 0) RalA NIH3T3 cells, v-Src and v-Ras-transformed cells, and the v-Src and v-Ras cells co-expressing S28N RalA were injected into the lateral tail veins of syngeneic mice and lungs from these mice were examined 21 days later. The incidence represents the percentage of mice with tumor lung colonies. Median represents the median number of nodules per lung and the range represents the maximum and minimum number of nodules in the lungs examined. The table shows a representative experiment repeated twice

Figure 5 v-Src- and v-Ras-induced tumorigenicity is dependent upon RalA. NIH3T3-pZip-neo cells, v-Src- and v-Ras-trans- formed cells, and v-Src and v-Ras-transformed cells expressing the S28N RalA mutant were injected in the subcutaneous ¯ank of syngeneic normal BALB/c mice at 46105 cells/mouse. Tumor size was monitored three times per week for 28 days at which time mice with tumors were sacri®ced. Tumor size represents the average tumor size for of least 10 mice for each group. Mice without tumors were monitored for tumors for 13 weeks. The ®gure shows a representative experiment which was repeated twice

injected subcutaneously and tumor formation was monitored. As shown in Figure 5, both v-Src and v- Ras cells developed subcutaneous tumors that could be detected after a 5 ± 7 day latency period, with an incidence of 50 and 55% respectively. In contrast, v- Src- and v-Ras-transformed cells expressing the dominant negative S28N RalA mutant did not develop subcutaneous tumors after injection when followed for 13 weeks. Tumor growth was evaluated up to 28 days when mice were sacri®ced and tumors removed. The histopathology revealed sarcoma-like tumors for both v-Src and v-Ras cell lines (data not Figure 6 v-Src- and v-Ras-induced tumorigenicity as measured shown). We also investigated whether the S28N RalA by colony formation in the lung is dependent upon RalA. mutant interfered with the ability of v-Src- and v-Ras- NIH3T3-pZip-neo cells, v-Ras-transformed NIH3T3-pZip-neo cells, and the v-Ras-transformed cells expressing RalA-S28N transformed cells injected into the tail vein to colonize were injected into the tail veins of syngeneic mice and the lungs in the lung. Upon injection of either v-Src- or v-Ras- were excised 21 days later. Representative lungs are shown here. transformed cells into the tails of syngeneic mice, large Quanti®cation of the data is presented in Table 1 colonies were detected in the lungs with high incidence (Table 1, Figure 6). The histopathological analysis showed the presence of not only subpleural but also parechymal nodules consisting of sarcomatoid cells and colonies was signi®cantly reduced in mice inoculated almost devoid of extracellular matrix (data not shown). with either v-Ras- or v-Src-transformed cells expressing The ability of v-Ras and v-Src cells to colonize the lung the S28N RalA mutant. These data in Figures 4 ± 6 and form nodules was dramatically impaired in cells indicate that, while colony formation in soft agar is overexpressing the S28N-RalA mutant. As shown in only partially dependent upon RalA, tumor formation Table 1, both the incidence and number of lung is completely dependent upon RalA. RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4723 Discussion modulates tumorigenicity. This result does not directly correlate the e€ect of S28N RalA on MMP expression In this report, we have investigated whether RalA plays in v-Ras-S28N-RalA cells with the tumorigenicity data. a role in: (i) the upregulation of proteases; and (ii) An explanation for this result may derive from recent tumor formation induced by the oncogenic v-Src and data showing that MMPs produced in cell culture or in v-Ras. We ®rst observed that uPA expression levels vivo are non-functional in the absence of the uPA/ were elevated in the CM from NIH3T3 cells plasmin system (Mazzieri et al. 1997; Carmeliet et al., transformed by v-Src and v-Ras. The upregulation of 1997). Additional low MW bands that may represent uPA could be reversed almost to the control levels by the active form of MMPs were observed for MMP-2 in the expression of a dominant negative RalA mutant. v-Ras cells, but were absent in v-Ras-S28N-Ral cells. We previously demonstrated that upregulation of uPA These data suggest that uPA downregulation in v-Ras- production is dependent upon PLD activity (Aguirre S28N-RalA cells may account for the lack of the Ghiso et al., 1997), and since PLD1 activation by v-Src MMP-2 low MW bands (active form) since the active and v-Ras is dependent upon RalA (Jiang et al., 1995b; form is generated in response to uPA. Luo et al., 1997), the data presented here suggest that Reduced protease production and/or activity in cells constitutive uPA overproduction in v-Src and v-Ras expressing the dominant negative RalA may contribute transformed cells is dependent upon a RalA-PLD1 to the more stringent e€ect of the dominant negative signaling pathway. RalA mutant on tumor formation relative to colony MMP activity was elevated in v-Src-transformed formation in soft agar and focus formation. We have cells as detected by zymography and MMP protein was recently found that ®bronectin downregulation in elevated as determined by Western blot analysis of cell NIH3T3 cells by v-Ras or v-Src is completely restored lysates. The increased MMP production induced by v- in v-Src or v-Ras cells expressing the S28N-RalA Src, like increased uPA production, was reduced in the mutant (our unpublished data). This result suggests cells expressing the dominant negative RalA. However, that reducing RalA signaling in v-Ras or v-Src the increased MMP activity observed in v-Ras- transformed cells may restore the proper expression transformed cells was insensitive to the dominant of not only uPA and MMPs in the case of v-Src, but of negative RalA. Interestingly, the increased MMP several other genes that in¯uence the tumorigenic and activity seen in zymograms was not observed at the metastatic phenotype explaining the dramatic e€ects MMP protein level in the v-Ras-transformed cells. This observed on tumorigenicity. suggested that MMP activity may be regulated This report provides: (i) new insight on the role of di€erently in the v-Src and v-Ras-transformed cells RalA in the signals controlling protease expression; and may explain why the dominant negative RalA and (ii) demonstrates a role for RalA in tumor prevents v-Src-induced increases in MMP2, but not v- formation in the NIH3T3 model. In addition, our Ras-induced increases in MMP2. These data suggest data are consistent with mounting information that the increased expression of MMP in the v-Src- (Silberman et al., 1997; Gum et al., 1996, 1997; transformed cells is dependent upon RalA and the Lengyel et al., 1996, 1997) indicating that mitogenic elevated speci®c activity observed in the v-Ras- signaling pathways stimulate the upregulation of uPA transformed cells is independent of RalA. Exton and and MMPs. In this regard, an understanding of the co-workers recently reported that MMP-9 secretion mechanism(s) that tumor cells use to become tumori- induced by phorbol esters was dependent on PLD and genic and govern the production of proteases, which phosphatidic acid production (Williger et al., 1999). It apparently includes the RalA-PLD1 pathway, may is unlikely that the mechanism was the same as that prove to be an important target for therapeutic reported here since phorbol esters activate PLD by intervention in tumor progression. activating protein kinase C, which can activate PLD directly (Exton, 1998). Thus, RalA is not likely to be involved in this case, however these data suggest the possibility that both RalA-dependent and independent Materials and methods mechanisms for MMP production exist. More importantly, the studies presented here clearly Materials indicate a role for RalA in v-Ras and v-Src induced tumorigenicity. Previous studies using cells in culture G418 and Dulbecco's modi®ed Eagle medium (DMEM) were obtained from GIBCO/BRL Laboratories. Trypsin and have suggested a role for RalA in mitogenic signaling amiloride were purchased from Sigma. Anti-murine uPA and transformation (Urano et al., 1996). However, antibody was kindly provided by Dr G Hoyer-Hansen dominant negative RalA mutants were less ecient in (Copenhagen, Denmark). Anti MMP-2 antibody was from blocking the transformed phenotype than dominant Neomarkers (Union City, CA, USA). Anti-RalA antibody negative mutants of Ras and Raf (Qureshi et al., 1993; was from Trandsuction laboratories (Lexington, KY, USA). Jiang et al., 1995a,b; Urano et al., 1996). The studies Biotinylated anti-rabbit IgG polyclonal antibody and provided here demonstrate conclusively that RalA streptavidin-alkaline-phosphatase were purchased from GIB- inhibits completely the ability to form tumors. CO ± BRL. Puri®ed human urokinase was provided by Extracellular matrix remodeling by tumor cells has Serono Laboratories (Buenos Aires). Plasminogen was been proposed to be a necessary process for invasion purchased from Chromogenics (Sweden). Nitrocellulose membranes were purchased from BioRad (USA). and metastases establishment (Liotta et al., 1991; Werb et al., 1996). The inhibitory e€ect of the dominant negative RalA on the upregulation of uPA expression Cells and cell culture conditions in v-Src and v-Ras-transformed cells may be one of the Cells were routinely maintained in DMEM supplemented mechanisms by which RalA-dominant negative mutant with 10% fetal calf serum (GEN, Buenos Aires). The v-Src RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4724 and v-Ras-transformed NIH3T3 cells and the v-Src- and v- Detection of MMP production Ras-transformed cells overexpressing the S28N RalA mutant Collagenolytic activity present in CM or cell lysates was were pooled clones generated as described previously (Jiang determined by electrophoresis in SDS-polyacrylamide gels co- et al., 1995b). Construction of the S28N RalA mutant was polymerized with 0.1% gelatin as described previously described previously (Urano et al., 1996). Increased expres- (Pittman, 1985). After electrophoresis, gels were washed in sion of the RalA (S28N) protein levels was veri®ed by 2% Triton X-100, washed with distilled water and incubated Western blot analysis (data not shown). For growth of cells for 48 ± 72 h in 0.25 M Tris-HCl: 1 M NaCl: 25 mM CaCl in soft agar, 16103 cells were suspended in top agar 2 (pH 7.4) for speci®c activity detection, or in the same (DMEM, 20% calf serum, 0.38% agar) and overlaid onto solution containing 40 m EDTA to detect non-speci®c hardened bottom agar (DMEM, 20% calf serum, 0.7% agar) M activity. Gels were ®xed and stained with Coomassie Blue. as described previously (Qureshi et al., 1993). In all Activity bands were visualized by negative staining. The experiments as a control NIH3T3 cells were vector (pZip- activity bands for MMP or uPA activity were quantitated neo)-transfected as described previously (Jiang et al., with a densitometer as described above. 1995a,b).

Mice Preparation of conditioned media (CM) and cell lysates Inbred BALB/c mice 2 ± 4 months old, were obtained from uPA activity was investigated in CM and cell lysates. Brie¯y, the Animal Production Division of the Research Area of the semicon¯uent cell monolayers were washed in phosphate Institute of Oncology `Angel H/Ro€o'. Mice were bred with bu€er saline to eliminate serum. Serum-free DMEM/0.5% a 12 h light-12 h darkness cycle and a constant temperature bovine serum albumin/25 m HEPES was added and M of 208C. Water and food were administered ad libitum. incubation was continued for 24 h. CM was harvested, centrifuged (600 g), concentrated with a Centricon ®lter (MW cut o€ 30 000), aliquoted, stored at 7408C, and used Tumorigenicity assays only once after thawing. The remaining monolayers were To evaluate local tumor growth, NIH3T3 cells, v-Src- and v- washed, scrapped and sonicated at 48C; protein content Ras-transformed cells, and v-Src- and v-Ras-transformed determinations were performed and the cell lysate samples cells expressing the S28N dominant negative RaIA mutant were then stored at 7408C and used only once after thawing. were trypsinized, washed three times with DMEM, and allowed to recover for 1 h. Cells were assayed for viability Zymography and radial caseinolysis for detection of uPA with the Trypan Blue exclusion test prior to injection. activity Tumorigenicity was then examined in two ways. Method 1: Cells were injected in the subcutaneous ¯ank of syngeneic SDS-polyacrylamide gel electrophoresis was performed as BALB/c mice (46105 cells in 0.2 ml of DMEM). Growth described previously (Aguirre-Ghiso et al., 1997), using 9% kinetics of tumors were established by measuring the average separating and 4% stacking gels. Gels were washed with tumor diameter with a caliper three times per week. Method 2.5% Triton X-100, and incubated on the surface of a 2: cells prepared as above were injected into the lateral tail plasminogen-rich casein-agarose underlay, as described vein of normal mice (36105 cells in 0.3 ml of DMEM). previously (Aguirre Ghiso et al., 1997, Saksela, 1981). To Growth of these cells was then investigated by histopatho- con®rm uPA activity in the zymograms, plasminogen-casein- logical analysis of the lungs and other organs 21 days after agarose underlays were prepared with an anticatalytic uPA injection. Lung subpleural tumor nodules were counted under antibody, amiloride (1 m ) with or without plasminogen. M a stereoscopic magni®er after ®xation for 5 h with Bouin's uPA activity was quanti®ed by a radial caseinolysis assay solution. (Saksela, 1981), using plasminogen-rich (2 mg/ml) casein- agarose plates. uPA activity was referenced to a standard urokinase curve (0.1 ± 100 IU/ml) and normalized to total cell protein.

Acknowledgements Western blot and densitometry This work was supported by grants from the University of CM was collected as described above, electrophoresed and Buenos Aires (UBACYT ME074) and CONICET, PICT- then transferred to nitrocellulose. The ®lter was probed with 0015-(BID 802/OC-AR) (to EB De Kier Jo€e) and by or without rabbit IgG anti-murine uPA antibody and grants from the National Institutes of Health (CA46677), revealed with a biotinylated-IgG anti-rabbit antibody. The the American Cancer Society (BE-243) (to DA Foster), and streptavidin-alkaline-phosphatase system was used to develop a Research Centers in Minority Institutions (RCMI) award the signal (Alonso et al., 1993). Protein bands were from the Division of Research Resources, National quantitated with a Molecular Analyst TM/PC Densitometer Institutes of Health (RR-03037) to Hunter College. We Model GS-700 (Bio-Rad USA) and analysed with the Image thank Phil Kaplan and Liliana Ossowski for comments on Analysis Software for Model GS-700 (Bio-Rad USA) the manuscript. We are particularly grateful to Alicia (Aguirre-Ghiso et al., 1997). Rivelli for technical assistance.

References

Aguirre-Ghiso JA, Alonso DF, Farias EF and Bal de Kier Blobe GC, Obeid ML and Hannun YA. (1994). Cancer Meta. Jo€e E. (1997). Biochim. Biophys. Acta, 1356, 171 ± 184. Rev., 13, 411 ± 431. Alonso DF, Farias EF and Bal de Kier Jo€e E. (1993). Boyd D. (1996). Cancer Meta. Rev., 15, 77 ± 89. Cancer Letts., 70, 181 ± 187. Burgering BMT and Bos JL. (1995). TIBS, 20, 18 ± 22. Bell SM, Connolly DC, Maihle NJ and Degen JL. (1993). Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Mol. Cell. Biol., 13, 5888 ± 5897. Tipping P, Drew A, Eckhout Y, Shapiro S, Lupu F and Bi K, Roth MG and Ktikstakis NT. (1997). Curr. Biol., 7, Collen D. (1997). Nat. Genet., 17, 439 ± 444. 301 ± 307. RalA involvement in uPA production and tumorigenicity JA Aguirre-Ghiso et al 4725 Carpenter CL and Cantley LC. (1996). Biochim. Biophys. Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Acta, 1288, M11 ± M16. Garbisa S and Mignatti P. (1997). EMBO J., 16, 2319 ± Exton JH. (1994). Biochim. Biophys. Acta, 1212, 26 ± 42. 2332. Exton JH. (1998). Biochim. Biophys. Acta, 1436, 105 ± 115. Moller LB. (1993). Blood Coagul. Fibrinol., 4, 293 ± 303. Foster DA. (1993). Cell Signalling, 5, 389 ± 399. Ossowski L. (1992). Cancer Res., 52, 6754 ± 6760. Gum R, Lengyel E, Juarez J, Chen JH, Sieki M and Boyd D. Ossowski L and Reich E. (1983). Cell, 35, 611 ± 619. (1996). J. Biol. Chem., 271, 10672 ± 10680. Pittman RN. (1985). Dev. Biol., 110, 91 ± 101. Gum R, Wang H, Lengyel E, Juarez J and Boyd D. (1997). Qureshi SA, Joseph CK, Gupta R, Hendrickson M, Song J, Oncogene, 14, 1481 ± 1493. Bruder J, Rapp U and Foster DA. (1993). Biochem. Hart IR, Goode NT and Wilson RE. (1989). Biochim. Biophys. Res. Comm., 192, 969 ± 975. Biophys. Acta, 989, 65 ± 84. Reich R, Blumenthal M and Liscovitch M. (1995). Clin. Exp. Jiang H, Lu Z, Luo JQ, Wolfman A and Foster DA. (1995a). Meta., 13, 134 ± 140. J. Biol. Chem., 270, 6006 ± 6009. Roth MG and Sternweis PC. (1997). Curr. Opin. Cell. Biol., Jiang H, Luo J-Q, Urano T, Frankel P, Lu Z, Foster DA and 9, 519 ± 526. Feig LA. (1995b). Nature, 378, 409 ± 412. Saksela O. (1981). Anal. Biochem., 111, 276 ± 282. Lengyel E, Gum R, Stepp E, Juarez J, Wang H and Boyd D. Sato H, Kita M and Seiki M. (1993). J. Biol. Chem., 268, (1996). J. Cell. Biochem., 61, 430 ± 443. 23460 ± 23468. Lengyel E, Wang H, Gum R, Simon C, Wang Y and Boyd D. Silberman S, Janulis M and Schultz RM. (1997). J. Biol. (1997). Oncogene, 14, 2563 ± 2573. Chem., 272, 5927 ± 5935. Liotta LA and Stetler-Stevenson WG. (1991). Cancer Res., Song J, Pfe€er LM and Foster DA. (1991). Mol. Cell. Biol., 51, 5054 ± 5059. 11, 4903 ± 4908. Liotta LA, Steeg PS and Stetler-Stevenson W. (1991). Cell, Urano T, Emkey R and Feig LA. (1996). EMBO J., 15, 810 ± 64, 327 ± 336. 816. Luo J-Q, Liu X, Frankel P, Rotunda T, Ramos M, Flom J, Werb Z, Ashkenas J, MacAuley A and Wiesen JF. (1996). Jiang H, Feig LA, Morris A, Kahn RA and Foster DA. Braz.J.Med.Biol.Res.,29, 1087 ± 1097. (1998). Proc. Natl. Acad. Sci. USA, 95, 3632 ± 3637. Williger B-T, Ho W-T and Exton JH. (1999). J. Biol. Chem., Luo J-Q, Liu X, Hammond SM, Colley WC, Feig LA, 274, 735 ± 738. Frohman MA, Morris AJ and Foster DA. (1997). Yu H and Schultz RM. (1990). Cancer Res., 50, 7623 ± 7633. Biochem. Biophys. Res. Comm., 235, 854 ± 859. Yu W, Kim J and Ossowski L. (1997). J. Cell. Biol., 137, Matrisian LM. (1992). BioEssays, 14, 455 ± 463. 767 ± 777.