RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin

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Abstract

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to α v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

Introduction

Metastasis is the major cause of treatment failure in breast cancer patients (Pisani et al., 1999). One molecule, osteopontin (OPN), can enhance neoplastic transformation, malignant cell attachment, migration/invasion in vitro (Furger et al., 2003; Das et al., 2005) and metastasis in vivo (Oates et al., 1996; Chen et al., 1997). Conversely, inhibition of OPN expression by antisense cDNA impedes cell growth and tumor-forming capacity (Su et al., 1995). In patients, inverse associations have been shown between tumor OPN and prognosis in breast (Rudland et al., 2002) and gastric cancers (Ue et al., 1998) and high-serum OPN levels have been associated with metastasis in patients with breast cancer (Singhal et al., 1997). Thus, OPN is important in the development and metastasis of breast and other cancers.

Screening for genes differentially expressed between the parental cell line—the benign rat mammary Rama 37 cell line (R37) (Dunnington et al., 1983)—and parental cells stably transfected with an expression vector for OPN (R37-OPN cells) has recently been undertaken using the suppressive subtractive hybridization (SSH) technique (Kurisetty et al., 2008). R37 cells produce low levels of OPN and are noninvasive and nonmetastatic (El-Tanani et al., 2006a), whereas R37-OPN cells produce high levels of OPN and are invasive and metastatic (Oates et al., 1996; El-Tanani et al., 2006a). One of the genes whose expression is substantially increased in association with overexpression of OPN is RAN GTPase (RAN), a small GTP-binding protein that is a member of the RAS super family (Wennerberg et al., 2005). In this paper we show that stable transfection of noninvasive R37 cells with an expression vector for RAN (R37-RAN) induces an invasive/metastatic phenotype in vitro and the development of metastases in vivo. Moreover, stable transfection of invasive cells (both R37-OPN and R37-RAN) with small-interfering RNA molecules directed at RAN GTPase (siRNA RAN) specifically inhibits the invasive/metastatic phenotype in vitro and in vivo. These data suggest that RAN may function as an effector of OPN-induced invasion and metastasis in an epithelial model of breast cancer dissemination, and we go on to identify some of the downstream signaling events in this process.

Results

Relationship between OPN and RAN levels: effect of OPN antisense cDNA (asOPN) and siRNA-RAN on OPN and RAN protein expression

Stable transfection of R37 cells with an expression vector for OPN (R37-OPN) resulted in increases in OPN and RAN mRNA and protein. R37-RAN cells displayed increased RAN levels, whereas OPN levels were unaffected. Moreover, expression of RAN in R37-OPN cells (R37-OPN-RAN cells) did not affect either mRNA and protein levels of OPN or RAN (Figure 1a).

Figure 1
figure1

Expression and biological effect of RAN in benign R37 cells and invasive R37-osteopontin (OPN) cells. (a) Northern (upper panel) and immuno (lower panel) blots for RAN, OPN and β-actin mRNA and protein in R37, R37-OPN and R37-RAN cells. (Upper panel) Expression of OPN and RAN mRNA levels. β-Actin mRNA was assessed as control. Major hybridizing bands are shown in kilobases (kb). The average fold increase for three different experiments for OPN is lane 1=1, lane 2=1.2±0.3, lane 3=7.5±1.5 and lane 4=8±1.1 and for RAN is lane 1=1, lane 2=6.5±0.5, lane 3=6.3±0.5 and lane 4=7.5±1.4. (Lower panel) Cell lysates were diluted and 5 μg for RAN and β-actin and 20 μg for OPN immunoblots were loaded on a sodium dodecyl sulfate (SDS) 10% (w/w) polyacrylamide gel. Specific proteins were detected using antibodies to OPN, RAN or β-actin. Bands were quantified using densitometric analysis and normalized against β-actin. The average fold increase for three different experiments for OPN is lane 1=1, lane 2=1±0.2, lane 3=5.8±0.4 and lane 4=5.8±0.7 and for RAN is lane 1=1, lane 2=4.9±0.7, lane 3=5.1±0.5 and lane 4=5.6±0.7. The images shown are representative of the three experiments. (b) Ability of transfected cell lines to adhere to a fibronectin-treated surface was assessed over a 30 min period and the number of adherent cells quantified. The results were the mean±s.e. from three independent experiments. (c) Soft agar assay was carried out to assess the ability of stably transfected cell lines to grow in an anchorage independent environment. The colony number was assessed after 5 days. The results were the mean±s.e. from three independent experiments. (d) R37 and R37-OPN stably transfected with and without RAN were plated on fibronectin-coated filters (500 μg/ml) in Boyden chambers. Cells that migrated through the filter after 48 h were counted by staining the inserts, and scanning them using a digital imaging system (Materials and methods). The results were the mean±s.e. from three independent experiments. (e) Growth of R37, R37-RAN, R37-OPN and R37-RAN/asOPN cell lines in culture. Absorbance of incorporated MTT after 24 h was used to normalize all other time points and the mean±s.d. of three independent experiments is shown. Each experiment consisted of six wells.

Immunoblot analysis demonstrated that both OPN and RAN proteins were reduced in R37-OPN/asOPN cells (cells with both OPN and antisense OPN expression vectors) compared with R37-OPN cells (Figure 2a; P0.0001). In contrast, only RAN protein levels were significantly reduced in R37-OPN/siRNA-RAN cells compared with the R37-OPN cells (P0.001); there was no significant difference in the levels of OPN protein (Figure 2b; P>0.5).

Figure 2
figure2

Expression and biological effect of inhibiting osteopontin (OPN) or RAN in R37 and R37-OPN cells. (a) Immunoblot showing OPN and RAN levels in cells containing asOPN. Cell lysates were diluted and 20 μg (for anti-OPN) or 5 μg (for anti-RAN) were loaded onto a sodium dodecyl sulfate (SDS) 10% (w/w) polyacrylamide gel as follows: lane 1 R37; lane 2 R37-OPN and lane 3 R37-OPN/asOPN. Specific proteins were detected using antibodies to OPN, RAN and β-actin. Bands were quantified using densitometric analysis and normalized with respect to those of β-actin. The average fold increase for three different experiments for OPN is lane 1=1, lane 2=4.2±0.3 and lane 3=0.8±0.15 and for RAN is lane 1=1, lane 2=2.8±0.3 and lane 3=1±0.2. Student's test of significance of levels of OPN and RAN proteins, respectively: R37 vs R37-OPN (P0.001), R37-OPN vs R37-OPN/asOPN (P0.0001), R37 vs R37-OPN/asOPN (P=0.082), and R37 vs R37-OPN (P=0.00048), R37-OPN vs R37-OPN/asOPN (P=0.00098), R37 vs R37-OPN/asOPN (P=1). (b) Immunoblot showing OPN and RAN levels in cells containing siRNA-RAN. Cell lysates were diluted and 20 μg (for anti-OPN) or 5 μg (for anti-RAN) were loaded onto an SDS 10% (w/w) polyacrylamide gel, as follows: lane 1 R37; lane 2 R37-RAN; lane 3 R37/siRNA-RAN; lane 4 R37-OPN; lane 5 R37-OPN-RAN and lane 6 R37-OPN/siRNA-RAN. Specific proteins were detected using antibodies to OPN, RAN or β-actin. Bands were quantified using densitometric analysis and normalized with respect to those of β-actin. The average fold increase for three different experiments for RAN is lane 1=1, lane 2=3.8±0.4, lane 3= 0.8 ±0.2, lane 4=2.6±0.2, lane 5=3.0±0.3 and lane 6=1.0±0.3 and for OPN is lane 1=1, lane 2=1±0.3, lane 3=0.9±0.2, lane 4=4.1±0.4, lane 5=4.2±0.5 and lane 6=4.3±0.6. (c) Ability of transfected cell lines to adhere to a fibronectin-treated surface was assessed over a 30 min period and the number of adherent cells quantified. R37 and R37-OPN cells were stably transfected with expression vectors for siRNA-RAN, for asOPN or for a combination of asOPN and RAN (asOPN+RAN). The results were the mean±s.e. of three independent experiments. (d) Soft agar assays were carried out to assess the ability of stably transfected cell lines to grow in an anchorage-independent environment. The colony number was assessed after 5 days. The results were the mean±s.e. of three independent experiments. (e) In vitro invasion through extracellular matrix. Cells were plated on fibronectin-coated filters (500 μg/ml) in Boyden chambers. The number of cells that migrated through the filter after 48 h was determined by staining the inserts and scanning using a digital imaging system (Materials and methods). The results were the mean±s.e. of three independent experiments.

Effect of RAN on in vitro cellular properties of R37 and R37-OPN cells: effect of inhibiting RAN and OPN expression

Adhesion

R37-RAN and R37-OPN cells displayed increased adhesion to fibronectin-coated dishes compared with parental R37 cells (P<0.01); R37-OPN-RAN cells were no more adhesive than the other two transfectants (Figure 1b). Transfection of R37-OPN cells with either siRNA-RAN or asOPN significantly reduced cell adhesion (P<0.01); there was little or no effect of these transfections on R37 cells alone. R37-RAN/asOPN cells had increased cell adhesion compared with R37/asOPN cells (P=0.0004), and expression of RAN in R37-OPN/asOPN cells partially overcame the inhibitory effect of asOPN on OPN-induced cell adhesion (Figure 2c; P<0.01).

Colony formation

R37-RAN and R37-OPN cells formed an increased number of colonies per plate compared with R37 cells; R37-OPN-RAN cells were not significantly different from the R37-RAN or R37-OPN cells (Figure 1c; P>0.05). Expression of siRNA-RAN or asOPN in R37-OPN cells significantly reduced colony formation in soft agar (P<0.01) (Figure 2d). Reintroduction of the RAN expression vector into R37-OPN/asOPN (R37-OPN-RAN/asOPN cells) caused colony formation to increase to similar levels to those observed with R37-OPN cells (P<0.01). Although there was little effect of siRNA-RAN expression on R37 anchorage-independent growth, R37-RAN/asOPN cell colony formation was increased compared with R37/asOPN cells (P=0.02).

Invasion

R37-RAN and R37-OPN cell invasion was increased over the parental R37 cells (P<0.01), but overexpressed RAN had no significant additional effect on cell invasion in R37-OPN cells (P=0.5) (Figure 1d). R37/siRNA RNA and R37/asOPN cells were not different from R37 cells (Figure 2e). R37-OPN/siRNA-RAN and R37-OPN/asOPN cells displayed reduced invasion over R37-OPN cells (P<0.01). Expression of RAN in an asOPN background resulted in increased invasion; R37-RAN/asOPN were more invasive compared with R37/asOPN cells (P=0.04), as were R37-OPN-RAN/asOPN cells when compared with R37-OPN/asOPN cells (Figure 2e) (P0.01).

Proliferation, cell-cycle analysis

To demonstrate the cellular viability of the phenotypes generated, cells were subjected to proliferation assays and cell-cycle analysis. The R37-RAN and R37-OPN cells divided with no significant increased rates of cell proliferation compared with the R37 parental cells (Student's t-test, P=0.5 and 0.4). R37-RAN/asOPN cells did not show a reduction in cell numbers after 72 and 96 h (Figure 1e). Propidium iodide staining showed that growing populations of the R37, R37-RAN, R37-OPN and R37-RAN/asOPN cell lines had similar distributions of cells throughout the cell cycle, with no observed arrest at any stage (Table 1).

Table 1 Cell-cycle analysis

Effect of permanent transfection of RAN on metastasis in vivo

Injection of pooled clones of R37-derived cell lines yielded no significant difference in incidences (Fisher's exact test, P0.8) or weights (Student's t-test, P0.2) of primary tumors. Injection of R37-RAN, R37-OPN and R37-RAN/asOPN cells all yielded tumor-bearing rats with metastases (Table 2). The differences in incidence of metastasis between R37-OPN, R37-RAN and R37-RAN/asOPN (P=0.51, 0.52 and 1) were not significant, but the differences in incidence between R37-pBK-CMV, R37-OPN/siRNA-RAN and R37-OPN, R37-RAN and R37-RAN/asOPN were highly significant (P0.00003). The majority of the metastases occurred in the lungs, with a few metastases in the lymph nodes, but no metastatic deposits were observed in other organs. The histological appearance of primary tumors and any metastases from all of the three groups were similar, primarily consisting of spindle cells admixed with more cuboidal, epithelial-like cells. The distribution between the cytoplasm and the nucleus of transgene-produced OPN was not significantly different in primary tumors generated by R37-OPN/siRNA-RAN cells and metastases generated from R37-OPN cells, nor between these lesions and the small fraction of R37-RAN cells stained positive for endogenous OPN (Student's t-test, P0.11). Similarly, the cellular distribution of RAN was not appreciably different in metastases produced by R37-RAN/asOPN and R37-OPN cells, both cell lines produced a significant reduction in cytoplasmic (P<0.0001) and increase in nuclear staining (P<0.0001), for cells at the center compared with those at the periphery of the metastases (Table 3).

Table 2 Comparison of the incidence of metastasis produced by permanently transfected cell lines
Table 3 Summary of immunocytochemical staining in primary tumors and metastases

Mechanistic effect of RAN GTPase inhibition on invasion in vitro

To test how the invasive phenotype induced by RAN was mediated, its GTPase activity was inhibited in R37-RAN cells. A double mutant of RAN was constructed which, when expressed in a reticulocyte cell-free protein-synthesizing system, produced a lower rate of GTP hydrolysis than wild type RAN (Table 4). R37-RAN (G19V/Q69L) cells contained no detectable phospho-Akt (Figure 3d), and their cell invasion was lower in comparison with R37-RAN cells (Figure 3a).

Table 4 Phosphate released by lysates containing RAN and RAN (G19V/Q69L) GTPase
Figure 3
figure3

Mechanistic effects of RAN on cell invasion in vitro. (a) R37 cells transfected with expression vectors for wild-type (R37-RAN), mutant RAN (R37-RAN (G19V/Q69L) or R37-RAN cells treated with or without 50 μM of phosphoinositol-3-kinase (PI3K) inhibitor LY294002 for 48 h were plated on fibronectin-coated filters (500 μg/ml) in Boyden chambers. The number of cells that migrated through the filter after 48 h was determined by staining the inserts and scanning using a digital imaging system (Materials and methods). The results were the mean±s.e. from three independent experiments. (b) Immunodetection of phosphorylated c-Met receptor. (Upper panel) Phosphorylated c-Met receptor: a total of 500 μg cell lysates were immunoprecipitated with antiphosphotyrosine and the redissolved precipitate loaded onto a sodium dodecyl sulfate (SDS) 7.5% (w/w) polyacrylamide gel as follows: lane 1 R37, lane 2 R37-pBK-CMV (R37-CMV), lane 3 R37-osteopontin (OPN) and lane 4 R37-RAN. The resultant gel was immunoblotted with an antiserum raised against the C-terminal sequence of c-Met. The arrow indicates a 145-kDa protein band corresponding to c-Met β-subunit. Bands were quantified using densitometric analysis and normalized to an equal amount of protein lysate before application to each lane of the gel. The average fold increase±s.e. for three different experiments for phosphorylated c-Met is lane 1=1, lane 2=1.6±0.1, lane 3=3.6±0.2 and lane 4=10.4±0.8. (Lower panel) Total c-Met: cell lysates were loaded directly onto the SDS polyacrylamide gel and the resultant gel was immunoblotted with antiserum to c-Met and to β-actin. The average fold increase±s.e. for three different experiments for total c-Met normalized to β-actin is lane 1=0.9±0.1, lane 2=1.2±0.2, lane 3=1.0±0.1 and lane 4=0.9±0.2. (c) Immunodetection of phosphorylated Akt in RAN and OPN expressing cells. A total of 10 μg cell lysates were loaded on to an SDS 10% (w/w) polyacrylamide gel as follows: lane 1 R37, lane 2 R37-pBK-CMV (R37-CMV), lane 3 R37-RAN, lane 4 R37-OPN, lane 5 R37-OPN treated with 25 μM LY290042 for 18 h (R37-OPN/LY) and lane 6 R37-RAN treated with 25 μM LY290042 for 18 h (R37-RAN/LY). (Upper panel) Phosphorylated Akt: proteins were detected in immunoblots using specific antibodies to phosphoserine 473 Akt or to β-actin, their authentic positions at 60 and 47 kDa, respectively, are shown. Bands were quantified using densitometric analysis and normalized with respect to those of β-actin. The average fold increase±s.e. for three different experiments for phosphorylated Akt on ser473 is lane 1=1, lane 2=2.2±0.4, lane 3=22.5±0.7, lane 4=21.1±0.5, lane 5=0±0 and lane 6=0±0. (Lower panel) Total Akt: proteins were detected as above using antibodies to total Akt and β-actin. The average fold increase±s.e. for three different experiments for total Akt normalized to β-actin is: lane 1=0.8±0.2, lane 2=1.0±0.1, lane 3=0.9±0.2, lane 4=1.0±0.2, lane 5=1.0±0.1 and lane 6=1.0±0.2. (d) Immunodetection of phosphorylated Akt in RAN and RAN (G19V/Q69L) expressing cells. (Upper panel) Phosphorylated Akt: a total of 10 μg of cell lysates were loaded, run, blotted on SDS–polyacrylamide gels and quantitated for phosphoserine 473 Akt, as described in c above, as follows: lane 1 R37-RAN (R37-RAN) and lane 2 R37 mutant RAN (R37-RAN (G19V/Q69L)). The average fold increase±s.e. for three different experiments for phosphoserine 473 Akt normalized to that in R37 cells (=1) is lane 1=22.0±0.6 and lane 2=0±0. (Lower panel) Total Akt: proteins were detected as above using antibodies to total Akt and to β-actin. The average fold increase±s.e. for three different experiments for total Akt normalized to that in R37 cells=1 is lane 1=1.0±0.1 and lane 2=1.0±0.1.

Mediators of induction of in vitro invasion by RAN

To test whether RAN signaled increased invasion through the c-Met receptor and activation of Akt, levels of phosphorylation of both these proteins were measured in cells using either a combination of antiphosphotyrosine, anti-c-Met antibodies or a specific antibody to phosphoserine 473 of Akt (Materials and methods). Phosphorylated c-Met β-subunit and phospho-Akt were present at much higher levels in the R37-OPN and R37-RAN cells than in untransfected R37 cells or empty vector transfected R37 cells when normalized to appropriate controls (Figures 3b and c). R37-Mut.RAN cells contained no detectable phospho-Akt (Figure 3d). Inhibition of PI3 kinase (PI3K) by exposure of the R37-RAN and R37-OPN cells to LY294002 completely abolished detectable levels of phosphorylated Akt (Figure 3c) and reduced in vitro invasion of R37-RAN cells (Figure 3a). The total levels of c-Met and Akt were relatively unchanged in the transfected cell lines (Figures 3b–d). Moreover, the cellular levels of endogenous c-Met and Akt showed only a slight increase in cytoplasmic staining in metastases produced by R37-OPN and R37-RAN cells compared with that in primary tumors produced by R37 cells (Student's t-test, 0.01<P<0.04); there were no significant differences in levels of nuclear staining (P0.11; Table 3). This slight increase in cytoplasmic staining was not observed in R37-RAN cells compared with R37 cells in immunofluorescently stained cultures (P0.34; Table 5).

Table 5 Summary of immunofluorescent staining for c-Met and Akt in cultured cells

Discussion

Progression of cancer to a more aggressive phenotype involves a heightened ability to invade tissues and metastasize to other sites. OPN expression is associated with this progression and is recognized as an effector of invasion and metastasis in breast cancer (Tuck et al., 2007). Higher levels of OPN (in either tissue or plasma) predict a poor prognosis for patients (Singhal et al., 1997; Tuck et al., 1998; Rudland et al., 2002; Bramwell et al., 2006), and inhibiting the OPN signal is an attractive target for cancer therapy (Johnston et al., 2008). However, the pathways downstream of OPN are not fully understood (Tuck et al., 2003), and in this paper we present RAS-related nuclear protein GTPase (RAN) as a novel mediator of OPN-induced invasion and metastasis.

Transfection of the R37 epithelial mammary cancer cell line with an OPN expression vector (R37-OPN cells) resulted in a more aggressive phenotype (data presented here and Oates et al., 1996). In vitro, R37-OPN cells were more adherent, had enhanced survival in anchorage-free conditions, and an increased ability to invade through extracellular matrix (Figures 1b–d). In vivo injection of cells expressing OPN resulted in the formation of metastases, in contrast to the parental cell line (Table 2). Using SSH technology, we have identified potential effectors of the OPN-induced signaling network (Kurisetty et al., 2008). Forced expression of OPN led to increases in RAN mRNA and protein (Figures 1a and e), and although inhibition of OPN expression results in inhibition of RAN (Figure 2a), knockdown of RAN by siRNA does not significantly reduce OPN expression (Figure 2b). Changes in the cellular phenotype are seen to parallel changes in the levels of RAN. When RAN was overexpressed in R37 cells, a transformation, similar to that induced by OPN, was seen in the cellular phenotype of R37-RAN cells, both in vitro and in vivo (Figures 1b–e; Table 2), and knockdown of RAN mRNA leads to decreases in invasive activity in vitro and metastasis in vivo that cannot be completely overcome by the increased expression of OPN in these cells (Figures 2c–e; Table 2). We sought to establish whether RAN was a direct intermediate in OPN-induced changes in phenotype, and to elucidate potential mechanisms that led to the increased potential for invasion and metastasis as a result of increased RAN expression.

We conclude that RAN is most likely to be positioned downstream of OPN in any metastatic signaling pathway. This conclusion is supported by the similar incremental decreases in expression of RAN protein (Supplementary data Figure 1A) and in cell adhesion (Supplementary data Figure 1B) and anchorage-independent growth (Supplementary data Figure 1C) when the R37-OPN cells are transfected singly or in binary combinations with the siRNA oligonucleotides targeted against RAN. One possible mechanism explaining RAN's role in the transduction of OPN induced signaling is that RAN is a specific target for OPN. Since no interaction between OPN and RAN could be identified in a mammalian two-hybrid system (Supplementary data Figure 1D), and R37-OPN cells contain elevated levels of RAN mRNA, we conclude that there is no direct interaction, but rather that enhanced transcription of RAN is a result of signaling pathway(s) activated by OPN. Whether this process is mediated through the integrin receptor alone, with the aid of CD44, or via intracellular OPN (El-Tanani et al., 2006b; Shinohara et al., 2008), is currently under investigation.

Expression of OPN in R37 (and other cells) increases phosphorylation of the c-Met/HGF receptor (Figure 3b; Tuck et al., 2003), leading to phosphorylation of Akt at serine residue 473 (Figure 3c). OPN activation of c-Met has been observed previously in human cell lines (Tuck et al., 2000); expression of this receptor is associated with progression in breast cancer (Beviglia et al., 1997), and the receptor and downstream pathways have been identified as a target for cancer therapy (Maulik et al., 2002; Peruzzi and Bottaro, 2006). In addition, activation of Akt is linked to increased invasiveness and metastasis (Shukla et al., 2007; Hara et al., 2008). One effect of overexpression of RAN is to increase phosphorylation of c-Met and Akt (although their total levels are unchanged; Figures 3b–d), and we have shown that it is possible to inhibit by drug treatment both the Akt phosphorylation and the cell invasion induced by RAN (Figures 3a and c). RAN GTPase activity was next examined. A double mutant was generated, containing G19V and Q69L substitutions. In agreement with observations of RAN mutated with single substitutions (Bischoff et al., 1994; Carey et al., 1996), the effect of these mutations is to inhibit hydrolysis of GTP in vitro (Table 4). When this mutated RAN protein was expressed in cells, the invasive phenotype and Akt phosphorylation seen with wild type RAN was significantly reduced, similar to the effect seen after treatment of R37-RAN cells with the PI3K inhibitor LY294002 (Figures 3a, c and d). These data suggest that the GTPase activity of RAN is required for RAN-mediated invasion.

RAN overexpression could influence the subcellular location of molecules involved in migration and invasion. However, in the cell and animal systems used here, this is not overtly the case, since RAN overexpressing tumors/metastases fail to display an appreciable redistribution of Akt between the cytoplasm and nucleus of cells (Tables 3 and 5). RAN itself would also appear to have little effect on OPN localization, as primary tumors produced by R37-OPN/siRNA-RAN cells and metastases produced by R37-OPN show the same subcellular distribution of OPN. Similarly, under- or overexpression of OPN fails to alter appreciably the distribution of RAN. In addition, R37-OPN cells permit endogenously overexpressed RAN to redistribute inside cells in the same way as when RAN is produced from its transfected transgene in R37-RAN/asOPN cells (Table 3). It is important to note that within the R37 cell context, OPN and RAN expression only affect specific aspects of the invasive phenotype. Proliferation rates are unaffected, in agreement with previous observations of OPN in R37 cells (Moye et al., 2004) (Figure 1e) and there is no significant effect on the distribution of cells in the cell cycle (Table 1).

It seems likely that the effect of RAN is exerted via an adapter complex for activation of the c-Met receptor, similar to that described previously in a human cell system (Wang et al., 2002). One candidate member of this adapter complex is RAN-binding protein 9 (RANBPM), a 90 kDa protein that is capable of binding to RAN (Nishitani et al., 2001) and that interacts with a variety of membrane-bound proteins including TrkA, Sky, Axl, c-Met and integrins (Wang et al., 2002; Hafizi et al., 2005; Yuan et al., 2006). RAN and RANBPM are expressed in many cells (Denti et al., 2004), including a reported association of RANBPM with breast carcinoma (Emberley et al., 2002), and we suggest that overexpression of RAN in the presence of RANBPM is sufficient to increase activation of c-Met, resulting in Akt phosphorylation and the invasive phenotype. RAN has many diverse functions in the cell (Sazer and Dasso, 2000; Wennerberg et al., 2005), and to this we add the activation of the c-Met receptor, leading to invasion and metastasis in the context of the R37 cell line.

Cells with an invasive or metastatic phenotype do not always display increased levels of RAN. For instance, RAN is not overexpressed in R37 cells overexpressing the metastasis-inducing protein S100A4 (Davies et al., 1993). However, RAN expression has been reported to be upregulated in renal, ovarian and HLA-A33+ epithelial cancers (Azuma et al., 2004; Ouellet et al., 2006; Abe et al., 2008), and inhibition of RAN by RNAi has been shown to be an effective approach to reduce growth and induce apoptosis in cancer cell lines (Morgan-Lappe et al., 2007). As no direct link between OPN and RAN has been established in these reports, the possibility therefore remains that RAN can be an independent effector of metastasis by other mechanisms related to its other functions in cell biology. In addition, RAN may act as an integration point where different signaling pathways involved in invasion/metastasis, including that for OPN, converge.

In conclusion, it would appear that OPN exerts its effect, at least in part, through RAN, operating through a signaling pathway via phosphorylation of the c-Met receptor and Akt, to stimulate cell invasion and ultimately metastasis. We believe this is a novel mechanism for the upregulation of RAN GTPase in metastasis, and that inhibition of this specific activity of RAN GTPase is a viable target for the development of an anticancer therapy in the future.

Materials and methods

Cell culture and production of stable transformant cell lines

See Supplementary details.

Plasmids and oligonucleotides

The expression vectors for rat OPN, OPN-pBK-CMV and for human RAN GTPase, RAN-pcDNA6 were prepared as described previously (El-Tanani et al., 2006a); those for siRNA to RAN, pRETROSUPER-siRNA RAN or siRNA-RAN and for antisense to OPN RNA, antisense to OPN-pcDNA4 or asOPN were prepared as before (Berns et al., 2004; El-Tanani et al., 2006a). DNA sequencing confirmed their authenticity. The siRNA oligonucleotide sequences for RAN (siRNA-RAN) were designed according to the human mRNA sequences (GenBank accession number NM_006325) (Supplementary data) and confirmed by DNA sequencing.

Mutated RAN

pcDNA-RAN encoding wild-type RAN cDNA mutated at positions 19 (glycine to valine G19V) (8955121) and 69 (glutamine to leucine Q69L) (Bischoff et al., 1994; Carey et al., 1996; Lounsbury et al., 1996) was generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) (El-Tanani et al., 2004), using appropriate primers (Supplementary data); the resulting expression construct was termed RAN (G19V/Q69L).

Northern blotting

Total RNA was extracted from cells and subjected to Northern blot analysis (El-Tanani et al., 2006a). All results were normalized using a housekeeping gene, β-actin and quantified using densitometry readings.

Western blotting for OPN and RAN proteins

Western blots were performed on extracts from a pool of cloned cells. Antibodies to OPN and RAN recognized proteins of Mr 65 000 and 25 000 Da, respectively (Figures 2a and b; El-Tanani et al., 2001; Rudland et al., 2002). OPN and RAN protein levels were established in the stably transfected cells by western blotting as described previously (El-Tanani et al., 2004). Monoclonal antibodies to OPN (1/500) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), RAN (8 ng/ml; BD Biosciences Pharmingham, Oxford, UK) and β-actin (1/5000) (Sigma, Poole, Dorset, UK) were used. Bands were quantified using a digital imaging system (Syngene, Genetool, Cambridge, UK). All results were normalized using β-actin as a housekeeping protein control.

In vitro tests for cell adhesion, invasion and colony formation

Assays for cell adhesion, colony formation and invasion through Matrigel in Boyden chambers were carried out as previously described (El-Tanani et al., 2006a; Supplementary data).

mRNA isolation

Cells were harvested at an exponential growth phase for RNA isolation. Total RNA was isolated with TriZol reagent (Life Technologies, Paisley, Scotland, UK) following the manufacturer's instructions. mRNA was isolated from total RNA with a NucleoTrap mRNA extraction kit (BD Clontech, Oxford, UK) following the manufacturer's protocol.

Proliferation assay

Proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in increasing numbers and measured after 24 h to ensure there was a linear, doubling relationship between cell number and assay result over the range of the assay (data not shown). For the assay, 30 000 cells were seeded into each of six wells of a 24-well plate (Nunc, Denmark) and incubated for 24, 48, 72 and 96 h. After the incubation, media was aspirated, and 500 μl of 0.5 μg/ml MTT in phosphate buffered saline (PBS) (Sigma) were added to each well and incubated in the dark at 37 °C for 3 h. The reaction was terminated by the addition of 500 μl of 10% SDS (Sigma, w/v dissolved in ddH2O) and stored overnight in the dark. Absorbance at 590 nm was measured using a GENios plate reader (Tecan).

Cell-cycle analysis

Cells were analysed for cell cycle using a protocol modified from Darzynkiewicz et al. (2001). Briefly, 1 million cells were seeded in 90 mm plates (Nunc) and incubated overnight. Cells were then harvested by trypsin digestion, washed twice in PBS (Gibco, Invitrogen, Paisley, UK) and fixed in cold (−20 °C) 70% ethanol and stored at 4 °C. Cells were stained in 0.1% (v/v) Triton X-100 in PBS containing 200 μg/ml RNase A (Qiagen Ltd, West Sussex, UK) and 20 μg/ml propidium iodide (Sigma) for 30 min before flow cytometry in a Becton Dickinson LSR II cytometer was carried out. Cell-cycle analysis was performed using Flowjo software (Tree Star Inc.).

In vitro recombinant RAN and mutant protein preparations

Products were generated in a coupled transcription–translation cell-free protein-synthesizing reticulocyte lysate for the expression vector of wild type (RAN) or mutant (RAN (G19V/Q69L)) RAN and unprogrammed lysate for the empty vector pcDNA, as previously described (El-Tanani et al., 2001, 2004). The proteins synthesized were confirmed by western blotting.

GTPase assay

The assay was performed as described in the manufacturer's instructions by measuring the release of inorganic phosphate using the malachite green phosphomolybdate colorimetric assay (Innova Biosciences, UK) (Margalit et al., 2004) (see Supplementary data).

C-Met autophosphorylation

Confluent monolayers (9-cm diameter Petri dishes) of R37, R37-pBK-CMV (R37-CMV), R37-OPN and R37-RAN cells were starved of serum overnight at 37 °C, lysed in antiphosphotyrosine RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, 1% (v/v) Nonidet P-40, plus protease inhibitors, pH 7.5), and 500 μg of lysate was immunoprecipitated with antiphosphotyrosine monoclonal antibody 4G10 bound to agarose beads (50 μl of beads/lysate; Upstate Biotechnology Inc., Lake Placid, NY, USA). Immunoprecipitated proteins were resolved by 7.5% (w/v) SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with mouse anti-c-Met (Santa Cruz; sc-161), as described previously (Sakata et al., 1997). Equal amounts of lysate were also resolved directly on SDS–PAGE gels and immunoblotted for c-Met to obtain total (phosphorylated and nonphosphorylated) c-Met.

Akt phosphorylation

The R37, R37-CMV, R37-RAN and R37-OPN cells were harvested at 80% confluence from 9-cm diameter dishes. The cells were lysed in 300 μl of 2 × loading buffer (20% glycerol (v/v), 4% SDS (w/v), 10% 2-mercaptoethanol (v/v), 126 mM Tris-HCl, pH 6.8 and colored with bromophenol blue). The lysates were sonicated, boiled at 100 °C for 5 min and then centrifuged at 15 000 r.p.m. for 10 min. A total of 10 μg of protein lysates were resolved by 10% (w/v) SDS–PAGE and immunoblotted for Ser473 phosphorylated Akt or separate lysates for Akt as described previously (Sarbassov et al., 2005).

Tumorigenicity and metastasis assays

Two ( × 106) cultured cells were injected s.c. into the mammary fat pad of syngeneic female Furth–Wistar rats (Dunnington et al., 1983). Estimation of the weight in grams and histology of tumors and tissues at autopsy after 2 months was carried out as previously described (El-Tanani et al., 2004). At least two sections of each tumor/tissue were examined by two independent observers. Animals containing microscopically visible metastases of malignant cells in the lungs were scored positive for metastasis (Chen et al., 1997). Animals were maintained according to UKCCCR guidelines under UK Home Office Project Licence No. 40/2395 to Professor PS Rudland.

Immunohistochemistry

Histological sections from tumors/lung metastases were dewaxed and incubated with optimal concentration of antibodies to one of OPN (mouse monoclonal antibody (MAb) MBIII B10; Developmental Studies Hybridoma Bank), RAN (mouse MAb 610340; BD Biosciences), c-Met (rabbit polyclonal sc-10; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and pan Akt (rabbit polyclonal ab8805; Abcam Plc, Cambridge, UK) (Rudland et al., 2002). Primary antibodies were located by incubation with biotinylated sheep anti-mouse Ig (Amersham Int., Bucks, UK) or donkey anti-rabbit (Amersham Int.) for 1 h followed by antibody–biotin complex (Dako Ltd., Ely, UK) for OPN, c-Met, Akt, and visualized with 3,3′ diaminobenzidine/H2O2 (Sigma, Poole, UK) to develop a brown coloration (Rudland et al., 2002). RAN was located using the Envision-Plus horseradish peroxidase system (Dako Ltd.) (Wang et al., 2006). Cellular nuclei were lightly counterstained blue with Mayer's hemalumn (Rudland et al., 2002). For immunofluorescence, cells were grown in eight-well slides (Nunc) (Kilty et al., 1999) until about 50% confluent and then fixed in neutral buffered formalin. Wells were rinsed in PBS, and incubated with rabbit anti-c-Met or anti-Akt. The first antibody was located by incubating with goat anti-rabbit Ig conjugated to fluorescein isothiocyanate (FO382; Sigma Chemical Co., Poole, Dorset, UK). The cultures were washed with PBS, the wells were peeled off, and the slides were then mounted using fluorescent mounting medium (Dako Ltd.) and stored in the dark at 4 °C before viewing. Immunocytochemically and immunofluorescently stained cells were observed in a Reichert Polyvar microscope at × 400 magnification using either bright field or epifluorescence optics (B4 filter block), the mean±s.d. of the percentage of the cells’ cytoplasm and/or nucleus which were stained from six fields of a minimum of 200 cells per field for each of two sections/slides was recorded.

Statistical treatment of results

All biological experiments were performed at least three times. The mean and standard error were calculated and P-values less than 0.05 were considered significant as calculated using the Student's t-test. Rats containing tumors and metastases were scored positive or negative and the significance of the difference between groups was assessed using Fisher's exact test, two-sided P-values less than 0.05 were considered significant.

Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

Mab:

monoclonal antibody

OPN:

osteopontin

Rama:

rat mammary

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Acknowledgements

This work was supported by grants from Research and Development Office, Queen's University Belfast, Northern Ireland, UK and The Cancer and Polio Research Fund, Wirral, UK. DGF's post is funded by the Northwest Cancer Research Fund, Liverpool, UK.

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Correspondence to M K El-Tanani.

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

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Kurisetty, V., Johnston, P., Johnston, N. et al. RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin. Oncogene 27, 7139–7149 (2008) doi:10.1038/onc.2008.325

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Keywords

  • osteopontin
  • RAN
  • breast cancer invasion and metastasis

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