Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer

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The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.


Bladder cancer arises from urothelial cells and is clinically manifested as two distinct subtypes, invasive or noninvasive.1, 2, 3 As the invasive subtype of bladder cancer is closely associated with metastatic spread and poor prognosis of patients,3 it is important to clarify the molecular mechanisms of development and progression of this lethal form of disease.

It has been reported that the Ral small GTPase, a protein of the Ras subfamily proteins, is highly activated in several human cancers, such as bladder,4 colon5 and pancreatic6 cancers. In bladder cancer cells, activated Ral is reported to augment cell proliferation, migration and metastasis.7, 8 Ral has two isoforms, RalA and RalB, which share 85% homology.9 Despite this high homology, RalA and RalB have been shown to have different roles in human cancer. RalA is involved in anchorage independent growth, whereas RalB is required for cell survival and migration.9, 10, 11

Inactive GDP-bound Ral is activated by Ral guanine nucleotide exchange factors (RalGEF), such as RalGDS, RGL1, RGL2, RGL3, RALGPS1 and RALGPS2.10, 12 Among them, RalGDS, RGL1, RGL2 (Rlf) and RGL3 contain the Ras association (RA) domain and are direct effectors of activated Ras. Importantly, GTP-bound RalA and RalB are inactivated by Ral GTPase-activating proteins (RalGAPs), which we have recently identified.13 RalGAPs are heterodimers of a common β subunit (RalGAPβ) and a catalytic α subunit (RalGAPα1 or RalGAPα2). The heterodimer composed of RalGAPα1 and RalGAPβ is designated as RalGAP1, and that of RalGAPα2 and RalGAPβ as RalGAP2. The α subunits of RalGAP share 54% identity at the amino acid level in overall structure and 85% identity at the C-terminal GAP domain.13 Complex formation is essential for their RalGAP activity and protein stability, and each form of RalGAP regulates both RalA and RalB.13

In Ras-driven cancers, such as pancreas and colon cancers, Ral activation is mediated by constitutively active Ras mutant and RalGEFs with the RA domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancers, in which Ras active mutation is relatively infrequent.14 In the present study, we demonstrated that RalGAPα2 regulated tumor progression of bladder cancer through inactivation of Ral by showing that (1) both RalA and RalB were aberrantly activated in invasive bladder cancer cell lines because of reduced expression of RalGAPα2, which is the dominant catalytic subunit in normal urothelium or noninvasive urothelial cancer cells, (2) exogenous expression of wild-type (wt) RalGAPα2 in invasive bladder cancer cells successfully suppressed lung metastasis, (3) genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model and (4) attenuated expression of RalGAPα2 was significantly associated with advanced clinical stage and poor survival of patients with bladder cancer.


RalA and RalB were aberrantly activated in human invasive bladder cancer cell lines harboring negligible RalGAPα2 expression

We first examined the activation status of RalA and RalB in both noninvasive (RT112, RT4 and DSH1) and invasive bladder cancer cell lines (KU7, 253J, TCCSUP and T24). Among the four invasive cell lines, the amount of GTP-bound RalA and -RalB was increased as compared with those in the three noninvasive ones (Figure 1a). Accordingly, the ratio of GTP-RalA/B to total RalA/B was higher in the invasive cell lines as compared with the cells with a noninvasive phenotype (Figure 1a). Unexpectedly, no apparent correlation was observed between invasion ability of cells and abundance of GTP-Ras except in T24 cells harboring mutant Ras-G12V (Figure 1a). Moreover, examination of the levels of six RalGEFs in these cells did not show any apparent correlation with the invasive property (Figure 1b and Supplementary Figure S1).

Figure 1

RalA and RalB were aberrantly activated in invasive bladder cancer cell lines in correlation to downregulation of RalGAPα2 and RalGAPβ. (a) GTP-bound Ras and RalA/B were evaluated by GST-Raf and GST-Sec5 pull down assays, respectively, in three noninvasive and four invasive bladder cancer cell lines. The ratios of GTP-bound RalA/B to total RalA/B are indicated. (b) mRNA expression of indicated RalGEFs in the seven bladder cancer cell lines was evaluated by reverse transcription PCR (RT–PCR). (c) The protein expression of RalGAP subunits was evaluated in various human tissues by immunoblotting. (d, e), Protein and mRNA expression of RalGAP subunits in these cell lines were evaluated by immunoblotting (d) and RT–PCR (e), respectively. Data are representative of three independent experiments.

Importantly, human urinary bladder predominantly expressed the RalGAPα2 catalytic subunit, but expressed little RalGAPα1 (Figure 1c). This was also the case in bladder cancer cell lines, in which RalGAPα1 was scarcely detected (Figure 1d). Notably, all four invasive cell lines expressed negligible amounts of RalGAPα2, while this protein was abundantly expressed in all three noninvasive cells. The expression level of the RalGAPβ subunit in the invasive cells was also moderately lower than those in the noninvasive ones. As complex formation of the RalGAPα and β subunits is critical for the stability of these proteins,13 the drastic reduction of RalGAPα2 expression in the invasive cell lines would also contribute, at least in part, to the lower expression of RalGAPβ. Further, reduced expression of both RalGAPα2 and β mRNA was observed, suggesting that transcriptional regulation was also involved in the low expression of these proteins in the invasive cells (Figure 1e).

Exogenous expression of RalGAPα2 attenuated migration of bladder cancer cells through inactivation of Ral

To clarify whether Ral activation was mainly regulated by RalGAPα2 in bladder cancer cells, we stably expressed wt-RalGAPα2 in KU7 and TCCSUP cells, which have scanty levels of endogenous RalGAPα2. Both GTP-bound active RalA and RalB were remarkably decreased by forced wt-RalGAPα2 expression, but not by that of the mutant RalGAPα2-N1742 K lacking RalGAP activity13 (Figure 2a). As an inverse experiment, when RalGAPα2 was suppressed by small interfering RNA (siRNA) in RT4 cells, which express endogenous RalGAPα2 abundantly, both GTP-RalA and -RalB were increased by 2.0- and 4.5-folds, respectively (Figure 2b). Collectively, these data indicate that RalGAPα2 is the predominant modulator of GTP-Ral levels in bladder cancer cells.

Figure 2

RalGAPα2 regulated Ral activation and suppressed invasive characteristics of human bladder cancer cells. (a) The abundance of GTP-RalA/B was evaluated by GST-Sec5 pull down assay in KU7 and TCCSUP cells stably expressing control vector, (wild-type) wt-RalGAPα2 (α2) or mutant RalGAPα2-N1742 K (α2-N1742 K) lacking RalGAP activity. (b) Activation of RalA and RalB was evaluated by GST-Sec5 pull down assay in RT4 cells, which were transiently transfected with the indicated siRNAs. (ce) KU7 and TCCSUP cells from (a) were used. Morphology and actin cytoskeleton organization of the cells were evaluated in (c). Phalloidin (red) for F-actin and DAPI (blue) for nuclei. Cell motility was evaluated by wound healing assay in (d) and by transwell migration assay in (e). (d) Subconfluent KU7 and TCCSUP cells were scratched and cultured for 24 h. The widths of the ‘wound’ (scratched areas) were measured by image J and proportion of the wound healed was calculated by the following formula: ‘the wounded area filled (%)’=100% (width after 24 h/width at the beginning) × 100% as shown in the histogram. Each experiment was performed three times in triplicate samples. (e) KU7 and TCCSUP cells were serum-starved for 6 h and suspended in serum-free RPMI 1680. The cell suspension (5 × 104 cells) was then added to the insert of a transwell cell culture chamber and was incubated for 24 h with medium containing 10% FBS in the bottom of the chamber. The cells that attached to the lower side of the membrane were fixed with 70% ethanol and stained with hematoxylin–eosin (HE) (See Supplementary Figure S2a for TCCSUP). Each experiment was performed three times in triplicate samples. The average number of cells in five microscopic fields ( × 200) was defined as the number of migrating cells. Scale bar in (d) and (e), 200 μm. Data are either representative or shown as mean±s.e. (error bars) of three independent experiments.

As Ral has been implicated in actin cytoskeleton organization,7 cell shape and structure were also examined. KU7 cells stably expressing wt-RalGAPα2 showed a rounded shape or less spread, whereas those expressing mutant RalGAPα2-N1742 K or control vector retained a spindle shape (Figure 2c). Similarly, TCCSUP cells stably expressing wt-RalGAPα2 showed suppressed stress fiber formation as compared with those expressing the mutant RalGAPα2 (Figure 2c).

To determine whether the change in cell shape and structure by RalGAPα2 expression had any effects on cell motility of bladder cancer cells, we performed migration tests. Exogenous expression of wt-RalGAPα2, but not mutant RalGAPα2-N1742 K, suppressed the migration of KU7 and TCCSUP cells as evaluated by the wound healing assay (Figure 2d) and the transwell migration assay (Figure 2e and Supplementary Figure S2a). Unexpectedly, exogenous expression of wt-RalGAPα2 did not affect the cell proliferation of KU7 nor TCCSUP cells (Supplementary Figure S2b). These findings suggest that the expression levels of RalGAPα2 specifically regulated cytoskeletal organization and the cell motility of bladder cancer cells presumably through the regulation of Ral activity.

Loss of RalGAPα2 promoted tumor development and disease progression of experimental bladder cancer in a mouse model

To clarify the functional relevance of RalGAPα2 to the development of bladder cancer in vivo, we generated homozygous Ralgapa2 knockout (Ralgapa2−/−) mice by disrupting exons 2 and 3 of the gene (Figure 3a and Supplementary Figure S3a-c). Ralgapa2−/− mice grew without any apparent abnormalities (Shirakawa et al. unpublished data). The RalGAPα2 subunit is predominant in murine bladder as in human bladder (Supplementary Figure S3d and Figure 1c). As expected, the level of GTP-RalA in the bladder tissues of Ralgapa2−/− mice showed a 2.5-fold increase as compared with that of wild-type (Figure 3b).

Figure 3

Genetic ablation of Ralgapa2 promoted the invasion of bladder cancer chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). (a) Protein expression levels of RalGAPα1, α2 and β subunits in urinary bladders were evaluated by immunoblotting in Ralgapa2+/+ (n=2) and Ralgapa2−/− (n=2) mice. (b) The RalA activation state in urinary bladder was evaluated by GST-Sec5 pull down assay in Ralgapa2+/+ (n=3) and Ralgapa2−/− (n=3) mice. The ratios of GTP-bound RalA/B to total RalA/B are indicated. (c) Six- to eight-week-old Ralgapa2−/− (n=14) and Ralgapa2+/+ (n=20) mice were continuously given drinking water containing 0.025% BBN. After 16 weeks, mice were killed and urinary bladders were harvested. In (c), typical macroscopic (left) and histopathological (middle) images (H and E stain) of bladder urothelial cancers in Ralgapa2+/+ (noninvasive cancer) and Ralgapa2−/− (muscle-invasive cancer) mice are shown. The distribution of histopathological grading (no malignancy, noninvasive or invasive cancer) is also shown (right). Scale bar (left), 10 mm.

Further, the effect of Ralgapa2 ablation on tumor development and disease progression was evaluated using a chemically induced bladder cancer model. When six- to eight-week-old mice were given 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine for 16 weeks continuously, 65% of wild-type mice developed bladder cancer although all of them showed a noninvasive phenotype. By contrast, all of the Ralgapa2−/− mice developed tumors, and 43% of them showed an invasive phenotype (Figure 3c). These results clearly indicate that RalGAPα2 has a critical role in tumor development and progression in an experimental murine bladder cancer model.

Lower expression of RalGAPα2 is associated with tumor invasion and poor prognosis of human bladder cancer

On the basis of these results, the clinical significance of RalGAPα2 was assessed in human bladder cancer. We first examined the expression of RalGAPα2 in bladder cancer specimens derived from the public database ONCOMINE. Tumors with an invasive phenotype exhibited a significantly lower expression of RalGAPα2 compared with those presenting a nonmuscle-invasive phenotype15 (Supplementary Figure S4). Furthermore, a similar pattern was also observed for the expression of RalGAPβ16 (Supplementary Figure S4).

For more detailed analysis, we evaluated the abundance of RalGAPα2 protein in human bladder cancer tissues by immunohistochemistry. Specificity of the anti-RalGAPα2 antibody was previously validated using human bladder cancer tissues, and it was confirmed that the signal intensity of immunohistochemical staining coincided with that obtained by western blot analysis (Supplementary Figure S5).

A total of 97 histopathologically diagnosed human bladder cancer tissues (Table 1) and 8 normal urothelial specimens were examined for RalGAPα2 expression. The staining intensity of RalGAPα2 was classified into strong-staining and weak-staining groups. As expected, all of the normal urothelium (n=8) showed strong staining (Figure 4a). On the other hand, 36 of 68 (52%) cases with no muscle invasion and 27 of 29 (93%) specimens with muscle-invasive bladder cancer exhibited weak staining (Figure 4b). The difference in incidence of weak staining between nonmuscle-invasive and muscle-invasive bladder cancers was statistically significant (Figure 4b; P<0.0001, Fisher's exact probability test). Notably, Kaplan–Meier analysis demonstrated that cases with strong RalGAPα2 staining (n=37) had favorable cancer-specific survival as compared with cases with weak staining (Figure. 4c; P=0.0029; log-rank test). These results suggest that a lower expression of RalGAPα2 is associated with disease progression and poor survival of patients with bladder cancer.

Table 1 Patients' characteristics
Figure 4

Low expression of RalGAPα2 is associated with invasion, metastasis and poor prognosis of human bladder cancer. (ac), Immunohistochemical staining for RalGAPα2 was performed in 105 human samples, consisting of 97 human bladder cancer tissues and 8 normal urothelial tissues. (a) Representative images of (i) normal urothelium and bladder urothelial cancer tissues representing nonmuscle-invasive cancer (ii) (iii) and muscle-invasive cancer (iv). Scale bar, 100 μm. In (b), distribution of samples with weak or strong intensity of RalGAPα2 staining is shown in noninvasive (n=68) and invasive (n=29) urothelial cancer cases (P<0.0001, Fisher's exact probability test). In (c), cancer-specific survival in bladder cancer patients was analyzed by Kaplan–Meier curves. Statistical significance between cases with strong staining (n=37) and cases with weak staining (n=60) was evaluated by log-rank test (P=0.0029). In (d), distribution of staining intensity with RalGAPα2 antibody in bladder cancer samples with (n=13) or without (n=79) metastasis at initial presentation is shown (P=0.0019, Fisher's exact probability test).

We then assessed the clinical significance of RalGAPα2 expression in tumor metastasis. Distant metastases were identified in 13 patients at initial presentation. Importantly, 12 of 13 specimens (92%) of primary lesions in the bladder derived from these patients exhibited weak expression of RalGAPα2 (Figure 4d). By contrast, only 45 of 79 specimens (57%) from patients without distant metastasis showed weak staining (Figure 4d). The difference in incidence of weak staining between these groups was statistically significant (Figure 4d P=0.015, Fisher's Exact Probability Test).

RalGAPα2 suppresses metastasis formation of bladder cancer cells

The significance of lower RalGAPα2 expression on tumor metastasis prompted us to conduct an experiment to clarify whether exogenous expression of the RalGAPα2 subunit could inhibit distant metastases in bladder cancer cells. For this purpose, we first generated KU7-luc cells that stably expressed control vector, wt-RalGAPα2, or mutant RalGAPα2-N1742 K lacking RalGAP activity. These cells were then injected intravenously into nude mice and lung metastasis was evaluated after 35 days by signal intensity of luciferase bioluminescence. As shown in Figure 5a, mice with exogenous expression of wt-RalGAPα2, but not mutant RalGAPα2-N1742 K, showed a significant reduction of photon flux in the lungs, indicating a lower incidence of metastasis (Figure 5a). The suppression of metastatic lung tumors was confirmed by histopathological evaluation (Figure 5b). These data suggest that RalGAPα2 suppresses lung metastasis of bladder cancer cells presumably through the inactivation of Ral activity.

Figure 5

Forced expression of RalGAPα2 in KU7 bladder cancer cells suppressed experimental lung metastasis formation. (a, b), KU7-luciferase (luc) cells stably expressing either control vector, wt-RalGAPα2 (α2) or mutant RalGAPα2-N1742 K (α2-N1742 K) were generated. (a) At 35 days after intravenous injection of these cells into nude mice (n=6 for each group), lung metastasis formation was quantified by in vivo imaging system (IVIS) (Xenogen). The lungs were evaluated histopathologically in (b). The average number of metastatic foci from four sections was quantified as described in the ‘Materials and Methods’. Data are either representative or shown as mean±s.e. (error bars). Scale bar, 10 mm (bottom) and 200 μm (top).


Accumulating evidence shows that Ral is involved in tumorigenesis and disease progression in various cancers including bladder cancer.4, 7, 8 In the present study, we revealed that Ral was highly activated in invasive bladder cancer cells. Several pancreatic cancer cell lines exhibited highly activated Ral because of overexpression of RGL2, one of the RalGEFs.17 However, none of the invasive bladder cancer cell lines examined in this study showed any increase of specific RalGEFs. By contrast, the expression of RalGAPα2 protein was remarkably decreased in the cells possessing an invasive phenotype (Figures 1d-e). These results highly suggest that aberrant activation of Ral in invasive bladder cancer cells is mainly caused by downregulation of the RalGAPα2 subunit, the dominant RalGAP catalytic subunit in bladder. To our knowledge, this is the first study to report the significance of RalGAPs on the regulation of Ral activation in human cancer cells.

RalA is involved in the transformation of normal cells,18 whereas RalB regulates cell survival,19 migration7 and metastasis8 of cancer cells. Ralgapa2−/− mice showed a higher incidence of tumor development with invasive phenotype than control Ralgapa2+/+ mice in a chemically induced murine bladder cancer model (Figure 3e), which was consistent with the finding that RalGAPα2 reduced the migration ability of human bladder cancer cells (Figure 2d-e). These results imply that downregulation of RalGAPα2 causes development of bladder cancer as well as enhancing disease progression through activation of Ral.

Recently, it has been shown that deregulated Ral activation is involved in lung metastasis of bladder cancer cells.8 Our data demonstrating suppression of lung metastasis by exogenous expression of wt-RalGAPα2 (Figure 5a-b) emphasize the significance of this molecule in the regulation of metastasis in human invasive bladder cancer cells. From this point of view, RALGAPA2, also designated as C20orf74, has been identified as a candidate gene for metastasis suppression in colon cancer.20 Thus, downregulation of RalGAPα2 may also have significant impact on metastasis in other types of cancers.

Previously, it has been reported that siRNA-mediated knockdown of RalA or RalB inhibited cell proliferation.6, 7 However, exogenously expressed RalGAPα2 did not grossly affect it in the present study. One possible explanation for this discrepancy may be that increasing RalGAP activity is physiologically less effective in attenuating Ral activity compared with siRNA-mediated knockdown of Ral.

The present study has several important implications with regard to the clinical setting. Tumors harboring downregulated RalGAPα2 appear to be predisposed to invasion or metastasis (Figures 4b and d). On the other hand, most noninvasive bladder tumors showing abundant RalGAPα2 by immunohistochemical analysis exhibited a favorable prognosis (Figures 4b–d). Thus, RalGAPα2 expression level may be a useful predictive biomarker in the management of those patients. This study also revealed that decreased RalGAPα2 expression might be a cause of aberrant Ral activation in a majority of human invasive bladder cancer cells. Therefore, these results highlight the importance of the RalGAPα2 and Ral signaling pathway as a potent therapeutic target in bladder cancer.

Besides bladder cancer, low expression of RalGAPα2 was also reported in cancers of various organs, such as colon21 (Supplementary Figure S6), pancreas22, 23 (Supplementary Figure S6), breast24, 25 and testis.26 Correlatively, low expression of RalGAPβ mRNA was also observed in breast,24 colon27 (Supplementary Figure S7), lung28 (Supplementary Figure S7), pancreas29 (Supplementary Figure S7), prostate30 and testicular26 cancers. These findings imply that suppression of RalGAP2 may also occur in these cancers. Further studies are required to elucidate the role and significance of RalGAP2 in other cancers.

In conclusion, we have shown that reduced expression of RalGAPα2 resulted in aberrant Ral activation, which could be a cause of the progression of human bladder cancer. As several kinds of human cancers exhibited decreased expression of RalGAPs, further understanding of the roles of RalGAPs and Ral would provide important insights for the prevention and treatment of a variety of human cancers.

Materials and methods

Cell lines and cell culture

A total of seven human bladder cancer cell lines were used: RT112,31 RT4,31 DSH1,32, 33 KU7,34 253J,31 TCCSUP31 and T24.31 It has been reported that 253J, TCCSUP and T24 were established from invasive tumor tissues31 and that RT112,31 RT431 and DSH132, 33 were established from well-differentiated noninvasive tumor tissues. Also, KU734, 35 253J,36 TCCSUP37, 38 and T2439 cell lines were classified as ‘invasive’ because the xenografts derived from these cells exhibited invasive and aggressive phenotype. On the other hand, RT112, RT4 and DSH1 were classified as ‘noninvasive’ cell lines, because the xenografts derived from these cells exhibited noninvasive and papillary morphology.31 All the cell lines were cultured in RPMI 1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 12.5 mM HEPES and penicillin–streptomycin at 37 oC in 5% CO2.

RT–PCR analysis

Total RNA was prepared from bladder cancer cells and reverse-transcribed using a First-Strand cDNA Synthesis Kit (GE Healthcare, Buckinghamshire, UK). Expression of the cDNAs for RalGAPs and RalGEFs was analyzed by PCR using FastStart Taq DNA Polymerase (Roche, Indianapolis, IN, USA). Primers used are shown in Supplementary Table S1. Experiments were performed at least three times independently.


Cells and tissues were lysed in RIPA buffer (Sigma, St Louis, MO, USA). Protein concentration was determined by the Lowry method using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting were carried out as previously described.40 Rabbit anti-RalGAPα1, α2 and β polyclonal antibodies were previously described.13 Other antibodies used were as follows: Anti-RalA (BD Biosciences, Franklin Lakes, NJ, USA), anti-RalB (Millipore, Billerica, MA, USA), anti-β-actin (Abcam, Cambridge, MA, USA) and anti-tubulin α (Cell Signaling, Beverly, MA, USA), horseradish peroxidase (HRP)-labeled secondary antibodies (GE Healthcare).

GST-Sec5 pull down assay

Amounts of GTP-bound Ral were measured as previously described.41 Cultured cells were serum-starved for 6 h and stimulated with medium containing 10% FBS for 2 min at 37 °C. The cells were rinsed twice with ice-cold phosphate-buffered saline and lysed in 0.3 ml ice-cold buffer A (50 mM HEPES/KOH, pH7.4, 100 mM NaCl, 4 mM MgCl2, 2 mM EGTA, 1 mM dithiothreitol, 1% (w/v) Triton X-100, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM Na3VO4) containing protease inhibitors. The cell lysates were rotated at 4 °C for 5 min and then centrifuged at 21 500 × g for 15 min. Supernatants containing 200 μg of proteins were incubated at 4 °C for 30 min with glutathione beads coated with 20 μg glutathione-S-transferase (GST)-Sec5 Ral binding domain (Sec5-RBD). After washing the beads three times with buffer A, bead-associated RalA and RalB were analyzed by immunoblotting to quantify the GTP-bound forms. Supernatants containing 20 μg (10% of the supernatants incubated with beads) of proteins was saved as input before the incubation and used for immunoblotting to quantify the total volume of RalA and RalB.13 The same method was used for GST-Sec5 pull down assays in murine bladder tissues. These experiments were performed at least three times independently.

Plasmid construction and lentiviral expression

The full length RalGAPα2 cDNA was amplified by PCR from human lung cDNA (Marathone-Ready cDNA, Clontech, Mountain View, CA, USA) and cloned into lentivirus vector. Mutant RalGAPα2-N1742 K, which lacks GAP activity, was created by site-directed mutagenesis. Lentiviral stocks were produced in 293FT cells by using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Virus-containing medium was collected at 48 h post-transfection and filtered through a 0.45-μm filter. Lentiviral infection into KU7 cells and TCCSUP cells was performed by replacing the medium with medium containing the virus and 8 μg/ml Polybrene (Millipore) followed by centrifugation at 1190 × g for 30 min at 32 °C. Stably infected cells were selected with blasticidin (4 μg/ml; Invitrogen) and used for the indicated assays.

RNA interference

We used two distinct siRNAs targeting human RalGAPα2 (Stealth RNAi, Invitrogen): HSS148422 (RalGAPα2 siRNA no. 1) and HSS148424 (RalGAPα2 siRNA no. 2). Universal control sequence (Invitrogen) was used as a negative control. RT4 cells were transfected with each siRNA at 20 nM using RNAiMAX transfection reagent (Invitrogen). The cells were harvested and analyzed by the pull down assay at 54 h after the transfection.

Cell proliferation assay in monolayer culture

KU7 and TCCSUP cells stably expressing RalGAPα2 (α2), RalGAPα2-N1742 K (α2-N1742 K) or control vector (control) were seeded in triplicate at 2 × 104 cells/well in six-well culture dishes and cultured under standard conditions. At the indicated time, cells were trypsinized and viable cell numbers were counted.

Wound healing assay and transwell-migration assay

For wound healing assays, subconfluent KU7 and TCCSUP cells in 24-well culture dishes were scratched with a plastic pipette tip and cultured for 24 h. The widths of the ‘wound’ (scratched areas) were measured by image J ( and proportion of the wound healing was calculated by the following formula: 100%−(width after 24 h/width at the beginning) × 100%. Each experiment was triplicated and performed three times independently.

For transwell migration assays, KU7 and TCCSUP cells were serum-starved for 6 h and suspended in serum-free RPMI 1680. The cell suspension (5 × 104 cells) was then added to the 8.0-μm pore polyethylene terephthalate filter insert of a 24-well transwell cell culture chamber (BD Falcon, Franklin Lakes, NJ, USA) and was incubated for 24 h with medium containing 10% FBS in the bottom of the chamber. Residual cells on the upper side of chambers were removed by scraping with cotton swabs and the cells that attached to the lower side of the membrane were fixed with 70% ethanol and stained with hematoxylin–eosin. Migrated cells were counted under microscopy. Each experiment was triplicated and performed three times independently.

Experimental lung metastasis model and in vivo imaging system

All the animal experiments were approved by the Animal Research Committee of Kyoto University. Mice were housed in a specific pathogen-free room. We generated KU7-luciferase (luc) cells stably expressing firefly luciferase, as described previously,42 and then generated KU7-luc cells that stably expressed control vector, RalGAPα2 or RalGAPα2-N1742 K. One million of these cells were injected into the lateral tail vein of 7-week-old female nude mice (Japan Clea) to evaluate lung colonization. Exact injection into circulation was confirmed by measuring photon flux from lungs using an in vivo imaging system (Xenogen, Alameda, CA, USA) 2 min after each injection. The extent of lung metastasis formation was evaluated at 35 days after the injection by injecting luciferin intraperitoneally and measuring photon flux from lungs. The mice were killed and the lungs were then embedded in paraffin. In all, seven step sections (5 μm thick) of each lung lobe were made at 300 μm intervals and stained with HE. Metastatic tumors were identified by light microscopic examination. The numbers of metastatic foci in the lungs were counted in every other section (four sections) and the average was calculated.

Generation of Ralgapa2−/− mice

Exon 2 and exon 3 of the mouse Ralgapa2 gene were disrupted by gene targeting. The targeting vector was constructed by modifying the bacterial artificial chromosome RP23-334D15 (Invitrogen) using defective prophage λ-Red recombineering system.43 Reagents and plasmids used for the recombination were provided by Dr NG Copeland (National Cancer Institute, Maryland, MD, USA). The linearized targeting vector was electroporated into C57BL/6J mouse ES cells (DS Pharma Biomedical) and positive clones were selected for resistance to Geneticin (Invitrogen). Homologous recombination was confirmed by PCR and Southern blotting. Successfully recombined ES cells were injected into blastocysts obtained from ICR strain mice, and the resulting chimeric males were mated with C57BL/6J females to obtain F1 mice carrying the targeted allele. Genotyping of knockout mice was carried out using the following three primer sequences in a single PCR: F2 (forward) 5′-CTTGGACATTGATGTGTGAGTGGTGCCCAC-3′, R2 (reverse) 5′-GAACTGCTTAAGATCGCTTGCATCCACG-3′ and RR (reverse) 5′-CAGGTTTCCGGGCCCTCACATTGCCAAAAG-3′. PCR product size is theoretically expected to be 208 b for wild-type allele (F2–R2) and 316 b for mutated allele (F2–RR).

Mouse bladder cancer model

In all, six- to eight-week-old Ralgapa2 knockout mice and wild-type mice were continuously given drinking water containing 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine (Tokyo Kasei Kogyo, Tokyo, Japan). After 16 weeks, mice were killed and urinary bladders were harvested, processed for paraffin sectioning and stained with hematoxylin and eosin. The histopathological evaluation of the bladder tissues was performed in a blind fashion by one pathologist in Kyoto Histopathology Study Group, Inc. and by two trained urologists (R Saito and HN), based on ‘the 2004 WHO Classification of Bladder Tumors’.

Immunohistochemical analysis of clinical samples

We used bladder urothelial cancer samples surgically obtained at Kyoto University Hospital between 2000 and 2006, under a protocol approved by the institutional review board. Formalin-fixed, paraffin-embedded tumor tissues from patients were retrieved from the archives of the center for anatomical study. The clinical and pathological stage was determined using tumor-node-metastasis classification and graded according to the tumor-node-metastasis classification of malignant tumors, seventh edition, UICC. Immunostaining was performed as described previously.44 Each RalGAPα2-stained bladder tissue was graded as strong staining and weak staining. All scoring was independently conducted by a trained pathologist (YT) and trained urologists (RSaito. and HN) in a blind fashion. Most of the judgments were identical among the three observers.

Statistical analysis

Results are reported as means and s.e. We used SPSSII (SPSS Japan Inc.) for analyzing statistical data. Student's t-test and Fisher's exact test were used to analyze statistical significance. Kaplan–Meier curves and log-rank test were used to analyze survival data. P-value <0.05 was considered significant.


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We would like to thank Aaron Mathew Coutts for proofreading of the manuscript. We appreciate all members of the Cancer Research Course for Integrated Research Training in Kyoto University Graduate School of Medicine for their helpful advice and discussion. This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 22591766 to HN and OO, 22890015, 22501009, 20013201 and 23113702 to R Shirakawa and HH) as well as grants from Uehara Memorial Foundation (to T Kobayashi and OO), Takeda Science Foundation, the Suzuken Memorial Foundation, Daiichi-Sankyo Foundation of Life-Science, Kurokawa Cancer Research Foundation (to R Shirakawa), and Novartis Foundation for the Promotion of Science (to HH).

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Correspondence to H Nishiyama or H Horiuchi.

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Supplementary Information accompanies the paper on the Oncogene website

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  • RalGAPα2
  • Ral small GTPase
  • bladder cancer
  • invasion
  • metastasis

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