Inactivation of the von Hippel–Lindau (VHL) tumor-suppressor gene causes both hereditary and sporadic clear-cell renal-cell carcinoma (ccRCC). Although the best-characterized function of the VHL protein (pVHL) is regulation of hypoxia-inducible factor-α (HIFα), pVHL also controls the development of pheochromocytoma through HIF-independent pathways by regulating JunB. However, it is largely unknown how these pathways contribute to the development and progression of ccRCC. In the present study, we confirmed that JunB was upregulated in VHL-defective ccRCC specimens by immunostaining. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in 786-O and A498 VHL null ccRCC cells suppressed their invasiveness. In addition, JunB knockdown significantly repressed tumor growth and microvessel density in xenograft tumor assays. Conversely, forced expression of wild-type, but not dimerization-defective, JunB in a VHL-restored 786-O subclone promoted invasion in vitro and tumor growth and vessel formation in vivo. Quantitative PCR array analysis revealed that JunB regulated multiple genes relating to tumor invasion and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2) in 786-O cells. JunB knockdown in these cells reduced the proteolytic activity of both MMPs in gelatin zymography and the amount of CCL2 in the culture supernatant. Moreover, shRNA-mediated knockdown of MMP-2 or inhibition of CCL2 activity with a neutralizing antibody repressed xenograft tumor growth and angiogenesis. Collectively, these results suggest that JunB promotes tumor invasiveness and enhances angiogenesis in VHL-defective ccRCCs.
Kidney cancer accounts for 2–3% of adult malignancies, and it causes about 102 000 deaths worldwide annually (Rini et al., 2009). Clear-cell renal-cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer, representing approximately 75–80% of RCCs (Linehan et al., 2007). von Hippel–Lindau (VHL) disease is caused by inactivating germline mutations of the VHL tumor-suppressor gene and is associated with an increased risk of ccRCC and other tumors, including pheochromocytoma, and hemangioblastoma of the retina, cerebellum and spinal cord (Woodward and Maher, 2006). In addition, it has been reported that biallelic inactivation of the VHL gene also occurs in the majority of sporadic ccRCCs (Young et al., 2009). These observations have motivated studies aimed at determining how the VHL protein (pVHL) suppresses tumor growth.
The best-characterized function of pVHL is its ability to regulate the hypoxia-inducible factor (HIF) through poly-ubiquitination and proteasomal destruction (Maxwell et al., 1999). HIF, especially HIF2α, appears to have a particularly important role in ccRCCs based on genotype–phenotype correlations in VHL disease and nude mouse xenograft assays using human VHL-defective ccRCC cell lines (Linehan and Zbar, 2004; Kaelin, 2008, 2009).
HIF activation caused by pVHL inactivation leads to excessive transcription of HIF target genes, including the angiogenic factor vascular endothelial growth factor (VEGF), which is responsible for higher vascularization of VHL-defective tumors. Indeed, molecules that target the HIF/VEGF pathway (mTOR inhibitors, sunitinib and sorafenib) are in clinical use and improve progression-free survival of patients with ccRCC significantly (Brugarolas, 2007; Rini et al., 2009).
Besides regulation of HIF, evidence is accumulating that pVHL also regulates extracellular matrix (ECM) formation, the microtubule cytoskeleton, apoptosis, cell motility and invasion (Frew and Krek, 2008; Roberts and Ohh, 2008; Calzada, 2010). In fact, it has been revealed that HIF-independent VHL functions affect the development and behavior of ccRCC (Yang et al., 2007; Frew and Krek, 2008; Kaelin, 2009). In this respect, we previously reported that pVHL regulated the abundance of JunB through atypical protein kinase-C (aPKC) in an HIF-independent manner, and that inappropriate accumulation of JunB through he inactivation of pVHL was responsible for the development of pheochromocytoma in VHL disease (Lee et al., 2005b; Nakamura and Kaelin, 2006; Woodward and Maher, 2006). However, the roles of JunB in the carcinogenesis of ccRCC remain to be elucidated.
JunB is a member of the activated protein-1 (AP-1) family that forms dimeric protein complexes with family members (jun, fos and ATF) (Eferl and Wagner, 2003; Piechaczyk and Farras, 2008). There is a growing body of evidence that AP-1 proteins have an important role in tumor invasion and angiogenesis (Eferl and Wagner, 2003; Ozanne et al., 2007; Schmidt et al., 2007). Therefore the aim of this study was to clarify the roles of JunB in the development and progression of VHL-defective ccRCC.
Here, we show that JunB is upregulated in sporadic ccRCC specimens with VHL inactivation. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in VHL-defective ccRCC cells inhibited the invasive ability, angiogenesis and tumor growth of xenograft tumors. Present study revealed that JunB regulated tumor invasion and angiogenesis of ccRCC through matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2). Our results suggest that the JunB pathway could be a promising therapeutic target in these tumors.
The JunB protein is highly expressed in tumor specimens from pVHL-defective ccRCC
To clarify whether JunB protein is upregulated in VHL-defective ccRCC, we performed immunohistochemical analysis of JunB for a total of 75 ccRCC specimens. The degree of immunopositivity in clinical samples was assessed as the percentage of cells positive for JunB. The patients’ characteristics and the status of VHL are shown in Supplementary Table 1. Of these 75 cases, VHL inactivation was observed in 43 cases (42 cases with VHL mutations and one case with promoter methylation). Positive staining was predominantly observed in the nuclei of tumor cells. RCC tumors harboring inactive VHL showed significantly higher JunB staining than those with wild-type (wt) VHL (Figures 1a and b). This result indicates that JunB protein accumulates in VHL-defective RCC cells.
As for upregulation of JunB in VHL-defective RCC cells in vitro, we previously reported that pVHL mainly regulates the abundance of JunB mRNA by regulating he kinase activity of aPKC (Lee et al., 2005b). An analysis of gene expression data in the Oncomine website accords with our results by indicating that human primary ccRCC tissues express significantly higher levels of JunB mRNA compared with those with other histological types of RCC (Higgins et al., 2003) (Figure 1c). Collectively, these results strongly indicate that the JunB protein accumulates in ccRCC specimens, especially those with mutant VHL, as a result of an increase in JunB mRNA.
JunB regulates the invasive ability of VHL-defective RCC cells in an HIF-independent manner in vitro
To confirm that expression of JunB is regulated by pVHL, we next examined whether restoration of wt VHL in VHL-defective 786-O and A498 ccRCC cell lines reduced the amount of JunB. As expected, in 786-O cells stably transfected with a plasmid encoding hemagglutinin (HA)-tagged wt pVHL (clone WT8) and in A498 cells stably expressing the same plasmid (clone WTD10), JunB expression, along with that of HIF2α and Glut1, decreased. However, JunB expression did not recover when a constitutive active mutant of HIF2α was introduced to WT8 cells (WT8/HIF2αP531A cells) (Figure 2a). Moreover, shRNA knockdown of HIF2α in 786-O cells did not grossly affect the expression of JunB (Supplementary Figure 1). These results are in keeping with previous results (Lee et al., 2005b) that suggest that JunB expression is regulated by pVHL, mainly independent of HIF.
To examine the effect of VHL and HIF in vivo, we used scanning electron microscopy to analyze the ultrastructure of xenograft tumors from 786-O subclones. Interestingly, the scanning electron microscopic images demonstrated clear differences in ECM structure. Tumors from pRC3 (empty vector clones) had a loosely assembled ECM with fibrillar meshwork, whereas tumors from WT8 and WT8/HIF2αP531A had a densely assembled ECM with no fibrillar meshwork, indicating that ECM remodeling was regulated by pVHL in an HIF-independent manner, consistent with a previous report (Kurban et al., 2006) (Supplementary Figure 2). These findings prompted us to test the invasive ability of 786-O and A498 subclones expressing or not expressing VHL and HIF, as ECM remodeling is closely associated with the invasive ability of tumor cells. In keeping with previous studies (Koochekpour et al., 1999; Kurban et al., 2006), restoration of wt VHL significantly suppressed the invasive ability of both highly invasive 786-O and poorly invasive A498 cells. However, introducing constitutively active HIF2α to WT8 cells (WT8/HIF2αP531A cells) failed to restore it, indicating that the invasive ability of these cells, like their expression of JunB, was regulated by VHL, but independent of HIF activity (Figure 2b).
We next examined whether pVHL controls invasive ability through JunB. For this purpose, we stably knocked down JunB expression by using two independent shRNAs (#1 and #2), both of which markedly reduced endogenous JunB in 786-O and A498 cells without altering the expression of c-Jun, HIF-2α or Glut1 (Figure 2c). Downregulation of JunB significantly suppressed the invasive ability of both cell lines in vitro (Figure 2d). Collectively, these results suggest that deregulation of JunB, in an HIF-independent manner, contributes to the increased invasiveness of pVHL-defective tumor cells.
shRNA knockdown of JunB in VHL-defective RCC cells suppresses tumor growth, invasion and angiogenesis in vivo
To further clarify the role of the JunB that is upregulated in VHL-defective ccRCC cells, the effect of downregulating this AP-1-family protein was examined by using nude mouse xenograft models. Remarkably, JunB knockdown in both 786-O and A498 cells suppressed the growth of tumors from these cells when they were ectopically (subcutaneously) implanted (Figure 3a). In addition, we also performed orthotopic xenograft assays using highly invasive 786-O cells to examine whether JunB regulates the invasive ability of VHL-defective RCC cells in vivo. Microscopic examination of the tissue revealed that control 786-O/scramble tumor cells invaded into the parenchyma of the normal kidney, whereas 786-O/JunB shRNA cells hardly infiltrated (Figure 3b), indicating that JunB regulates the invasive ability of those cells in vivo.
As shRNA-mediated knockdown of JunB did not grossly affect in vitro proliferation and cell-cycle progression (Supplementary Figures 3 and 4), we next explored the roles of JunB in regulating tumor growth in vivo. For this purpose, we first examined blood vessel formation by staining subcutaneous xenograft tumors from 786-O and A498 cells for CD31 (Figure 3c). Unexpectedly, tumors from subclones with JunB shRNAs showed markedly lower vessel formation. As no significant differences were observed in the Ki-67 index between tumors infected with scramble or JunB shRNAs (Figure 3c), it was suggested that poor vascularization might inhibit tumor growth. Samples were then stained with an anti-VEGF antibody. However, no apparent difference was observed in the intensity of VEGF staining among them (Figure 3c). Accordingly, 786-O/JunB shRNA cells secreted comparable amounts of VEGF in vitro (Supplementary Figure 5).
As a converse experiment, a 786-O subclone stably expressing VHL (WT8) was infected with a retrovirus encoding HA-tagged wt JunB or dimerization-defective JunB (ΔbZip JunB) (Figure 4a). Forced expression of wt JunB or ΔbZip JunB affected the expression of neither c-Jun nor HIF-2α (Figure 4a), nor cell proliferation in vitro (Supplementary Figure 3). Introduction of wt JunB did, however, increase the in vitro invasive ability of WT8 cells, and promoted tumor growth accompanied by augmented vessel formation in vivo (Figures 4b–d). Again, forcefully expressed JunB neither grossly affected the intensity of VEGF staining, the Ki-67 index nor in vitro VEGF secretion (Figure 4d and Supplementary Figure 5). As WT8 cells transfected with the ΔbZip JunB mutant failed to restore the phenotypes promoted by wt JunB, these results indicate that JunB transcripts control, at least in part, invasiveness and angiogenesis in VHL-defective ccRCCs.
shRNA-mediated JunB suppression in VHL-defective RCC cells alters the expression of invasion- and angiogenesis-related genes
To identify genes under the control of JunB, the expression profile of 168 genes involved in angiogenesis and tumor invasion in 786-O/scramble and 786-O/JunB shRNA cells (#1 and #2) was compared by using a quantitative real-time reverse transcription–PCR array. As shown in Table 1, we identified 12 genes whose expression was changed at least 1.5-fold by both shRNAs.
Of genes relating to angiogenesis, CCL2 was downregulated by JunB shRNAs. This result was also confirmed by quantitative PCR using another set of primers (Figure 5a). In fact, the amount of CCL2 protein was significantly decreased in cell culture supernatant from 786-O/JunB shRNA cells (Figure 5b). Immunohistochemical studies also showed that the amount of CCL2 was reduced in xenograft tumors from 786-O/JunB shRNA cells (Figure 5c). As CCL2 has been demonstrated to act as a direct mediator of angiogenesis in in vivo models (Salcedo et al., 2000; Keeley et al., 2008), we next examined the effect of this chemokine in the tumorigenesis and angiogenesis of ccRCC. Inhibiting CCL2 activity with a neutralizing antibody significantly suppressed xenograft tumor growth in 786-O cells. Inactivation of CCL2 also significantly reduced microvessel density (MVD) 2 weeks after inoculation, although the effect was diminished at the time point of 6 weeks (Figure 5d).
As for genes involved in tumor invasion, it was noted that expression of both MMP-2 and MMP-9 was reduced by JunB shRNAs (Table 1). Results of the PCR array analysis were confirmed by quantitative PCR (Figure 6a), and gelatin zymography revealed that shRNA-mediated knockdown of JunB reduced the proteolytic activity of both MMPs in vitro (Figure 6b). Moreover, in situ gelatin zymography applied to xenograft tumors showed that gelatinolytic activity was attenuated in tumors from 786-O/JunB shRNA cells (Supplementary Figure 6). To confirm the effects of endogenous or exogenous JunB on transcriptional activation of MMP-2, we next examined MMP-2 promoter activity using a luciferase reporter assay. As expected, JunB knockdown suppressed MMP-2 promoter activity in 786-O cells, and introduction of wt JunB, but not Δbzip JunB, promoted MMP-2 promoter activity in WT8 cells (Figures 6c and d). In addition, a site-specific mutagenesis of the AP-1-binding site in the MMP-2 promoter significantly attenuated its transcriptional activity in 786-O cells (Figure 6e), indicating that transcriptional activities were regulated, at least in part, by the AP-1-family protein.
In fact, shRNA-mediated knockdown of MMP-2 or MMP9 significantly inhibited in vitro cell invasion of 786-O cells by reducing gelatinolytic activity, which phenocopies the effect of JunB knockdown (Figures 7a and b). Similarly, a specific gelatinase (MMP-2 and MMP-9) inhibitor SB-3CT suppressed invasion of those cells (Supplementary Figure 7a). Notably, JunB-induced cell invasion was blocked by either knockdown of MMP-2 or treatment with SB-3CT in WT8 cells (Figure 7c and Supplementary Figure 7b). Previously, it was reported that pVHL regulated the expression of MMP-2 and MMP-9 (Koochekpour et al., 1999; Struckmann et al., 2008). In addition, Kurban et al. (2006) clearly showed that MMP-2 was regulated by pVHL in an HIF-independent manner in 786-O cells. These results, combined with our own, suggest that pVHL regulates the expression of MMP-2/9 and concomitant tumor invasion mostly through JunB in these cells. Importantly, shRNA knockdown of MMP-2 suppressed the tumor growth in xenograft model with decreased MVD (Figures 8a and b). No remarkable change was observed in either the intensity of VEGF staining or the Ki-67 index (Figure 8b). Collectively, these results highly suggest that MMP-2/9, particularly MMP-2, have significant roles on JunB-mediated tumor invasion and progression of pVHL-defective RCCs.
It is well known that JunB has an anti-oncogenic function that it exerts by antagonizing c-Jun-dependent proliferation and transformation (Piechaczyk and Farras, 2008; Shaulian, 2010). On the other hand, it was reported that homodimers of JunB positively regulated cell proliferation, whereas c-Jun:JunB heterodimers negatively regulated it in a mouse model, using a knock-in strategy and a transgenic complementation approach (Passegue et al., 2002). Indeed, it has been reported that JunB has oncogenic roles in particular cancer cells such as Hodgkin's lymphomas and anaplastic large cell lymphomas (Watanabe et al., 2005; Staber et al., 2007; Shaulian, 2010). The results in the present study indicate that JunB upregulated in VHL-defective ccRCCs augments the angiogenesis and invasion of tumors in vivo. Consistent with our result, there is a growing body of evidence that AP-1 subunits have an important role in tumor angiogenesis and invasion (Eferl and Wagner, 2003; Ozanne et al., 2007; Schmidt et al., 2007).
In terms of angiogenesis in VHL-defective ccRCCs, we clarified that JunB knockdown suppressed CCL2 expression in 786-O cells. CCL2 has a critical role in the recruitment of macrophages and the induction of angiogenesis (Balkwill, 2004; Keeley et al., 2008). As such, it was revealed that this chemokine regulated the angiogenesis of xenografts from pVHL-defective RCCs, although the effect of treatment with a neutralizing antibody was limited to the early phase of tumor formation. The pro-angiogenic effects of CCL2 should be further examined in VHL-defective ccRCCs by using shRNA-mediated knockdown of this chemokine in a future study. Importantly, it is already known that pVHL controls CCL2 transcripts in the livers of mice through inhibitory phosphorylation of CARD9, the nuclear factor-κB agonist (Yang et al., 2007). Moreover, it has been reported that transcriptional activation of the CCL2 promoter is regulated by both nuclear factor-κB and AP-1 in human endothelial cells (Martin et al., 1997). These results, combined with our own, indicate that pVHL might regulate the expression of CCL2 through JunB and the nuclear factor-κB pathway.
As for molecules relating to tumor invasion, it was revealed that JunB regulated MMP-2 and MMP-9 in highly invasive 786-O cells. In fact, shRNA-mediated knockdown of these MMPs significantly suppressed in vitro cell invasion in 786-O cells. Notably, treatment with the specific gelatinase (MMP-2 and MMP-9) inhibitor SB-3CT, or shRNA knockdown of MMP-2, overrides the invasion conferred by forcefully expressed JunB in WT8 cells, indicating that JunB upregulates the invasion ability in these cells mainly through MMP-2/9, particularly MMP-2.
Importantly, shRNA knockdown of MMP-2 suppressed tumor growth in xenograft model with decreased MVD. This result might be explained by reference to the literature: it has been reported that MMP-2 and MMP-9 promote angiogenesis by releasing VEGF stored in the ECM, or by directly activating the bioavailability of VEGF (Lee et al., 2005a; Roy et al., 2009). Additionally, it has been shown that MMPs are able to degrade ECM components. In fact, pVHL-deficient cells fail to form ECM properly, which may facilitate neoangiogenesis and invasion of these cells, mostly through HIF-independent pathways (Ohh et al., 1998; Frew and Krek, 2008; Kurban et al., 2008; Calzada, 2010).
In terms of the possible regulation of ECM assembly by JunB, scanning electron microscopic images demonstrated that tumors from 786-O/JunB shRNAs had a densely assembled ECM with no fibrillar meshwork, which is similar to that in WT8 (pVHL-positive) cells. By contrast, tumors from 786-O/scramble cells had a loosely assembled ECM with fibrillar meshwork, which is similar to that in pRC3 (pVHL-negative) cells (Supplementary Figure 2). Recently, it was reported that the promoter region of COL14A1 was frequently methylated in RCC specimens (Morris et al., 2010). Interestingly, the expression profile analysis in the present study revealed that JunB represses the expression of COL14A1. Therefore, we suspect that this gene is down-regulated in RCC specimens. In fact, this suspicion is confirmed by data from the Oncomine website (Supplementary Figure 8). Similarly, expression of COL1A1, a target of upregulation by JunB, is overexpressed in RCC specimens (Supplementary Figure 8). These results indicate that JunB regulates several ECM components, linking the effects of this AP-1 protein to ECM remodeling and this link deserves examination to further our understanding of the roles of JunB in VHL-defective ccRCCs.
As for the potential clinical relevance of JunB target genes, it was reported that RCC patients with higher amounts of MMP-2 or MMP-9 in tumor tissues showed a trend of poor prognosis (Kallakury et al., 2001; Takahashi et al., 2002). It was also reported that the baseline serum level of MMP-9 was significantly higher among patients who did not respond to sunitinib treatment for metastatic ccRCC (Perez-Gracia et al., 2009). It is true that anti-angiogenic therapies, such as those targeting VEGF, demonstrate anti-tumor effects for patients with ccRCC (Brugarolas, 2007; Rini and Atkins, 2009). However, tumors receiving this therapy frequently acquire resistance or show initial resistance. Although the mechanisms of initial and acquired resistance remain to be elucidated, it is speculated that redundant angiogenic factors other than VEGF are related to the development of refractory disease (Rini and Atkins, 2009). Indeed, it is thought that VEGF-mediated signaling is the predominant stimulator of angiogenesis in VHL-defective ccRCC, but parallel angiogenesis-related pathways such as CCL2 can also drive tumor growth. In this sense, there is a possibility that expression of JunB-regulated genes affects the development of the above refractory disease.
Based on these results, we propose that the JunB pathway may be a promising therapeutic target for ccRCCs. Likewise, aPKC could be a useful target, as JunB is regulated through aPKC, although further investigation of JunB regulation by pVHL is required (Frew and Krek, 2008; Kaelin, 2008). Indeed, treatment of 786-O and A498 cells with the PKC inhibitor GF109203X efficiently reduced the invasive ability of those cells as well as markedly reducing JunB protein (Supplementary Figure 9).
In conclusion, we propose that JunB upregulation caused by inactivation of pVHL leads to tumor progression in VHL-defective ccRCCs. The aPKC/JunB pathway could be a target for suppressing the invasion and angiogenesis of these tumors.
Materials and methods
Patients and RCC samples
Tumor specimens from 75 patients who received radical or partial nephrectomy were obtained from a tissue bank in the Department of Urology at Kyoto University Hospital under the protocols approved by the University's institutional review board.
VHL genotyping and hypermethylation assays
Genotyping of VHL and determination of the methylation status of the VHL CpG island were performed as described previously (Nakamura et al., 2006).
After deparaffinization and antigen retrieval, endogenous peroxidase activity was blocked by 0.3% H2O2 in methyl alcohol. The glass slides were washed in phosphate-buffered saline and mounted with 1% horse normal serum in phosphate-buffered saline. Subsequently primary antibody was applied overnight at 4 °C. They were incubated with biotinylated horse anti-mouse serumin phosphate-buffered saline, followed by washes in phosphate-buffered saline. Avidin–biotin–peroxidase complex (ABC; ABC-Elite; Vector Laboratories, Burlingame, CA, USA) was applied together with the nuclear counterstaining with hematoxylin. JunB immunohistochemical analysis of 75 clinical ccRCC samples was performed on paraffin-embedded, formalin-fixed tissues. The degree of immunopositivity in clinical samples was assessed as the percentage of cells positive for JunB, and the assessment was performed by a pathologist (YM) with no prior knowledge of the VHL status of the samples. VEGF, Ki-67 and CCL2 staining of xenograft tumors was performed on paraffin sections and CD31 staining on frozen sections. Vessel counts were performed as described previously (Weidner, 1995). Numbers of total or Ki-67-positive nuclei were automatically quantitated by using the ImageJ software. Labeling index for Ki-67 was calculated as the percentage of positive tumor nuclei divided by the total number of tumor cells examined.
Antibodies were purchased commercially: JunB (for immunohistochemistry, Abcam, Cambridge, MA, USA; for western blotting, Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Jun (Cell Signaling, Beverly, MA, USA), HIF2α (Novus Biologicals, Littleton, CO, USA), Glut1 (Alpha Diagnostic Inc., San Antonio, TX, USA), HA (Covance, Berkeley, CA, USA), β-actin (Abcam), histone H2A (Cell Signaling), mouse CD31 (Dako, Carpinteria, CA, USA), VEGF (Santa Cruz Biotechnology.), Ki-67 (Novocastra, Newcastle Upon Tyne, UK) and CCL2 (R&D Systems, Minneapolis, MN, USA).
VHL-deficient 786-O and A498 RCC cells were purchased from the American Type Culture Collection (Rockville, MD, USA). 786-O and A498 subclones stably transfected with pRc-CMV (clone pRC3 and pRCJ17, respectively) or pRc-CMV-HA-VHL (clone WT8 and WTD10, respectively), and WT8 subclones further stably transfected with either pIRES-puro-HA (WT8/mock) or pIRES-puro-HA-HIF2α P531A (WT8/HIF2α P531A), were described previously (Kondo et al., 2002, 2003; Nakamura et al., 2006). 786-O and A498 cells stably infected with shRNA retroviruses were selected in the presence of 1 mg/ml G418. WT8 cells stably infected with pBabe-puro retroviruses and 786-O cells stably infected with shRNA lentiviruses were selected in the presence of 1.5 μg/ml puromycin.
JunB and c-Jun were detected in nuclear extracts, and HIF2α and Glut-1 were detected in whole-cell extracts. Immunoblotting was performed as described previously (Lee et al., 2005b).
In vitro tumor cell invasion was measured using BD Biocoat Matrigel Invasion Chambers (Becton Dickinson, Franklin Lakes, NJ, USA). Cells (786-O and A498 subclones, 4 × 104) in serum-free Dulbecco's modified Eagle's medium (500 μl) were plated in the upper chamber and incubated with Dulbecco's modified Eagle's medium containing 10% fetal calf serum in the bottom of the chamber at 37 °C in 5% CO2, 786-O for 24 h and A498 for 48 h.
The oligonucleotide sequences used in the construction of the shRNA vector were as follows: JunB#1 (5′-IndexTermGATCCAATGGAACAGCCCTTCTACCATAGTGCTCCTGGTTGTGGTAGAAGGGCTGTTCCATTTTTTTTAT-3′) and (5′-IndexTermCGATAAAAAAAATGGAACAGCCCTTCTACCACAACCAGGAGCACTATGGTAGAAGGGCTGTTCCATTG-3′) (Watanabe et al., 2005); JunB#2 (5′-IndexTermGATCCGGTGAAGACACTCAAGGCTTAGTGCTCCTGGTTGAGCCTTGAGTGTCTTCACCTTTTTTAT-3′) and (5′-IndexTermCGATAAAAAAGGTGAAGACACTCAAGGCTCAACCAGGAGCACTAAGCCTTGAGTGTCTTCACCG-3′) (Staber et al., 2007); and control scramble (5′-IndexTermGATCCGTACAGCGGTCCAATCATAGTAGTGCTCCTGGTTGCTATGATTGGACCGCTGTACTTTTTTAT-3′) and (5′-IndexTermCGATAAAAAGTACAGCGGTCCAATCATAGCAACCAGGAGCACTACTATGATTGGACCGCTGTACG-3′). The oligonucleotides were annealed and then ligated into the BamHI/ClaI sites of the pSINsi-hU6 vector (Takara Bio, Shiga, Japan).
Previously, we made pDEST 47-JunB and pDEST-ΔbZip JunB (dimerization-defective JunB) (Lee et al., 2005b). To make pBABE-puro-HA-JunB and pBABE-puro-HA-ΔbZip JunB, the former two were PCR-amplified using the primers 5′-IndexTermCGGGATCCATGTGCACTAAAATGGAACAGCCCT-3′ and 5′-IndexTermCGGAATTCTGGGACAACTCCAGTGAAA-3′, digested using BamHI and EcoRI, and ligated into pBABE-puro-HA cut with these two enzymes.
G3T-hi packaging cells were infected with retroviral plasmids by using a Retrovirus Packaging Kit (Ampho; Takara Bio), according to the manufacturer's instructions.
Lentivirus-based plasmids containing shRNAs to human MMP-2 and MMP-9 were purchased from Open Biosystems (Huntsville, AL, USA). Non-silencing shRNAs (Open Biosystems) were used as negative control. The experimental procedures for shRNA transfection were according to the Open Biosystems technical manual.
Nude mouse xenograft assays
All experiments involving laboratory animals were performed in accordance with the Guideline for Animal Experiments of Kyoto University. For subcutaneous xenograft assays, 107 cells were injected subcutaneously into both flanks of 6-week-old female BALB/cAJcl nude (nu/nu) mice (CLEA Inc., Tokyo, Japan). Orthotopic xenograft was performed as described previously (Yang et al., 2007). Presence of tumor mass was finally confirmed by hematoxylin and eosin staining.
VEGF and CCL2 ELISA
The VEGF and CCL2 proteins in the supernatant were quantified using VEGF and CCL2 ELISA kits (R&D Systems). Concentration was normalized to total cell protein.
Real-time PCR and PCR arrays
cDNA synthesis and real-time PCR were performed as described previously (Terada et al., 2010). The primer sequences were as follows: CCL2, 5′-IndexTermCCCCAGTCACCTGCTGTTAT-3′ (sense) and 5′-IndexTermAGATCTCCTTGGCCACAATG-3′ (antisense); MMP-2, 5′-IndexTermATAACCTGGATGCCGTCGT-3′ (sense) and 5′-IndexTermAGGCACCCTTGAAGAAGTAGC-3′ (antisense); MMP-9, 5′-IndexTermGAACCAATCTCACCGACAGG-3′ (sense) and 5′-IndexTermGCCACCCGAGTGTAACCATA-3′ (antisense); and GAPDH, 5′-IndexTermGAAGGTGAAGGTCGGAGTC-3′ (sense) and 5′-IndexTermGAAGATGGTGATGGGATTTC-3′ (antisense).
RT2 Profiler human angiogenesis and Extracellular Matrix and Adhesion Molecules PCR Arrays (SA Biosciences, Frederick, MD, USA), containing 84 × 2 angiogenesis- and invasion-related genes, plus housekeeping genes and controls, were used according to the manufacturer's protocols.
Blocking CCL2 with neutralizing antibody
Neutralizing antibodies against human CCL2 (MAB679; R&D Systems) or control mouse IgG were administered to mice with subcutaneous tumors from 786-O cells, by injecting intraperitoneally every 4 days from the date of tumor cell inoculation to the end of the mouse experiment at a dose of 10 μg per mouse (Lu and Kang, 2009).
Gels for gelatin zymography were purchased from Invitrogen (Carlsbad, CA, USA) and used according to the manufacturer's instructions. Cells were cultured in Opti-MEM for 24 h, and the medium was collected and concentrated using Amicon Ultra-4 (Millipore, Billerica, MA, USA). The samples were normalized for intracellular protein concentration.
Luciferase reporter assays
Full-length human MMP-2 promoter-luciferase constructs (pGL3-MMP2p-Luc) were made as described previously (Bian and Sun, 1997). A site-specific mutation (1270-TGACTTCT-1263→TGGATTCT) was introduced into the AP-1-binding site in the MMP-2 promoter as described previously (Song et al., 2006). Cells grown in 12-well tissue culture plates were transfected with 0.4 g of pGL3-MMP2p-Luc and 4 ng of pTK-RL using Lipofectamine 2000 (Invitrogen). After 24 h of incubation, the luciferase activity of the cell lysate was measured in triplicate by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), using a luminometer (MicroLumat Plus LB96V; Berthold Technologies, LLC, TN, USA).
Data were expressed as mean±s.e., except real-time PCR (mean±s.d.), and Student's t-test was used to analyze the differences between means. Mann–Whitney non-parametric U-test was performed to compare differences in JunB immunopositivity between VHL mutant groups and wt VHL groups. Inhibition of tumor growth with a CCL2 neutralizing antibody was analyzed by two-way repeated-measures analysis of variance. Statistical analyses were all performed using StatView ver. 5.0 (SAS Institute, Cary, NC, USA).
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We thank Dr William G Kaelin, Jr, for critical comments and for providing us with the different RCC cell lines. We appreciate all members of Cancer Research Courses for Integrated Research Training at Kyoto University Graduate School of Medicine for helpful advice and discussion. We also thank Tomoko Matsushita, Megumi Kuraguchi and Yuko Fujieda for technical assistance. Grant support: Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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