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Introduction

Gliomas are the most common primary tumors of the central nervous system. Despite recent advances in surgical approaches and available adjuvant therapies, a poor prognosis is imminent due to the highly migratory and invasive nature of glioma cells (1,2). Their active migration and invasion ultimately lead to tumor recurrence and reduced life expectancy (3). Nevertheless, the molecular mechanisms underlying glioma cell migration and invasion remain unclear. Therefore, it is vital to investigate the signaling pathways mediating glioma pathogenesis and progression in order to formulate efficient therapeutic regimes.

Ubiquitination is essential in many biologic processes, while aberrant expression of the enzymes involved in the regulation of the ubiquitination process plays a key role in malignant transformation (4). Protein ubiquitination is achieved in the ubiquitin-conjugating process via the covalent attachment of enzymes E1, E2 and E3 on the specific-target of substrate protein. The E3 ubiquitin ligases are the key regulatory components, which recognize specific substrates (5). Emerging evidence indicates that the E3 ubiquitin ligase Nedd4-1 is involved in cancer pathogenesis (6,7), and may be a target protein for cancer therapy (8–10), as the overexpression of Nedd4-1 in colorectal and gastric cancers has been found to promote malignant cell proliferation (11,12). FoxM1-regulated Nedd4-1 expression was reported to destabilize phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and to promote astrocyte transformation and glioma formation (5). To date, PTEN is the substrate which has received the most attention in Nedd4-1 ubiquitination (6,13,14). PTEN is argued to be dispensable for Nedd4-1 (15,16), which is supported by numerous studies (17–21). In addition, Nedd4-1 has been reported in several cases of human tumors, whereas the functional significance of Nedd4-1 in glioma remains unclear.

Rap, the closest homolog of the small G protein Ras, plays a variety of biological functions in human cells, such as signal transduction, migration and proliferation (22,23). In addition to the recognized multiple mammalian proteins as Nedd4-1 substrates (24), the mono-ubiquitination of Rap2a by Nedd4-1 reportedly controls neurite development and decreases Rap2a activity in mouse neurons (24). Recent studies indicate that upregulation of Rap2a expression is correlated with tumor formation and progressive malignancy in human prostate and follicular thyroid cancer (25,26), whereas our previous study indicated that Rap2a is modestly expressed in glioma (27). Thus, further investigations are warranted to ascertain the effects of Nedd4-1-mediated Rap2a ubiquitination on the migration and invasion of glioma cells.

In the present study, we investigated the role of Nedd4-1 and the involvement of intracellular pathways in glioma. We validated that Nedd4-1 protein was overexpressed in glioma tissues vs. that noted in non-cancerous tissues. Moreover, Nedd4-1 promoted the migration and invasion of glioma cells, mediated by Nedd4-1/Rap2a ubiquitination.

Materials and methods

Glioma and non-cancerous brain tissue samples

The present study was approved by the Institutional Review Boards of The Affiliated Hospital of Xuzhou Medical College. Glioma tissue samples, obtained from patients undergoing resection for glioma tumors at the Department of Neurosurgery of The Affiliated Hospital of Xuzhou Medical College, were histologically confirmed by experienced pathologists based on the World Health Organization (WHO) grading system (28). Non-cancerous brain tissues were collected from patients undergoing decompression surgery for intracranial traumas.

Ethical approval

All procedures performed in the present study involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all individual participants or their legally authorized representatives prior to enrollment in the study.

Cell culture

Human glioma cell lines U251 and U87 (Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences) were cultured in Dulbecco's modified Eagle's medium and F-12 (DMEM/F-12) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sijiqing, Hangzhou, China), followed by incubation at 37̊C in a humidified incubator with 5% CO2.

Plasmids and antibodies

The mammalian expression plasmid encoding full-length human Nedd4-1 was a gift from Dr Xuejun Jiang (Memorial Sloan-Kettering Cancer Center, New York, USA). The shNedd4-1 plasmid was constructed by Shanghai GenePhama, and the human Nedd4-1 residues 217–549 was a gift from Dr Nile Brose (Max Planck Institute, Göttingen, Germany). The Myc-tagged Rap2a plasmids, including wild-type (WT-Rap2a), dominant-active (DA-Rap2a) and dominant-negative (DN-Rap2a), were gifts from Dr Kenichi Kariya (University of the Ryukyus, Okinawa, Japan). DA-Rap2a and DN-Rap2a were constructed by mutating Gly 12 to Val and Ser 17 to Asn, respectively. Hemagglutinin (HA)-tagged ubiquitin and its mutants including K63R-ubiquitin and K48 R-ubiquitin were gifts from Shanghai Institute of Neuroscience, Chinese Academy of Sciences. The bacterial expression plasmid for the glutathione S-transferase (GST)-fusion protein of Ras-binding region of RalGDS was cloned by reverse transcription polymerase chain reaction (RT-PCR) using a human brain cDNA library as a template. The following antibodies were commercial products: rabbit polyclonal antibodies to Nedd4-1 and Rap2a (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-TNIK (BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal antibodies P4D1 and FK1 to ubiquitin (Santa Cruz Biotechnology, Inc.), rabbit monoclonal antibodies to ubiquitin Apu2 and Apu3, and mouse monoclonal (9E10) antibody to Myc (Millipore, Billerica, MA, USA).

Reverse transcription-PCR

Total RNA was extracted from U251 and U87 cells using TRIzol reagent (Tiangen, Beijing, China). The RT-PCR protocol was performed according to the manufacturer's instructions using reverse transcription reagents (Takara, Dalian, China). Primers (Invitrogen, Shanghai, China) for Nedd4-1 were as follows: sense, 5′-ACTTTATCCATTACCGACAG-3′ and antisense, 5′-TGGTGGCTTCATCTTCTC-3′, yielding a 320-bp product. Primers for the internal standard β-actin were: sense, 5′-GCGCGGCTACAGCTTCAC-3′ and antisense, 5′-GGGGCCGGACTCGTCATA-3′, yielding a 500-bp product. PCR conditions were as follows: the first-strand DNA template was heated at 94̊C for 3 min before amplification, a total of 30 cycles (for Nedd4-1) or 23 cycles (for β-actin) were used including 94̊C for 30 sec (denaturation), 63̊C for 30 sec (annealing), 72̊C for 35 sec (extension), and 72̊C for 7 min (extension).

Western blotting

Standard western blotting was performed using antibodies against Nedd4-1, Rap2a, TNIK, Myc and β-actin. Cell extracts were prepared 48 h after transfection and subjected to 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transference to 0.45-µm pore size polyvinylidene fluoride membranes (Millipore) and blotted with the antibodies as indicated.

Immunohistochemistry

Immunohistochemistry was performed as previously described, with minor modifications (29). Paraffin-embedded tissue sections were obtained from the Department of Pathology, The Affiliated Hospital of Xuzhou Medical College, including 11 cases of WHO grade II astrocytomas, 12 cases of WHO grade III anaplastic astrocytomas, and 12 cases of WHO grade IV glioblastomas, with non-cancerous brain tissues as controls. Slices were incubated at 4̊C overnight with the anti-Nedd4-1 antibody (1:100 dilution; Abcam, Cambridge, MA, USA). Subsequent to microphotography, the percentages of glioma cells with positive staining were presented and the Nedd4-1 expression levels were thereby categorized.

Transfection assay

Cell transfection was performed using PolyJet™ In Vitro DNA Transfection Reagent (SignaGen, Rockville, MD, USA) when U251 cells reached 90% confluence on 10-cm plates. Plasmids encoding shRNA-Nedd4-1, full-length Nedd4-1, HA-tagged ubiquitin, and Myc-tagged Rap2a and its mutants were transfected in the respective experiments. Forty-eight hours after transfection, the cells were harvested, rinsed with phosphate-buffered saline (PBS) and lysed in 1% SDS or 1% NP40 buffer.

In vitro scratch assay

Cell motility was examined by scratch assay as previously described, except for minor modifications (30). Twelve hours after transfection, an artificial gap was created on the confluent cell monolayer using a plastic pipette tip. Migrated cells were quantified at 48 h (magnification, ×200) in order to compare with the cell counts at base line with a computer-aided microscopy imaging system. All experiments were performed in triplicates.

Migration and invasion assays

The capabilities of cell migration were assessed by Transwell chamber assay. The invasion assay was performed as previously described with minor modifications (31). Glioma cells were harvested and resuspended in serum-free medium at a concentration of 1×105 cells/ml, and 200 µl was added to the top chamber. The chambers were incubated for 24 h at 37̊C in an incubator with 5% CO2. The noninvasive cells on the upper surface of the membrane were removed by wiping with cotton-tipped swabs. The 8-µm pore-size membrane was fixed and stained, and the population of the invaded cells was counted by light microscopy. All the experiments were performed in triplicates.

Affinity pull-down assay with GST-Nedd4-1. The pull-down assay was performed as previously described with minor modifications (24). Total proteins were extracted from a 1% NP-40 extract of U251 cells and separated by 12% SDS-PAGE followed by western blotting using anti-TNIK (1:500) and anti-Rap2a (1:500) antibodies.

Chemical cross-linking of the endogenous Nedd4-1/TNIK/Rap2 complex

U251 cells in 10-cm culture dishes were incubated with 1 mM dithiobis(succinimidyl propionate) (DSP) (Pierce, Rockford, IL, USA) for 3 min on ice. Excess DSP was quenched with 20 mM Tris-Cl (pH 7.5 at 4̊C) for 15 min at room temperature, and then the proteins were extracted with RIPA buffer [50 mM Tris-Cl (pH 8.0) at 4̊C, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin and 0.5 µg/ml leupeptin]. Nedd4-1 was immunoprecipitated with anti-Nedd4-1 polyclonal antibodies coupled to protein A-sepharose beads (GE Healthcare, Milwaukee, WI, USA) at 4̊C for 14 h. The beads were rinsed with RIPA buffer, and then the precipitated proteins were eluted and boiled in 2X loading buffer containing 50 mM DTT, and separated by SDS-PAGE followed by western blotting using anti-Nedd4-1, anti-TNIK and anti-Rap2 antibodies, respectively.

GST-RalGDS pull-down assay

A total of 40 µg of GST fusion proteins of RalGDS encoding the Ras-binding domain (RBD) was purified and immobilized on GST-sepharose beads to pull down the GTP-bound form of Myc-Rap2a extracted from the U251 cells transfected with Myc-Rap2a, Nedd4-1 and HA-ubiquitin. After extensive washing at 4̊C overnight with a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2.5 mM MgCl2, 10% glycerol and 1% NP-40, the bound proteins were eluted by boiling in 2X loading buffer followed by SDS-PAGE and western blotting.

Statistical analysis

Data are presented as mean ± standard error of mean (SEM). Statistical significance was determined using Student's t-tests for comparisons between two groups and ANOVA for intergroup comparisons. P-values of <0.05 were considered statistically significant (P<0.05).

Results

Nedd4-1 expression in human glioma tissue samples

To investigate the possible effect in glioma pathogenesis, we determined Nedd4-1 protein expression in clinical glioma tissues and non-cancerous brain samples. A representative immunoblot is shown in Fig. 1A. Analysis of the band density revealed that Nedd4-1 was significantly upregulated in the glioma tissues vs. that observed in the non-cancerous samples (Fig. 1B). We further verified Nedd4-1 expression by immunohistochemistry. As shown in Fig. 1C, Nedd4-1 immunoreactivity was predominant in the cytoplasm. As shown in Fig. 1D, the percentage of Nedd4-1-positive cells reached 57.35±2.02% (WHO grade II) and 75.82±3.06% (WHO grade III–IV) in glioma tissues vs. the percentage of 22.30±2.17% in non-cancerous brain samples. Thus, Nedd4-1 expression was significantly correlated with glioma malignancy. Fig. 1 shows significant upregulation of Nedd4-1 in glioma tissues vs. non-cancerous brain samples. These results indicated the potential involvement of Nedd4-1 in the pathogenesis of human glioma.

Figure 1. Expression profile of Nedd4-1 in human glioma tissues. (A) Representative western blotting of the total protein extracts isolated from human glioma (7 samples shown) specimens and human non-cancerous (5 samples shown) brain using rabbit Nedd4-1 antibody. β-actin served as a loading control. (B) Relative protein levels of Nedd4-1 in glioma speimens (n=35) and non-cancerous brain tissue (n=15). Data are expressed as the mean ± SEM from three independent experiments; **P<0.01 compared with non-cancerous brain tissue. (C) Representative IHC analyses of Nedd4-1 expression in non-cancerous brain and human glioma tissues of various (WHO) grades. a, Normal brain tissue; b, diffuse astrocytoma, grade II; c, anaplastic astrocytoma, grade III; and d, GBM, grade IV are shown. Scale bar, 20 µm. (D) Quantitative analysis of the percentage of Nedd4-1-positive cells; **P<0.01 compared with non-cancerous brain tissue. Grade II, low-grade glioma. Grade III–IV, high-grade glioma.

Effects of Nedd4-1 on cell migration

Given the overexpression of Nedd4-1 in glioma in contrast to non-cancerous tissues, we employed a gain-of-function strategy to assess the effect of Nedd4-1 on glioma cell migration. As shown in Fig. 2A and B, exogenous shNedd4-1 and Nedd4-1 were added to interfere with or overexpress Nedd4-1 in the U251 and U87 cell lines. The effect of Nedd4-1 on cell migration was assessed by scratch assay. It was shown that the migratory capacity of glioma U251 cells was significantly reduced upon downregulation of Nedd4-1 as compared with the scramble group. Conversely, the scratch in the Nedd4-1-overexpressing group was rapidly replenished compared with the vector group (Fig. 2C and D). We also performed cell migration assays using Transwell chambers to investigate the migratory capacities of the U251 and U87 cells. Likewise, the shNedd4-1 group showed significantly reduced migration compared with that noted in the scramble group (Fig. 2E and F), whereas the migratory behavior in the Nedd4-1-overexpressing group was evidently enhanced compared with that noted in the vector group in both the U251 and U87 cell lines (Fig. 2G and H). Collectively, we demonstrated that either the downregulation or overexpression of Nedd4-1 exerted relevant causality on the migratory capacity of both cell lines.

Figure 2. Effects of Nedd4-1 on cell migration. (A) Exogenous shNedd4-1 and Nedd4-1 were markedly expressed 48 h after transient transfection of U251 cells as assessed by RT-PCR and western blotting. (B) Exogenous shNedd4-1 and Nedd4-1 were markedly expressed 48 h after transient transfection of U87 cells as assessed by RT-PCR and western blotting. (C) Scratch migration assay of U251 cells at time 0 and 48 h after transient transfection with the plasmids shNedd4-1 and Nedd4-1. Scale bar, 100 µm. (D) Cell migration was quantified after 48 h; **P<0.01 compared with the scramble group; #,**P<0.01 compared with the vector group. (E and G) U251 and U87 cell migration was assessed by Transwell assay 24 h after transient transfection with the shNedd4-1 and Nedd4-1 plasmids. Scale bar, 100 µm. (F and H) Quantification of cell migration calculated from the Transwell assays; **P<0.01 compared with the scramble group; #;**P<0.01 compared with the vector group. The number of migratory cells was normalized to that of the NC group. Data are expressed as the means values ± SEM of three independent experiments.

Effects of Nedd4-1 on cell invasion

In addition to the regulation of cell migration by Nedd4-1, we investigated the effect of Nedd4-1 on cell invasion with Matrigel-precoated Transwell chambers. The results indicated that the invasion of U251 and U87 cells was significantly diminished by the downregulation of Nedd4-1 vs. the scramble group subsequent to 24 h of incubation (Fig. 3A and B), while the invasion in the Nedd4-1-overexpressing group was markedly enhanced vs. that noted in the empty vector group in both cell lines (Fig. 3C and D). Thus, both downregulation and overexpression of Nedd4-1 regulated the invasive capacities of the cell lines.

Figure 3. Effects of Nedd4-1 on cell invasion. (A) Representative micrographs of U251 and U87 cell invasion 24 h after Nedd4-1 downregulation. Invaded cells were fixed in formaldehyde and stained with crystal violet. Scale bar, 100 µm. (B) Cell invasion was calculated from Transwell chambers precoated with Matrigel. (C) Representative micrographs of U251 and U87 cell invasion 24 h after Nedd4-1 overexpression. (D) Cell invasion was calculated from Transwell chambers precoated with Matrigel. The number of invasive cells was normalized to that of the NC group. Data are expressed as the means values ± SEM of three independent experiments; **P<0.01 compared with the scramble group; #,**P<0.01 compared with the vector group.

Rap2a as a Nedd4-1 substrate

To verify the role of Nedd4-1 targets in glioma cells, we selected the Nedd4-1 WW domains (32) (residue 217–547) as an affinity matrix and U251 cell lysate (1% NP40) to pull down TNIK and Rap2a. The results exhibited the presence of TNIK and Rap2a in the affinity-purified complex, confirming the binding of Nedd4-1 to either of the substrates (Fig. 4A). We further identified that endogenous Nedd4-1, TNIK and Rap2a constituted a complex (Fig. 4B). U251 cell lysate was incubated with thiol-cleavable cross-linker prior to immunoprecipitation with an anti-Nedd4-1 antibody. Under this condition, TNIK and Rap2a were coimmunoprecipitated with Nedd4-1.

Figure 4. Rap2a is a Nedd4-1 substrate. (A) Complex of Rap2a with TNIK and GST-Nedd4-1 (residues 217–549). Protein was extracted from U251 cells and blotted for TNIK (top) and Rap2a (bottom) after washing GST-Nedd4-1 beads. GST beads were used as a negative control. (B) Endogenous Rap2a, TNIK and Nedd4-1 form a complex. Nedd4-1 was immunoprecipitated using a rabbit polyclonal anti-Nedd4-1 antibody from U251 cell extract after treatment with a thiol-cleavable chemical cross-linker. Western blot analyses were probed with anti-Nedd4-1, anti-TNIK and anti-Rap2a antibodies. Rap2a and TNIK co-immunoprecipitated with Nedd4-1, but no band was observed with the negative control IgG. (C) Nedd4-1 selectively ubiquitinates Rap2a. Nedd4-1 (+) or (−) was co-expressed with WT, dominant-active (DA), or dominant-negative (DN) mutants of Myc-tagged Rap2a. Myc-Rap2a was immunoprecipitated using a mouse monoclonal anti-Myc antibody. Immunoprecipitates were blotted for Myc (bottom panel) and ubiquitin (P4D1, top panel). Note that Nedd4-1 selectively ubiquitinated WT and DA mutants of Myc-tagged Rap2a but not the DN mutant of Myc-Rap2a. (D) Mono- and di-ubiquitination of Rap2a by Nedd4-1. Myc-Rap2a was immunoprecipitated from U251 cells expressing Myc-Rap2a (WT) with or without Nedd4-1. Myc-Rap2a was eluted from anti-Myc antibody-coupled beads and immunoblotted with different monoclonal mouse or rabbit anti-ubiquitin antibodies: P4D1, FK1, Apu3 and Apu2. P4D1 recognizes both poly- and mono-ubiquitin-conjugated proteins, and FK1 recognizes only poly-ubiquitin-conjugated ones. Apu3 and Apu2 recognize K63- and K48-linked poly-ubiquitin chains, respectively. Lysate from U251 cells overexpressing Nedd4-1 was also blotted with the antibodies described above to show that the antibody titers are comparable. Asterisks in (C and D) indicate bands corresponding to Myc-Rap2a conjugated with two ubiquitin moieties. Triangular symbols in (C and D) indicate the light chain of the anti-Myc used for immunoprecipitation. (E) The function of Rap2a by Nedd4-1 ubiquitination. WT Myc-Rap2a expressed in U251 cells with or without overexpressed Nedd4-1 was precipitated using anti-Myc antibodies. GTP-bound Rap2a was assessed with GST-RalGDS pull-down assays compared with 20% total and GST as negative control. (F) Relative GTP-Rap2a activity. Note that GTP-bound active Rap2a was obviously decreased in the overexpressed Nedd4-1 condition (sixth lane). Data are expressed as the means ± SEM of three independent experiments; **P<0.01 compared without Nedd4-1 overexpression.

Rap2a rather than TNIK was found to be ubiquitinated by Nedd4-1 in HEK cells, which decreased the activity of the GTP-bound form of Rap2a (24). To ascertain the potential effect of Nedd4-1 on Rap2a ubiquitination in glioma cells, expression plasmids for the full-length Nedd4-1, HA-tagged ubiquitin as well as Myc-tagged WT-Rap2a, DA-G12V Rap2a or DN-S17N Rap2a were co-transfected in the U251 cells, with lysates extracted under denaturing conditions (100̊C, 1% SDS) and Myc-tagged proteins immunoprecipitated from cell extracts using anti-Myc antibodies. As shown in Fig. 4C, immunoprecipitates from WT and DA-G12V instead of DN-S17N-expressing U251 cells showed a gradient of protein bands visible to an anti-ubiquitin antibody (P4D1) only in the presence of Nedd4-1 overexpression. These data demonstrated that Nedd4-1 selectively ubiquitinated WT-Rap2a and DA-Rap2a, but not DN-Rap2a.

To investigate the exact form of ubiquitination of Rap2a (i.e. mono-ubiquitination, di-ubiquitination or poly-ubiquitination), expression plasmids for full-length Nedd4-1, HA-tagged ubiquitin and Myc-tagged WT-Rap2a were co-transfected in U251 cells. Four types of anti-ubiquitin monoclonal antibodies which discriminate between mono-ubiquitinated and poly-ubiquitinated proteins were employed: P4D1 for poly-ubiquitin and mono-ubiquitin conjugates; FK1 only for poly-ubiquitin conjugates; Apu2 and Apu3 for K48-linked and K63-linked poly-ubiquitin chains, respectively. As shown in Fig. 4D, the escalation of ubiquitinated Rap2a was only identified by P4D1, indicating mono-ubiquitination of Rap2a by Nedd4-1. Apu3 recognized a single faint band representing ubiquitinated Rap2a by Nedd4-1 via K63-linked ubiquitination. No bands were determined by Apu2, exempting the Rap2a ubiquitination by Nedd4-1 from K48-linked polyubiquitin chain, which may lead to proteasome-dependent degradation. A total lysate of Neddd4-1-expressing U251 cells was immunoblotted using these antibodies as controls with blurred bands.

Effect of Nedd4-1 ubiquitination on Rap2a function

To further investigate the effect of Nedd4-1-mediated ubiquitination on Rap2a function in glioma U251 cells, we purified and immobilized a GST fusion of the Rap2a-binding region of RalGDS, which served to pull down active GTP-bound Rap2a. Nedd4-1 and Myc-tagged Rap2a were thereby co-transfected in the U251 cells, and the lysate was loaded onto GST-RalGDS or onto GST as a control. Only active GTP-bound Rap2a was pulled down, indicating that ubiquitination blocked Rap2a function by inhibiting the activity of the GTP-bound Rap2a (Fig. 4E and F).

Effects of Nedd4-1-mediated Rap2a ubiquitination on cell migration and invasion

To verify the role of Nedd4-1-mediated mono-ubiquitination of Rap2a in cell migration and invasion, expression plasmids for full-length Nedd4-1, HA-tagged WT-ubiquitin and its mutants (including HA-tagged K63R-ubiquitin, HA-tagged K48R-ubiquitin, HA-tagged ubiquitin and Myc-tagged WT-Rap2a) were co-transfected in the U251 and U87 cell lines, with MG-132 added to exclude the interference of multiple-ubiquitination in all groups. We assessed migration and invasion parameters in each group of both U251 and U87 cells. As shown in Fig. 5A and B, Nedd4-1 and Rap2a were overexpressed in the U251 and U87 cell lines. The Transwell assay results demonstrated that the migration of glioma cells was significantly enhanced in the WT-ubiquitin and K48R-ubiquitin groups vs. the vector and K63R-ubiquitin groups (Fig. 5C and D). The invasive capacity of glioma cells was also significantly increased in the WT-ubiquitin and K48R-ubiquitin groups vs. the vector and K63R-ubiquitin groups in the Matrigel Transwell assay (Fig. 5E and F). Furthermore, as illustrated (Fig. 5G), the results of immunoprecipitation revealed the Rap2a mono-ubiquitination of Nedd4-1 via K63-ubiquitin. These results indicated that Nedd4-1 may regulate the migration and invasion of glioma cells via the inhibition of GTP-Rap2a.

Figure 5. Effects of the ubiquitination of Rap2a by Nedd4-1 on cell migration and invasion. (A) Relative protein levels of Nedd4-1 and Rap2a in the U251 cell line. (B) Relative protein levels of Nedd4-1 and Rap2a in the U87 cell line. (C) U251 and U87 cell migration 24 h after transient transfection with the indicated plasmids. Scale bar, 100 µm. (D) Quantification of cell migration calculated from Transwell assays. (E) Invasion of U251 and U87 cells 24 h after transient transfection with the indicated plasmids. Scale bar, 100 µm. (F) Quantification of cell invasion was calculated from Transwell chambers precoated with Matrigel. (G) Rap2a mono-ubiquitination by Nedd4-1 was determined by immunoprecipitation combined with western blotting. #Indicates the mono-ubiquitinated Myc-Rap2a. Data are expressed as the means ± SEM of three independent experiments; **P<0.01 compared with the vector group.

Discussion

In the present study, we confirmed that Nedd4-1 promotes the migration and invasion of glioma cells via the inhibition of GTP-Rap2a, a member of the Ras family of small G proteins. This finding clarified the role of Nedd4-1 in migration, invasion and other important pathogenetic processes in glioma.

Nedd4-1, an E3 ligase, plays essential regulatory roles in physiological processes, which is exemplified by its direct binding to and ubqiuitination of activated FGFR1, thus regulating the endocytosis and signaling pathways of FGFR1 in neural differentiation and embryonic development (32). Nevertheless, increasing evidence shows that Nedd4-1 is also involved in a variety of cancers such as bladder, lung and brain carcinomas (5,6,33). In the present study, we identified that the level of Nedd4-1 protein in glioma tissues was approximately 2- to 3-fold higher than that in non-cancerous tissues (Fig. 1A and C). The downregulation of Nedd4-1 reduced the migration and invasion of human glioma cell lines U251 and U87, while Nedd4-1 overexpression reversed these phenomena (Figs. 2C-F and 3A-D). These findings suggest a pivotal role of Nedd4-1 in glioma pathogenesis.

Nedd4-1 reportedly serves as a ubiquitin ligase for multiple proteins (6,12,34,35), with several different domains including C2, two to four WW domains and a HECT domain. Nedd4-1 is reported to bind rather than ubiquitinate TNIK in regulating the ubiquitination of GTP-Rap2a in neuron development. In addition, Rap2a, in lieu of Rap1a, H-Ras and K-Ras, is a specific substrate of Nedd4-1 (24). It is unclear whether Nedd4-1 ubiquitinates Rap2a in glioma. In the present study, Nedd4-1 was found to bind to TNIK and Rap2a, forming a ternary complex in both the GST-Nedd4-1 pull-down assays and co-immunoprecipitation in cell line U251 (Fig. 4A and B). These results suggest that the ubiquitination of Rap2a by Nedd4-1 may be involved in the regulation of migration and invasion of glioma cells. Moreover, the fate of the ubiquitinated proteins is often determined by the chains of ubiquitin conjugation (36). Poly-ubiquitination of substrate proteins by K48-linked ubiquitin chains leads to degradation within the 26S proteasome, whereas K63-linked poly-ubiquitin chains, mono-ubiquitination and multiple mono-ubiquitination usually regulate protein function, gene transcription, endocytosis and DNA repair (36,37). Additionally, there is a wealth of evidence that selective mono-ubiquitination or alternative ubiquitin chains can regulate protein activity (38–42). Herein, WT-Rap2a and DA-Rap2a, but not DN-Rap2a (Fig. 4C) were robustly ubiquitinated by Nedd4-1 by K-63-linked ubiquitin chains (Fig. 4D), with inhibition of GTP-Rap2a activity (Fig. 4E). The present study was supported by the study of Kawabe et al (24), in which Nedd4-1 ubiquitinated Rap2a and inhibited GTP-Rap2a activity in neurons. Based on these findings, we speculate that Rap2a ubiquitination by Nedd4-1 may contribute to glioma pathogenesis.

Rap proteins belong to the Ras superfamily and have been implicated in cell cycle control, cell adhesion and cell migration (25,43,44). To date, the role of Rap1 in tumorigenesis and progression remains controversial, with some researchers hypothesizing that aberrant Rap1 activation promotes cancer cell proliferation and tumorigenesis (45–47) and others reporting that inactivation of DOCK4, a Rap1 activator, rendered osteosarcoma cells with a higher invasiveness (48), and the expression of Rap1GAP was correlated with increased invasion in vitro in squamous cell carcinoma (49). Similar to Rap1, the function of Rap2a remains elusive despite its history of cloning (50). In the present study, overexpression of Nedd4-1 promoted the migration and invasion of human glioma cell lines U251 and U87 via Rap2a ubiquitination (Fig. 5C and D). Furthermore, only WT-ubiquitin and K48R exhibited mono-ubiquitination (Fig.5G). Taken together, these findings confirm the hypothesis that Nedd4-1 regulates the migration and invasion of glioma cells via Rap2a ubiquitination. Three Rap2 proteins (Rap2a/b/c) have been cloned, but we only focused on the function of Rap2a protein. Additionally, E3 ubiquitin ligase was also found to be involved in the regulation of cell cycle, apoptosis and differentiation (51,52), which may illuminate our forthcoming investigation of glioma.

In summary, our findings suggest that Nedd4-1 plays a pivotal role in promoting the migration and invasion of glioma cell lines U251 and U87 via the inhibition of Rap2a activity, and may qualify as a candidate therapeutic target in glioma.

Acknowledgements

The authors are grateful to Dr Kenichi Kariya, Dr Nile Brose and Dr Kawabe Hiroshi for their benevolent donation of the Rap2a plasmids. We are also indebted to Mr. Pan Li from Xuzhou Medical College, for guidance in the style and manuscript editing. The present study was funded by the Natural Science Foundation of Jiangsu Province of China (BK20151165), and the National Natural Science Foundation of China (81472345).

Glossary

Abbreviations

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References