Glioblastoma is a diffusely growing malignant brain tumor and among the most aggressive of all tumors. Wilms' tumor 1-associating protein (WTAP) is a nuclear protein that has been associated with regulation of proliferation and apoptosis. Although its dynamic expression and physiological functions in vascular cells have been reported, those in other cells are largely unknown. Here, we show for the first time that WTAP is overexpressed in glioblastoma. Moreover we found that WTAP regulates migration and invasion of glioblatoma cells. Specific knockdown by siRNA or overexpression by cDNA regulated migration and invasion of cancer cells. In xenograft study, WTAP overexpression made cancer cells more tumorigenic. In the investigation for its underlying mechanism, we found that the activity of epidermal growth factor receptor can be regulated by WTAP. These results reveal a novel function of WTAP and suggest its clinical application.
Glioblastoma is a diffusely growing malignant brain tumor and among the most aggressive of all tumors.[1, 2] The overall survival time for patients is 15 months although they were treated with the current standard chemoradiotherapy. Primary glioblastoma, which accounts for most glioblastomas, develops rapidly with a clinical history of only a few days or weeks. This poor prognosis of glioblastoma may be ascribed to its high aggressiveness and invasiveness. Active cell migration and diffuse invasion into the surrounding tissue ultimately lead to tumor recurrence and patients' death. Molecular mechanisms underlying glioblastoma migration and invasion have been poorly characterized. Identification of molecules crucial for glioblastoma invasion will help improve the prognosis of patients.
Wilms' tumor 1-associating protein (WTAP) is a nuclear protein that associates with the Wilms' tumor 1 suppressor gene product, WT1. In addition to its association and interference with WT1,[5, 6] its involvement in RNA splicing[4, 7-9] and stabilization have been suggested. The importance of this protein is implied by knockout experiments. The WTAP-null and heterozygous mice died at E6.5 and at E10.5, respectively.[10, 11] Despite its ubiquitous expression, dynamic change in its expression has been described. When vascular smooth muscle cells shift from a synthetic, proliferative state to a nonproliferative and contractile state, its expression was substantially upregulated. Moreover, during the early period after an injury in rat carotid artery, the expression of WTAP is low; however, at the later period when smooth muscle cells stop to proliferate, its expression is significantly high.
Roles and expression of WTAP have been mainly described in vascular cells. In the present study, we found for the first time that WTAP was overexpressed in glioblastoma. Moreover, we showed that WTAP regulates migration and invasion of glioblastoma cells by controlling epidermal growth factor (EGF) signaling.
Materials and Methods
Glioblastom cells (U87MG cells and GBM05 cells) were cultured in DMEM (High Glucose; Thermo Fisher Scientific, Logan, UT, USA), supplemented with 10% FBS (Thermo Scientific, Albany, North Shore City, New Zealand) and 100 U/mL penicillin/streptomycin (Thermo Scientific) at 37°C and 5% CO2 incubator.
siRNA and transfection
Wilms' tumor 1-associating protein siRNA (Bioneer, Daejeon, Korea) and scrambled siRNA (SCR; Bioneer) were purchased. Cells were transfected with 100 nM of WTAP siRNA, or SCR siRNA with DharrmaFECT reagent (Dharrmacon, Lafayette, CO, USA) in accordance with the manufacturer's protocol. SCR siRNA was used as negative control. The sequences of WTAP siRNA duplex were as follows: 5′-CAG AUC UUA ACU CUA AUG A-3′, 5′-CCU UGU AAU GCG ACU AGC A-3′, and 5′-GAG AUG CAA GAG UGU ACU A-3′.
Cells were seeded into 96-well plate at a density of 3 × 103 per well allowed to attach for 24 h. Then, they were transfected with WTAP siRNA or SCR siRNA. Four days after transfection, 10 μL of water soluble tetrazolium salt (Ez-Cytox reagent, Daeillab, Seoul, Korea) was added into each well and incubated at 37°C for 2 h. Cell viability was measured at 450 nm using an ELISA reader (TECAN, Mannedorf, Swizerland).
Boyden chamber assay
A modified Boyden chamber (Neuro Probe, Gaithersburg, MD, USA) migration assay was used. The 8.0 μm pore polycarbonate filter was coated with type-I collagen (10 μL/mL). Two days after transfection with SCR or WTAP siRNA, Boyden chamber assays were performed. The detailed experimental procedure is described in Appendix S1.
The pore size of 8.0 μm of PET track-etched membranes were used. The upper surface was pre-coated with 0.5 mg/mL of Matrigel (BD Biosciences, San Jose, CA, USA) and lower surface was pre-coated with 0.5 mg/mL of fibronectin (Sigma-Aldrich, St. Louis, MO, USA). Two days after transfection with WTAP or SCR siRNA, invasion assays were performed. The detailed experimental procedure is described in Appendix S1.
The primary antibodies against anti-WTAP (1:5000, Sigma-Aldrich), phospho-EGFR (1:1000, Cell Signaling, Danvers, MA, USA), total EGFR (1:1000, Cell Signaling), phospho-AKT (1:1000, Cell Signaling), and total AKT (1:1000, Cell Signaling) were used for western blotting. The detailed experimental procedure is described in Appendix S1.
Overexpression of WTAP in U87MG cells
pΔEGFP-N1-WTAP (variant 1, Origene, Rockville, MD, USA) was used to generate U87MG cells stably overexpressing WTAP (U87 + WTAP cells). Transfection was performed by using FuGENE HD transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer's protocol. G418 (140 μg/mL) was included as a selection antibiotic.
Patients' tissues for real time PCR were provided by the Chonnam National University Hwasun Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The detailed experimental procedure is described in Appendix S1.
Glioblastoma tissues for immunohistochemistry were obtained with informed consent from patients who underwent surgical resection at Pusan National University Hospital. The study was approved by the institutional review boards of the hospital. The following primary antibodies were used: anti-WTAP (1:300, Sigma-Aldrich), anti-GFAP (Glial Fibrillary Acidic Protein) (1:200, Merck Chemicals, Nottingham, UK), and anti-CD45 (1:50, BD Biosciences). The detailed experimental procedure is described in Appendix S1.
Cells (5 × 106 cells, mixed with Matrigel) stably expressing WTAP or control vector, were injected subcutaneously into nude mice (n = 6). Hundred percentage of injected mice formed tumors. Tumor volumes were measured every week from week 3 to week 6 and calculated by the following formula: 0.5236 × L1 × (L2)2, where L1 is the long axis and L2 is the short axis of the tumor. Six weeks later, mice were killed. Procedures related to animal care were in accord with the guideline of the “Guideline for the Welfare of Animals in Experimental Neoplasia”. The Pusan National University Institutional Animal Care and Use Committee (PNUIACUC) approved the experimental procedures.
Total RNA was extracted from U87 + WTAP and U87 + Mock cells using RNeasy Mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's protocol. Quantified RNA was then used for microarray analysis on Human HT-12_v4_Bead chips (Illumina, San Diego, CA, USA). The rest of the experimental procedure is described in Appendix S1.
All data are presented as means ± SD. All experiments were repeated at least four times. The difference between the mean values of two groups was evaluated using the Student's t-test (unpaired comparison). For comparison of more than three groups, we used one-way anova test followed by Tukey's multiple comparison. A P-value of <0.05 was considered statistically significant.
WTAP is overexpressed in glioblastoma
To determine expression status of WTAP in glioblastoma, we performed immunohistochemistry using patients' tissues (n = 10). In contrast to low level of expression in normal brain tissues, high level of expression is observed in glioblastoma tissues (Fig. 1a). To confirm whether WTAP-positive cells are glioblastoma cells, we stained serial sections for GFAP, which was known to be expressed in glioblastoma cells, and CD45, a marker for leukocytes. Most WTAP-positive cells were GFAP-positive, however CD45-negative (Fig. 1a). To quantitate the expression of WTAP in glioblastoma tissues, real time PCR was performed using specific primers for WTAP. Many, but not all glioblastoma tissues overexpressed it compared to normal brain tissues (Fig. 1b).
Role of WTAP in proliferation of glioblastoma cells
To examine roles of WTAP in glioblastoma, we knocked-down or overexpressed WTAP using specific siRNA or cDNA, respectively. We checked the efficiency of the modulation by western blotting. WTAP siRNA (100 nM) decreased the protein level of WTAP in U87MG cells and in GBM05 cells compared to SCR RNA by 46.1% and 56.7%, respectively (Fig. 2a,b). The protein level of WTAP in the stable cells overexpressing WTAP is increased compared to mock cells by 79.4% (Fig. 2c,d). Subsequently, we examined roles of WTAP in proliferation of glioblastoma cells. Four days after transfection with SCR siRNA (100 nM) or WTAP siRNA (100 nM), cell survival assay was performed. As shown in Figure 2, WTAP knockdown did not affect proliferation in the presence of 10% FBS in the culture media. However, in the presence of 1% FBS, WTAP knockdown decreased proliferation of U87MG cells by 53.3% although it did not significantly affect proliferation of GBM05 cells (Fig. 2f). Overexpression of WTAP in U87MG cells increased the proliferation by 27.6% compared to control vector.
WTAP regulates migration of glioblastoma cells
To determine the role of WTAP in migration of glioblastoma cells, we carried out studies using a Boyden chamber assay. Two days after siRNA transfection, Boyden chamber assays were performed as described in the Materials and Methods section. To remove effects of proliferation, mitomycin C (0.01 μg/mL, Sigma) was added. As shown in Figure 3, WTAP siRNA (100 nM) reduced FBS- and EGF-induced migration of U87MG cells compared to SCR siRNA by 50.5% and 79.2%, respectively (Fig. 3a,b). Moreover, WTAP siRNA (100 nM) reduced FBS- and EGF-induced migration of GBM05 cells compared to SCR RNA by 40.7% and 91.2%, respectively (Fig. 3c). Furthermore, overexpression of WTAP in U87MG cells increased FBS-induced migration compared to control vector by 159.8% (Fig. 3d,e). These results suggest critical roles of WTAP in migration of glioblastoma cells.
WTAP regulates invasion of glioblastoma cells
Next, we examined the role of WTAP in invasion of glioblastoma cells using a Matrigel invasion assay. Two days after siRNA transfection, Matrigel invasion assays were carried out as described in the Materials and Methods section. To remove the effects of proliferation, mitomycin C (0.01 μg/mL, Sigma) was added. As shown in Figure 4, WTAP siRNA (100 nM) reduced FBS- and EGF-induced invasion of U87MG cells compared to SCR siRNA by 60.4% and 47.6%, respectively. Moreover, WTAP siRNA (100 nM) reduced FBS-induced invasion of GBM05 cells compared to SCR RNA by 29.3% (Fig. 4c). Furthermore, overexpression of WTAP in U87MG cells increased FBS-induced invasion compared to control vector by 153.6% (Fig. 4d,e). These results suggest critical roles of WTAP in invasion of glioblastoma cells.
WTAP increases tumorigenicity of glioblastoma cells in xenograft
Next, we examined the effects of WTAP overexpression on in vivo behavior of glioblastoma cells. Glioblastoma cells overexpressing WTAP or control vector were subcutaneously injected into nude mice and tumor growth was measured. Notably, the growth rate of xenograft was significantly faster in WTAP overexpression group compared to control group (Fig. 5).
WTAP regulates activity of EGFR
How does WTAP regulate migration and invasion of glioblastoma cells? To reveal the underlying mechanism, we examined the activity of epidermal growth factor receptor (EGFR) because roles of EGF signaling are well established in migration and invasion of cancer cells. One day after transfection with SCR or WTAP siRNA (100 nM), cells were subjected to serum starvation for one day. Treatment with EGF (100 ng/mL) for 10 min significantly increased the level of phospho-EGFR (Fig. 6) in control cells. However, WTAP siRNA inhibited the EGF-induced phosphorylation of EGFR compared to SCR siRNA in U87MG or GBM05 cells by 43.4% or 24.0%, respectively, (Fig. 6). Moreover, overexpression of WTAP enhanced the EGF-induced phosphorylation of EGFR compared to control vector by 29.7%. Total amount of EGFR was not significantly changed by WTAP knockdown or overexpression. Because EGFR can enhance the activity of AKT, which plays important roles in migration of cancer cells, we examined the phosphorylation status of AKT. The phosphorylation of AKT was also regulated by WTAP in a similar pattern with that of EGFR (Fig. 6).
WTAP-induced changes in mRNA expression
To reveal other possible mechanisms of WTAP-regulated motility, we performed a cDNA microarray. Expressions of many genes that are involved in migration of cancer cells were changed by the overexpression of WTAP (Table 1). Among the significantly changed genes by WTAP, we focused on chemokine ligand 2 (CCL2), chemokine ligand 3 (CCL3), hyaluronan synthase 1 (HAS1), lysyl oxidase-like 1 (LOXL1), matrix metallopeptidase 3 (MMP3), and thrombospondin 1 (THBS1) because these genes have been significantly associated with motility of cancer cells. Real-time PCR was used to confirm significant changes in mRNA levels of each gene (Fig. 7). Overexpression or knockdown of WTAP increased or decreased the mRNA levels of these genes, respectively (Fig. 7).
Table 1. Wilms' tumor 1-associating protein (WTAP)-induced change in gene expression
FC, fold change.
Migration and invasion of cancer cells greatly affect prognosis of glioblastoma patients. So, understanding the molecular mechanism underlying motility of glioblastoma cancer cells can contribute to development of new diagnostics and therapeutics. In the present study, we showed for the first time that WTAP is overexpressed in glioblatoma and regulate motility of glioblastoma cells. Moreover, we also showed that the regulation of motility by WTAP is mediated by controlling the activity of EGFR.
A novel role of WTAP in regulation of migration and invasion of glioblastoma cells was demonstrated for the first time in the present study. Previous studies have shown main roles of WTAP in cell biology were regulation of proliferation and apoptosis.[6, 10, 14] Boyden chamber chemotaxis assay and Matrigel invasion assay in the present study showed that knockdown or overexpression of WTAP decreased or increased migration and invasion of glioblastoma cells, respectively.
How can WTAP regulate migration and invasion of cancer cells in glioblastoma? In the present study, we found that WTAP can regulate the activity of EGFR. Accordingly, the activity of AKT was also regulated by WTAP in a similar pattern. However the activity of EGFR was only partially regulated by WTAP siRNA, suggesting that the activity of EGFR plays a minor role in WTAP-regulated migration. Epidermal growth factor receptor can be regulated at several levels: expression, degradation and activity. Overexpression of EGFR occurs in 40–60% of primary glioblastoma by chromosomal amplification.[15-18] Degradation of EGFR can be regulated by cbl protein, whose overexpression promoted ubiquitination and degradation of EGFR. However, in the present study, expressions of ErbB family members were not significantly changed by overexpression or knockdown of WTAP (data not shown). Activity of EGFR can be regulated by modulator proteins including herstatin, mitogen-inducible gene 6 (Mig-6) and fibroblast growth factor receptor substrate 2 β (FRS2β) without change in the amount of EGFR protein. In future studies, how the activity of EGFR is regulated by WTAP needs to be further studied.
Although the activity of EGFR was inhibited by WTAP siRNA, the inhibition was not complete (Fig. 6b,d,f). Through the cDNA microarray experiments, we found several candidate genes (CCL2, CCL3, HAS1, LOXL1, MMP3, THBS1) that may mediate effects of WTAP (Table 1, Fig. 7). Expressions of those genes were increased or decreased by cDNA overexpression or siRNA knockdown of WTAP, respectively (Fig. 7). Chemokines including CCL2 and CCL3 have been shown to promote migration of cancer cells[22, 23] although they have been originally reported to be involved in migration of leukocytes. Interestingly a role of CCL2-chemokine receptor 2 (CCR2) interaction was suggested in glioblastastoma. Extracellular matrix components and related proteins are critical for migration and invasion of cancer cells. LOXL, which oxidizes lysine residues in collagens and elastin, can promote metastasis through active remodeling of tumor microenvironment. Hyaluronic acid family proteins including HAS1 have been shown to regulate metastasis. In glioma cells, hyaluronic acid affected their migration. Thrombospondin 1 is a multifunctional matrix protein implicated in migration of cancer cells. Malignant glioma cells can secrete THBS1, which regulates their motility.
Wilms' tumor 1-associating protein played some minor roles in proliferation of U87MG glioblastoma cells in the present study. In the presence of 1% FBS, knockdown of WTAP decreased proliferation of U87MG cells. However, in the presence of 10% FBS, we did not observe the significant decrease in proliferation (Fig. 2). Moreover, knockdown of WTAP did not affect proliferation of GBM05 cells (Fig. 2). Less inhibition of EGFR activity by WTAP siRNA in GBM05 than U87MG cells (Fig. 6) may explain the difference in effects of WTAP on proliferation. However, the inhibition of EGFR activity by WTAP is not complete. So, it is difficult to explain different effects of WTAP on proliferation simply using the effects on EGFR activity. Therefore, other mechanisms may be involved in regulation of proliferation by WTAP. The previous studies suggested the association of WTAP with cell proliferation by its expression pattern. However its roles in cell proliferation are controversial depending on cell type. In the vascular smooth muscle cell, WTAP negatively regulates proliferation. Knockdown of its expression by RNA interference increased DNA synthesis and proliferation. In contrast, its overexpression showed opposite effects. However, in endothelial cells, overexpression of WTAP did not change cell proliferation. Moreover, its knockdown decreased cell proliferation. These results suggest that the role of WTAP in proliferation might be cell-type specific.
Overexpression of WTAP in glioblatoma was found for the first time in the present study. Although an early study showed that WTAP is widely expressed in adult tissues including brain, thymus, heart, lung, liver, spleen and skeletal muscle, its dynamic change in expression was reported in vascular smooth muscle cells. In the present study, we observed the basal expression of WTAP in normal human brain tissues. However, overexpression of WTAP was clearly observed in glioblastoma tissues. In future study, how the expression of WTAP in glioblastoma tissues is regulated needs to be studied. Moreover, its relationship with patients' prognosis needs to be investigated.
This work was supported by Medical Research Institute Grant (2009-23), Pusan National University Hospital, the medical research centre program of Ministry of Education, Science and Technology/Korea Science and Engineering Foundation (2011-0006190), a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0920050). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.