Open Access

TIGAR promotes growth, survival and metastasis through oxidation resistance and AKT activation in glioblastoma

  • Authors:
    • Zhi Tang
    • Zhengwen He
  • View Affiliations

  • Published online on: July 5, 2019     https://doi.org/10.3892/ol.2019.10574
  • Pages: 2509-2517
  • Copyright: © Tang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Glioblastoma has a poor prognosis and is one of the most lethal types of cancer in the world. TP53 induced glycolysis regulatory phosphatase (TIGAR) is upregulated in various types of cancer. Therefore, the present study investigated the role of TIGAR in glioblastoma. TIGAR expression was measured in glioma samples and cell lines using immunohistochemistry and western blotting. Reduced nicotinamide adenine dinucleotide phosphate (NADPH), glutathione, malondialdehyde and intracellular reactive oxygen species levels were detected to measure oxidative stress in U‑87MG cells following short hairpin RNA (shRNA)‑mediated knockdown of TIGAR. Cell viability was determined using an MTT assay for TIGAR‑overexpression vector‑ and TIGAR‑shRNA‑transfected U‑87MG cells. Apoptosis was assessed to evaluate whether TIGAR knockdown sensitized cells to the antitumor effects of temozolomide (TMZ). Migration, invasion and epithelial‑mesenchymal transition (EMT) were further assessed using Transwell and western blotting assays. A co‑immunoprecipitation assay was used to detect the interaction between TIGAR and protein kinase B (AKT). The results of the present study revealed that TIGAR was positively associated with poor survival and was upregulated in glioblastoma. TIGAR knockdown significantly increased oxidative stress, decreased cell proliferation and exacerbated TMZ‑induced apoptosis in U‑87MG cells. Additionally, TIGAR knockdown decreased migration, invasion and EMT, and treatment of TIGAR‑shRNA‑transfected cells with NADPH had no effect on metastasis. In addition, TIGAR promoted AKT activation and bound to AKT. In conclusion, the present study demonstrated that TIGAR may promote glioblastoma growth and progression through oxidation resistance and AKT activation.

Introduction

Glioblastoma is the most common type of malignant brain cancer in adults around the world (1). Despite the rapid development of surgical techniques and radiotherapy, and the widespread use of chemotherapy drugs, such as temozolomide (TMZ), glioblastoma continues to be associated with poor prognosis, with a 5-year survival rate of 5–13%, along with a high recurrence rate (2). Therefore, it is important to explore the molecular mechanisms underlying the occurrence and progression of glioblastoma, and to identify novel therapeutic targets to improve glioblastoma treatment and prognosis.

TP53 induced glycolysis regulatory phosphatase (TIGAR) is located on chromosome 12p13.3 and includes six coding exons and two p53 binding sites (3). A previous study has demonstrated that TIGAR is similar to phosphoglucose mutagenesis enzyme and has biphosphatase activity to decompose fructose 2,6-bisphosphate (FB) (4). FB is the allosteric activator of phosphofructose kinase (PFK). Therefore, TIGAR inhibits PFK activity by decreasing FB and directs metabolic flow from glycolysis to the pentose phosphate pathway (PPP) (4). The PPP provides phosphate ribose for nucleic acid synthesis. Reduced nicotinamide adenine dinucleotide phosphate (NADPH), as a byproduct of the PPP, is the main reactive oxygen species (ROS) scavenger in cells. Additionally, TIGAR is overexpressed in several types of cancer, including leukemia (5), lung cancer (6), breast cancer (7), liver cancer (8) and colon cancer (9). TIGAR is considered to protect cancer cells against ROS-induced apoptosis and to induce DNA damage repair (10).

To the best of our knowledge, a role for TIGAR in glioblastoma has not been reported. The present study revealed that TIGAR was overexpressed in glioblastoma and is a potential novel prognostic and migration marker in patients with glioblastoma. Additionally, TIGAR decreased oxidative stress in glioblastoma cells through PPP-mediated NADPH generation. The present study demonstrated that TIGAR knockdown led to significant inhibition of proliferation, migration and invasion in U-87MG cells in an oxidative stress-independent manner. In addition, TIGAR interacted with protein kinase B (AKT) and promoted AKT activation. The results of the present study suggested that TIGAR, as an important mediator of glioma progression, may be a potential therapeutic target in glioblastoma.

Materials and methods

Reagents

Bovine serum albumin (BSA), NADPH (cat. no. ST360), dichloro-dihydro-fluorescein diacetate (DCFH-DA; cat. no. S0033-1) and rabbit immunoglobulin G (IgG; cat. no. A7016) were purchased from Beyotime Institute of Biotechnology. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific, Inc. Lipofectamine® 2000 reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The GTVisin™ anti-mouse/anti-rabbit immunohistochemical analysis kit was purchased from Gene Company, Ltd. Anti-TIGAR (cat. no. sc-166290; 1:1,000 dilution), anti-B cell lymphoma 2 (Bcl2; cat. no. sc-509; 1:1,000 dilution), anti-Bcl2-associated X protein (BAX; cat. no. sc-20067; 1:1,000 dilution), anti-α-smooth muscle actin (α-SMA; cat. no. sc-53142; 1:1,000 dilution), anti-E-cadherin (cat. no. sc-71009; 1:1,000 dilution), anti-N-cadherin (cat. no. sc-59987; 1:1,000 dilution), anti-Snail (cat. no. sc-271977; 1:1,000 dilution), anti-Vimentin (cat. no. sc-80975; 1:1,000 dilution) and HRP-conjugated goat anti-mouse IgG (m-IgGκ BP-HRP; cat. no. sc-516102; 1:4,000 dilution) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphoinositide 3-kinase (PI3K; cat. no. ab191606; 1:1,000 dilution) and anti-phosphorylated (p)-PI3K p85 α (cat. no. ab182651; 1:1,000 dilution) were obtained from Abcam. Anti-AKT (cat. no. 4685; 1:1,000 dilution), anti-p-AKT (Ser473; cat. no. 4060; 1:1,000 dilution), anti-β-Actin (cat. no. 3700; 1:3,000 dilution) and HRP-conjugated goat anti-rabbit IgG (cat. no. 7074; 1:4,000 dilution) antibodies, as well as a mouse IgG control (cat. no. 5415), rabbit IgG control (cat. no. 3900) and radioimmunoprecipitation assay (RIPA) buffer (cat. no. 9806), were purchased from Cell Signaling Technology, Inc. Dimethyl sulfoxide (DMSO), isopropanol, ethanol and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd.

Cell culture

The human glioblastoma cell lines LN-18, LN-229, U-87MG, U-251MG and SNB-19 were purchased from the Shanghai Institute of Biochemistry and Cell Biology and were authenticated by short tandem repeat profiling. The U-87MG American Type Culture Collection cell line used in the present study is most likely a glioblastoma cell line, but of unknown origin. The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.) without any antibiotics, supplemented with 10% FBS (Hangzhou Sijiqing Bio-engineering Material Co.) at 37°C in a humidified atmosphere with 5% CO2.

RNA interference and transfection

A total of 2×104 U-87MG cells were infected with 4 µl of 1×109 viral particles of LV-TIGAR-short hairpin RNA (shRNA; 5′-GATTAGCAGCCAGTGTCTTAG-3′) or negative control (nc)-shRNA (5′-TTACCGAGACCGTACGTAT-3′), which were synthesized by Shanghai GenePharma Co., Ltd. After 2 days, the transfected cells were selected for using DMEM containing 2 µg/ml puromycin in subsequent experiments.

pcDNA3.1 and pcDNA3.1-TIGAR (human) were purchased from Cyagen Biosciences, Inc. A total of 1.8×105 U-87MG cells in 6-well plates were transfected with 2 µg of either plasmid using 4 µl of Lipofectamine® 2000, and complete medium was added to the cells 6 h after transfection. After further culturing for 36 h, subsequent experiments were performed. For TIGAR dependent AKT activation, 1.8×105 U-87MG cells in 6-well plates were transfected with 1, 2 or 3 µg pcDNA3.1-TIGAR using 4 µl of Lipofectamine® 2000, and complete medium was added to the cells 6 h after transfection. After a further 36 h, cells were lysed, and AKT and p-AKT were detected using western blot assay.

Cell viability

U-87MG/NC cells, U-87MG/sh-TIGAR cells and U-87MG cells transfected with pcDNA3.1 or pcDNA3.1-TIGAR were seeded into a 96-well culture plate at a density of 3×104 cells/well. Cell proliferation was measured at 24, 48, 72 and 96 h. MTT (1 mg/ml dissolved in PBS; 100 µl/well; Sigma-Aldrich; Merck KGaA) was added, and the cells were cultured for 4 h at 37°C prior to adding 100 µl DMSO. The absorbance was determined using a multiplate reader (SpectraMax 190; Molecular Devices, LLC) at a wavelength of 570 nm. For temozolomide (TMZ)-induced cellular toxicity, cells were treated with 100 µM of TMZ (Selleck, cat. no. S1237) for 24 h and apoptosis was detected using western blotting.

Colony formation assay

U-87MG/NC, U-87MG/sh-TIGAR cells, U-87MG/pcDNA3.1 and U-87MG/pcDNA3.1-TIGAR cells (200 cells/well) were plated into a 6-well plate and cultured for 14 days at 37°C. The media were then removed, and cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 1% crystal violet in 2% ethanol at room temperature for 20 min. Visible colonies were counted by eye.

Western blotting assay

Total protein was extracted from whole cell lysates using RIPA buffer, and the protein concentration was measured using a bicinchoninic acid assay. The protein samples (30 µg/lane) were separated by 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore) at 300 mA for 90 min. Subsequently, the membranes were blocked at room temperature for 1 h with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% dry milk and incubated overnight with primary antibodies at 4°C. Membranes were washed three times with TBST and incubated for 2 h with secondary antibodies at room temperature. After washing for three times with TBST, blots were detected using enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, Inc) and captured by a Luminescent Image Analyzer LAS-3000 (Fujifilm Holdings Corporation), and the optical densities of antibody-specific bands were analyzed using ImageJ version 1.37 (National Institutes of Health).

Co-immunoprecipitation (Co-IP)

Cells were harvested by centrifugation at 500 × g for 5 min at 4°C and lysed in RIPA lysis buffer for 30 min. Following centrifugation at 13,000 × g for 15 min 4°C, the 1/10 volume of supernatant was collected as input, the 1/2 volume of remaining supernatant was incubated with 1 µg anti-TIGAR (1:200 dilution) or anti-AKT (1:100 dilution) antibody and 40 µl 50% protein A/G agarose slurry at 4°C overnight. The other remaining supernatant was incubated with 1 µg control IgG (1:200 dilution) and 40 µl 50% protein A/G agarose slurry at 4°C overnight. The protein A/G agarose was recovered by centrifugation at 3,000 × g for 5 min at 4°C and washed four times with ice-cold lysis buffer. Proteins were eluted with 2× loading buffer by boiling for 10 min and subjected to immunoblot analysis according to the aforementioned western blotting assay.

ROS, NADPH, glutathione (GSH) and malondialdehyde (MDA) detection

Intracellular ROS were determined using DCFH-DA. A total of 4×105 cells were washed with 0.01 M PBS and incubated with 20 µM DCFH-DA at 37°C for 30 min. The cells were visualized using an inverted fluorescence microscope (magnification, ×20; Olympus Corporation), and their fluorescence intensity was measured via fluorescence spectrometry (Spectra MaxGemini; Molecular Devices LLC). An NADPH detection kit (cat. no. ECNP-100) was obtained from BioAssay Systems, and GSH (cat. no. S0053) and MDA (cat. no. S0131) were purchased from Beyotime Institute of Biotechnology. The NADPH level, GSH content and MDA level were measured according to the manufacturers' protocols.

Migration and invasion

A Transwell plate (Costar; Corning Inc.) with an 8-µm pore insert was utilized to measure migration and invasion. DMEM containing 10% FBS (0.6 ml) was added to the lower chamber. A total of 5×104 U-87MG/nc or U-87MG/sh-TIGAR cells in serum-free DMEM were directly added to the upper chamber and incubated at 37°C for 12 h for the migration assay. To measure invasion, 100 µl diluted Matrigel (1 mg/ml; BD Biosciences) in serum-free cold DMEM was placed in the upper chamber and in the lower chamber, DMEM containing 15% FBS was added, and the cells were incubated at 37°C for 4 h to allow it to set. A total of 5×104 U-87MG/nc or U-87MG/sh-TIGAR cells in serum-free DMEM were added directly to the upper chamber and incubated at 37°C for 24 h. Cells in the lower chamber were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 1% crystal violet in 2% ethanol at room temperature for 20 min. The numbers of cells in the lower chamber were counted under a light microscope (magnification, ×20). At least five fields were analyzed per section.

Immunohistochemistry

Immunohistochemistry was used to detect TIGAR expression levels in surgical resections of glioblastoma tissues collected from 15 male patients (45–55 years old) first diagnosed and admitted to Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine (Changsha, China) between January 2018 and May 2018. The use of glioblastoma samples was approved by the Ethics Committee of Hunan Cancer Hospital and Affiliated Cancer Hospital of Xiangya School of Medicine (no. 20180104). Written informed consent was obtained from all patients prior to enrollment.

The glioblastoma sections were fixed in 4% paraformaldehyde for 2 days at room temperature followed by paraffin-embedding. From the embedded tissue, 5-µm thick sections were cut and deparaffinized in dimethylbenzene, rehydrated in ethanol solutions of 100, 95, and 70% ethanol, and subsequently washed in PBS for 10 min each. Antigen retrieval was performed at 95°C for 20 min, followed by washing with PBS for three times and blocking with 3% BSA for 1 h at room temperature. Sections were incubated with an anti-TIGAR antibody (1:100 dilution) at room temperature for 2 h, and the slides were washed three times with PBS and incubated with components of the GTVisin™ anti-mouse/anti-rabbit immunohistochemical analysis kit, according to the manufacturer's protocols. Glioblastoma tissues and paired normal-appearing tissues in sections were confirmed by Pathology department. Images were captured under a light microscope (magnification, ×20). At least five fields were analyzed per section and quantify staining was analyzed using ImageJ version 1.37 (National Institutes of Health)

Bioinformatics

Kaplan-Meier survival analysis between TIGAR high expression and low expression in glioma was carried out. Overall survival time based on GSE4412-GPL96 dataset in PrognoScan was performed on PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/) (11). A total of 74 patients were divided into TIGAR high expression group (n=36) and TIGAR low expression group (n=38) and Cox P-value (P=0.007189) was generated automatically.

Statistical analysis

Experimental data are expressed as the mean ± standard deviation of at least three independent experiments. Statistical analyses were performed using SPSS v18.0 statistical software (SPSS, Inc.). Paired or unpaired Student's t-test were used to compare the significance between two paired groups and two independent groups respectively, and a one-way ANOVA followed by a post-hos Tukey's test was used to determine the significance between three or more independent groups. P<0.05 was considered to indicate a statistically significant difference.

Results

TIGAR is overexpressed in glioblastoma tissues and positively associated with poor prognosis

It has been reported that TIGAR is upregulated in several types of tumor (59). To determine whether TIGAR was upregulated in glioblastoma, glioblastoma sections were obtained and subjected to immunohistochemistry. As shown in Fig. 1A and B, TIGAR was significantly overexpressed in glioblastoma tissues compared with paired normal-appearing tissues. TIGAR expression in glioblastoma cell lines, including LN-18, LN-229, U-87MG, U-251MG and SNB-19, was detected. The results revealed that TIGAR was overexpressed in U-87MG cells (Fig. 1C). Furthermore, PrognoScan-based Kaplan-Meier survival analysis (11) revealed an association between elevated TIGAR expression levels and shorter surv]ival duration in the 74 patients with glioblastoma from the database. (Fig. 1D). These results indicated that TIGAR was overexpressed in glioblastoma, and that its expression was negatively associated with survival time.

TIGAR maintains NADPH to alleviate oxidative stress in U-87MG cells

It has been reported that TIGAR promotes NADPH generation through PPP (3). The ratio of NADPH to NADP+ in TIGAR-knockdown U-87MG cells was measured. As shown in Fig. 2A, TIGAR content was decreased in TIGAR-knockdown cells. As shown in Fig. 2B, TIGAR knockdown significantly decreased NADPH content. Glutathione reductase catalyzes the NADPH-driven reduction of oxidized glutathione (GSSG) to GSH (12). Therefore, the ratio of GSH to GSSG was measured in TIGAR-knockdown U-87MG cells. The conversion of GSH from GSSG was significantly reduced in TIGAR-knockdown cells (Fig. 2C). NADPH and GSH are involved in cellular antioxidant responses. Levels of MDA (13), a byproduct of nonenzymatic lipid peroxidation and a principal marker of oxidative stress, were assessed. It has been reported that NADPH addition can protect against DNA damage in TIGAR-knockdown cells (14). Similarly, in the present study, it was observed that TIGAR knockdown significantly increased MDA levels in groups without NADPH treatment, and NADPH inhibited MDA content in TIGAR-knockdown U-87MG cells compared with negative control U-87MG cells although these changes were not significant (Fig. 2D). Additionally, TIGAR knockdown increased intracellular ROS in U-87MG cells compared with the negative control U-87MG cells, as detected by higher fluorescence following incubation with DCFH-DA. The elevated ROS levels in TIGAR-knockdown U-87MG cells were attenuated by the addition of NADPH (Fig. 2E and F). These results suggested that TIGAR maintained the NADPH level to protect U-87MG cells from oxidative stress.

TIGAR promotes proliferation and inhibits apoptosis in U-87MG cells

To test the effect of TIGAR in glioblastoma, U-87MG cells were transfected with pcDNA3.1-TIGAR, and viability rates were measured. As shown in Fig. 3A-B, TIGAR content was overexpressed after transfection, and TIGAR overexpression significantly increased U-87MG cell viability, and the numbers of cell clones in TIGAR-overexpressing U-87MG cells were higher than in control U-87MG cells (Fig. 3C). The viability of TIGAR-knockdown U-87MG cells was measured, and cell viability was decreased in TIGAR-knockdown U-87MG cells. To ascertain whether the decreased viability of TIGAR knockdown cells was associated with oxidative stress, exogenous NADPH was added. The results revealed that added NADPH did not increase cell viability in TIGAR-knockdown U-87MG cells (Fig. 3D). Additionally, the formation of cell clones was inhibited in TIGAR-knockdown U-87MG cells, and additional NADPH did not promote cell growth (Fig. 3E). TMZ-based therapy is the standard of care for patients with glioblastoma, and resistance to TMZ in glioblastoma is a universal phenomenon (15). TIGAR knockdown promoted the TMZ-induced decrease in Bcl2 and increase in BAX protein expression levels, which was indicative of an increase in apoptosis. Furthermore, NADPH significantly alleviated apoptosis in the TMZ-treated U-87MG cells and TIGAR-knockdown U-87MG cells (Fig. 3F). The results indicated that TIGAR promoted viability, and decreased TMZ-induced apoptosis through NADPH-mediated antioxidative activity in U-87MG cells.

TIGAR promotes metastasis in U-87MG cells

Glioblastoma is an aggressive intracranial tumor (16). The number of migratory cells in the Transwell assay was significantly lower in TIGAR-knockdown U-87MG cells, and addition of NADPH did not promote cell migration (Fig. 4A). Additionally, the number of invasive cells was significantly reduced in TIGAR-knockdown U-87MG cells; addition of NADPH failed to promote cell invasion in U-87MG cells (Fig. 4B). The migration and invasion of cancer cells are associated with the epithelial-mesenchymal transition (EMT) (17). Therefore, expression levels of EMT indicators, including N-cadherin, snail, E-cadherin, α-SMA and vimentin, were assessed. As shown in Fig. 4C, significantly increased E-cadherin, and decreased N-cadherin, α-SMA, snail and vimentin expression levels were observed in TIGAR-knockdown cells, indicating that metastasis was decreased in TIGAR-knockdown U-87MG cells. Notably, the addition of NADPH had no effect on the metastasis of TIGAR-knockdown U-87MG cells, which highlighted the pro-metastatic effect of TIGAR beyond NADPH production in glioblastoma.

TIGAR promotes AKT phosphorylation and interacts with AKT in U-87MG cells

The PI3K/AKT signaling pathway is activated in glioblastoma (18). The activation of AKT promotes cancer growth and metastasis and inhibits autophagy and apoptosis (19). Therefore, PI3K/AKT activation was assessed in TIGAR-knockdown and TIGAR-overexpressing U-87MG cells. As shown in Fig. 5A, no significant differences between total PI3K and p-PI3K levels were identified in TIGAR-knockdown and TIGAR-overexpressing U-87MG cells compared with their respective controls, whereas p-AKT was significantly decreased in TIGAR-knockdown cells and increased in overexpressing cells. To determine whether TIGAR promoted AKT activation in U-87MG cells, TIGAR was transfected into U-87MG cells in a dose-dependent manner. The present study demonstrated that p-AKT/AKT markedly increased as TIGAR expression increased (Fig. 5B). Additionally, Co-IP was performed to verify the interaction between TIGAR and AKT. As shown in Fig. 5C, TIGAR interacted with AKT in U-87MG cells. These results indicated that TIGAR promoted AKT activation and interacted with AKT in glioblastoma.

Discussion

Glioma is a type of tumor that occurs in the brain, and 75% of gliomas are astrocytomas (20). Glioblastoma represents the highest grade of astrocytoma, and the overall 5-year survival rate is only 5% (21). Furthermore, glioblastoma is associated with migration, invasion and chemotherapy resistance (22).

TMZ, a second-generation oral alkylating agent that causes DNA damage via methylation of the O(6) position of guanine, is commonly used to treat human malignant glioma. O(6)-methylguanine-DNA methyltransferase mitigates the effectiveness of TMZ and has been used as a marker to predict the efficacy of TMZ treatment (23). In addition, DNA damage response activates p53. However, the high incidence of TP53 mutations leads to p53 loss the function to prevent tumor formation in cancers (24). As a well-known tumor suppressor, the prevailing function of p53 is the transcriptional control of target genes that regulate numerous cellular processes, including the cell cycle, apoptosis, autophagy and metabolism (25). TMZ treatment induces p53 activation and subsequent upregulation of p21, Noxa and BAX (26).

As a downstream target protein of p53, TIGAR expression is dependent on p53, whereas p73, p63, hypoxia inducible factor 1 and Sp1 transcription factor also promote TIGAR expression (27,28). A number of studies have demonstrated that chemotherapy is accompanied by increased TIGAR expression, and TIGAR-derived NADPH protects cancer cells from chemotherapy-induced damage (29,30). Additionally, nuclear localized TIGAR exhibits antioxidant properties, and provides ribose-5-phosphate for DNA repair following epirubicin treatment (31). TIGAR is localized in the cytoplasm, endoplasmic reticulum, and the mitochondrial membrane and matrix. In addition to nuclear translocation, TIGAR may translocate to mitochondria under hypoxia in cancer (28). Following cerebral ischemia reperfusion in mice, increased levels of TIGAR are observed in the mitochondrial membrane and matrix, where TIGAR maintains the mitochondrial membrane potential under oxidative stress conditions (32). Therefore, effects of TIGAR beyond oxidation resistance, and the cellular distribution of TIGAR, require further investigation.

The PI3K/AKT signaling pathway serves an important role in the occurrence and development of tumors. This pathway can be activated by various factors, including platelet-derived growth factor, epidermal growth factor and insulin-like growth factor, to promote cell proliferation, differentiation, adhesion and migration, and inhibit apoptosis. Tumor biology studies have mainly concentrated on Class IA PI3K, which is composed of a heterodimer comprising a p110 catalytic subunit and a p85 regulatory subunit (33,34). PI3K binds to the upstream tyrosine receptor kinase via the SH2 region of p85, causing activation of PI3K. Additionally, PI3K is activated by G protein-coupled receptor (GPCR)-activated Ras upon stimulation with the GPCR ligand cyclic adenosine monophosphate (35). Once PI3K is activated, its activated substrates phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) and PI (3,4,5)-trisphosphate (PIP3) act as second messengers to activate and form a signaling cascade complex, leading to the phosphorylation of AKT.

PI3K phosphorylates AKT at Thr308 and Ser473 to activate AKT. PI3K catalyzes the phosphorylation of PI 4-phosphate and PI(4,5)P2 at their third positions, and converts them into PI(3,4)P2 and PIP3 to recruit AKT (36). AKT can be directly activated by PI(3,4)P2. However, PIP3 activates phosphoinositide-dependent kinase (PDK)-1 to phosphorylate AKT at Thr308, and AKT is further phosphorylated at Ser473 and fully activated in the presence of PDK-2. The tumor suppressor PTEN reverses the transformation of PI(4,5)P2 to PIP3 in the PI3K/AKT pathway, and maintains a low level of PIP3, thereby inhibiting the phosphorylation of AKT. Additionally, it has been confirmed that mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AKT at Ser473, and that this modification requires PIP3 (37).

Activated AKT is involved in the occurrence and development of glioblastoma; the PI3K-AKT-mTOR pathway is frequently activated in glioblastoma to regulate cancer survival (38). Additionally, AKT contributes to glioblastoma formation through the recruitment of existing mRNAs to polysomes (39). The direct and indirect inhibition of AKT activity promotes apoptosis and suppresses glioblastoma growth (40,41).

Due to the small sample size, the association between TIGAR expression and survival time could not be calculated for the patients included in the present study; therefore, the association was analyzed using PrognoScan. As TIGAR is overexpressed in glioblastoma, the role of TIGAR in oxidative stress was investigated. NADPH levels decreased and ROS indicators increased in the TIGAR-knockdown U-87MG glioblastoma cell line. Furthermore, cell viability, TMZ-induced apoptosis, migration, invasion and EMT were investigated in TIGAR-knockdown U-87MG cells. The results demonstrated that TIGAR-mediated antioxidative effects inhibited apoptosis but did not affect viability, migration, invasion or EMT. In addition, TIGAR promoted AKT phosphorylation in a dose-dependent manner and interacted with AKT in U-87MG cells. It has been reported that PFK/FB4, an enzyme similar to TIGAR, phosphorylates nuclear receptor coactivator 3 at Ser857 to enhance its transcriptional activity and promote breast cancer growth and metastasis (42). The manner in which TIGAR, as a phosphatase, promotes AKT phosphorylation requires further investigation.

In conclusion, the results of the present study demonstrated that TIGAR inhibited apoptosis and promoted proliferation, migration and invasion in glioblastoma through NADPH-mediated antioxidative effects and activation of AKT. Therefore, TIGAR may be considered as a potential therapeutic target in glioblastoma.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

ZT and ZH performed the experiments. ZT analyzed the data. ZH designed the experiments and wrote the manuscript.

Ethics approval and consent to participate

The use of glioblastoma sections was approved by the Ethics Committee of Hunan Cancer Hospital and Affiliated Cancer Hospital of Xiangya School of Medicine (no. 20180104; Changsha, China). All patients provided informed consent.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Wen PY and Kesari S: Malignant gliomas in adults. N Engl J Med. 359:492–507. 2008. View Article : Google Scholar : PubMed/NCBI

2 

Stupp R, Taillibert S, Kanner A, Read W, Steinberg D, Lhermitte B, Toms S, Idbaih A, Ahluwalia MS, Fink K, et al: Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: A randomized clinical trial. JAMA. 318:2306–2316. 2017. View Article : Google Scholar : PubMed/NCBI

3 

Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, Gottlieb E and Vousden KH: TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell. 126:107–120. 2006. View Article : Google Scholar : PubMed/NCBI

4 

Green DR and Chipuk JE: P53 and metabolism: Inside the TIGAR. Cell. 126:30–32. 2006. View Article : Google Scholar : PubMed/NCBI

5 

Agnoletto C, Melloni E, Casciano F, Rigolin GM, Rimondi E, Celeghini C, Brunelli L, Cuneo A, Secchiero P and Zauli G: Sodium dichloroacetate exhibits anti-leukemic activity in B-chronic lymphocytic leukemia (B-CLL) and synergizes with the p53 activator Nutlin-3. Oncotarget. 5:4347–4360. 2014. View Article : Google Scholar : PubMed/NCBI

6 

Zhou X, Xie W, Li Q, Zhang Y, Zhang J, Zhao X, Liu J and Huang G: TIGAR is correlated with maximal standardized uptake value on FDG-PET and survival in non-small cell lung cancer. PLoS One. 8:e805762013. View Article : Google Scholar : PubMed/NCBI

7 

Ko YH, Domingo-Vidal M, Roche M, Lin Z, Whitaker-Menezes D, Seifert E, Capparelli C, Tuluc M, Birbe RC, Tassone P, et al: TIGAR metabolically reprograms carcinoma and stromal cells in breast cancer. J Biol Chem. 116:7402092016.

8 

Zou S, Gu Z, Ni P, Liu X, Wang J and Fan Q: SP1 plays a pivotal role for basal activity of TIGAR promoter in liver cancer cell lines. Mol Cell Biochem. 359:17–23. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Cheung EC, Athineos D, Lee P, Ridgway RA, Lambie W, Nixon C, Strathdee D, Blyth K, Sansom OJ and Vousden KH: TIGAR is required for efficient intestinal regeneration and tumorigenesis. Dev Cell. 25:463–477. 2013. View Article : Google Scholar : PubMed/NCBI

10 

Lee P, Vousden KH and Cheung EC: TIGAR, TIGAR, burning bright. Cancer Metab. 2:12014. View Article : Google Scholar : PubMed/NCBI

11 

Mizuno H, Kitada K, Nakai K and Sarai A: PrognoScan: A new database for meta-analysis of the prognostic value of genes. BMC Med Genomics. 2:182009. View Article : Google Scholar : PubMed/NCBI

12 

Griffith OW: Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 106:207–212. 1980. View Article : Google Scholar : PubMed/NCBI

13 

Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR and Grandjean P: Plasma malondialdehyde as biomarker for oxidative stress: Reference interval and effects of life-style factors. Clin Chem. 43:1209–1214. 1997.PubMed/NCBI

14 

Xie JM, Li B, Yu HP, Gao QG, Li W, Wu HR and Qin ZH: TIGAR has a dual role in cancer cell survival through regulating apoptosis and autophagy. Cancer Res. 74:5127–5138. 2014. View Article : Google Scholar : PubMed/NCBI

15 

Lee SY: Temozolomide resistance in glioblastoma multiforme. Genes Dis. 3:198–210. 2016. View Article : Google Scholar : PubMed/NCBI

16 

Zeng WF, Navaratne K, Prayson RA and Weil RJ: Aurora B expression correlates with aggressive behaviour in glioblastoma multiforme. J Clin Pathol. 60:218–221. 2007. View Article : Google Scholar : PubMed/NCBI

17 

Heerboth S, Housman G, Leary M, Longacre M, Byler S, Lapinska K, Willbanks A and Sarkar S: EMT and tumor metastasis. Clin Transl Med. 4:62015. View Article : Google Scholar : PubMed/NCBI

18 

Li X, Wu C, Chen N, Gu H, Yen A, Cao L, Wang E and Wang L: PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget. 7:33440–33450. 2016.PubMed/NCBI

19 

Agarwal E, Brattain MG and Chowdhury S: Cell survival and metastasis regulation by Akt signaling in colorectal cancer. Cell Signal. 25:1711–1719. 2013. View Article : Google Scholar : PubMed/NCBI

20 

Gladson CL, Prayson RA and Liu WM: The pathobiology of glioma tumors. Annu Rev Pathol. 5:33–50. 2010. View Article : Google Scholar : PubMed/NCBI

21 

Delgado-Lopez PD and Corrales-Garcia EM: Survival in glioblastoma: A review on the impact of treatment modalities. Clin Transl Oncol. 18:1062–1071. 2016. View Article : Google Scholar : PubMed/NCBI

22 

Xie Q, Mittal S and Berens ME: Targeting adaptive glioblastoma: An overview of proliferation and invasion. Neuro Oncol. 16:1575–1584. 2014. View Article : Google Scholar : PubMed/NCBI

23 

Chen X, Zhang M, Gan H, Wang H, Lee JH, Fang D, Kitange GJ, He L, Hu Z, Parney IF, et al: A novel enhancer regulates MGMT expression and promotes temozolomide resistance in glioblastoma. Nat Commun. 9:29492018. View Article : Google Scholar : PubMed/NCBI

24 

Blagosklonny MV: Loss of function and p53 protein stabilization. Oncogene. 15:1889–1893. 1997. View Article : Google Scholar : PubMed/NCBI

25 

Fischer M: Census and evaluation of p53 target genes. Oncogene. 36:3943–3956. 2017. View Article : Google Scholar : PubMed/NCBI

26 

Zhang WB, Wang Z, Shu F, Jin YH, Liu HY, Wang QJ and Yang Y: Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J Biol Chem. 285:40461–40471. 2010. View Article : Google Scholar : PubMed/NCBI

27 

Lee P, Hock A, Vousden K and Cheung E: P53-and p73-independent activation of TIGAR expression in vivo. Cell Death Dis. 6:e18422015. View Article : Google Scholar : PubMed/NCBI

28 

Cheung EC, Ludwig RL and Vousden KH: Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death. Proc Natl Acad Sci USA. 109:20491–20496. 2012. View Article : Google Scholar : PubMed/NCBI

29 

Zhang Y, Chen F, Tai G, Wang J, Shang J, Zhang B, Wang P, Huang B, Du J, Yu J, et al: TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport. Sci Rep. 7:429282017. View Article : Google Scholar : PubMed/NCBI

30 

Zhang H, Gu C, Yu J, Wang Z, Yuan X, Yang L, Wang J, Jia Y, Liu J and Liu F: Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation. Free Radic Biol Med. 69:239–248. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Yu HP, Xie JM, Li B, Sun YH, Gao QG, Ding ZH, Wu HR and Qin ZH: TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway. Sci Rep. 5:98532015. View Article : Google Scholar : PubMed/NCBI

32 

Li M, Sun M, Cao L, Gu JH, Ge J, Chen J, Han R, Qin YY, Zhou ZP, Ding Y and Qin ZH: A TIGAR-regulated metabolic pathway is critical for protection of brain ischemia. J Neurosci. 34:7458–7471. 2014. View Article : Google Scholar : PubMed/NCBI

33 

Oda K, Stokoe D, Taketani Y and McCormick F: High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma. Cancer Res. 65:10669–10673. 2005. View Article : Google Scholar : PubMed/NCBI

34 

Luo J, Manning BD and Cantley LC: Targeting the PI3K-Akt pathway in human cancer: Rationale and promise. Cancer Cell. 4:257–262. 2003. View Article : Google Scholar : PubMed/NCBI

35 

Vanhaesebroeck B, Guillermet-Guibert J, Graupera M and Bilanges B: The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol. 11:329–341. 2010. View Article : Google Scholar : PubMed/NCBI

36 

Zhao L and Vogt PK: Class I PI3K in oncogenic cellular transformation. Oncogene. 27:5486–5496. 2008. View Article : Google Scholar : PubMed/NCBI

37 

Ebner M, Sinkovics B, Szczygiel M, Ribeiro DW and Yudushkin I: Localization of mTORC2 activity inside cells. J Cell Biol. 216:343–353. 2017. View Article : Google Scholar : PubMed/NCBI

38 

Fan QW and Weiss WA: Autophagy and Akt promote survival in glioma. Autophagy. 7:536–538. 2011. View Article : Google Scholar : PubMed/NCBI

39 

Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X and Holland EC: Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell. 12:889–901. 2003. View Article : Google Scholar : PubMed/NCBI

40 

Gao T, Furnari F and Newton AC: PHLPP: A phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell. 18:13–24. 2005. View Article : Google Scholar : PubMed/NCBI

41 

Assad Kahn S, Costa SL, Gholamin S, Nitta RT, Dubois LG, Fève M, Zeniou M, Coelho PL, El-Habr E, Cadusseau J, et al: The anti-hypertensive drug prazosin inhibits glioblastoma growth via the PKCdelta-dependent inhibition of the AKT pathway. EMBO Mol Med. 8:511–526. 2016. View Article : Google Scholar : PubMed/NCBI

42 

Dasgupta S, Rajapakshe K, Zhu B, Nikolai BC, Yi P, Putluri N, Choi JM, Jung SY, Coarfa C, Westbrook TF, et al: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer. Nature. 556:249–254. 2018. View Article : Google Scholar : PubMed/NCBI

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September 2019
Volume 18 Issue 3

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Online ISSN:1792-1082

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Copy and paste a formatted citation
APA
Tang, Z., & Tang, Z. (2019). TIGAR promotes growth, survival and metastasis through oxidation resistance and AKT activation in glioblastoma. Oncology Letters, 18, 2509-2517. https://doi.org/10.3892/ol.2019.10574
MLA
Tang, Z., He, Z."TIGAR promotes growth, survival and metastasis through oxidation resistance and AKT activation in glioblastoma". Oncology Letters 18.3 (2019): 2509-2517.
Chicago
Tang, Z., He, Z."TIGAR promotes growth, survival and metastasis through oxidation resistance and AKT activation in glioblastoma". Oncology Letters 18, no. 3 (2019): 2509-2517. https://doi.org/10.3892/ol.2019.10574