IDH1 mutation decreases the invasiveness of glioma by downregulating the expression and activity of TAZ

Ningning Li; Rui Zhang; Yi Sun; Chenyue Xu; Yin Wang; Ji Xiong; Qi Chen; Ying Liu

doi:10.4103/glioma.glioma_46_18

ORIGINAL ARTICLE

Year : 2019 | Volume : 2 | Issue : 1 | Page : 37-45

IDH1 mutation decreases the invasiveness of glioma by downregulating the expression and activity of TAZ

Ningning Li¹, Rui Zhang², Yi Sun², Chenyue Xu², Yin Wang³, Ji Xiong³, Qi Chen², Ying Liu²
¹ Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai; Department of Pathology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang Province, China
² Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China
³ Department of Pathology, Huashan Hospital affiliated to Fudan University, Shanghai, China

Date of Web Publication

1-Apr-2019

Correspondence Address:
Dr. Ying Liu
School of Basic Medical Sciences, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/glioma.glioma_46_18

Abstract

Background and Aim: Gliomas carrying mutated isocitrate dehydrogenase 1 (IDH1) have an improved prognosis, but how this mutation improves survival is not known. In this study, we evaluated the correlation of expression of the gene transcriptional coactivator with PDZ-binding motif (TAZ) with IDH1 mutation in astrocytomas of different grades. Materials and Methods: We analyzed the expression of TAZ by immunohistochemistry in a cohort of 90 formalin-fixed paraffin-embedded human astrocytoma samples. A human glioblastoma cell line (U87) was transfected with mutated IDH1^R132H; the expression and subcellular location of TAZ were analyzed by western blot assay, quantitative real-time polymerase chain reaction, and immunofluorescence staining. We detected activation of the Hippo signaling pathway by western blot. Octyl-2-hydroxyglutarate (Octyl-2-HG), an analog of 2-HG, was used to treat IDH1 wild-type U87 cells to determine its influence on the expression of TAZ. Cell viability assay, flow cytometry, Transwell migration assay, and scratch assay were used to analyze cell proliferation and invasive capacity. To verify that those changes were caused by the expression of TAZ, we did a rescue experiment by transfecting TAZ in the IDH1^R132H cells. The study was approved by the Ethics Committee of Fudan University (approval No. 2016-Y013) on January 18, 2016. Results: TAZ expression was significantly lower in IDH1-mutated astrocytoma than the wild type in the same tumor grade. In IDH1-mutant cells, the nuclear TAZ location was decreased and the Hippo signaling pathway was activated as determined by TAZ phosphorylation and increased 14-3-3e expression. Treatment with Octyl-2-HG reduced the expression of TAZ. IDH1^R132H cells showed decreased invasion proliferation compared with IDH1 wild-type cells. Overexpression of TAZ rescued cell migration and invasion capacity. Conclusion: In glioma, IDH1^R132H mutation decreases TAZ expression and significantly reduces the invasive character of glioma cells.

Keywords: 14-3-3, 2-hydroxyglutarate, astrocytoma, Hippo signaling pathway, isocitrate dehydrogenase 1 mutation, malignant grade, proliferation, transcriptional coactivator with PDZ-binding motif

How to cite this article:
Li N, Zhang R, Sun Y, Xu C, Wang Y, Xiong J, Chen Q, Liu Y. IDH1 mutation decreases the invasiveness of glioma by downregulating the expression and activity of TAZ. Glioma 2019;2:37-45

How to cite this URL:
Li N, Zhang R, Sun Y, Xu C, Wang Y, Xiong J, Chen Q, Liu Y. IDH1 mutation decreases the invasiveness of glioma by downregulating the expression and activity of TAZ. Glioma [serial online] 2019 [cited 2023 May 28];2:37-45. Available from: http://www.jglioma.com/text.asp?2019/2/1/37/255152

Introduction

Diffuse astrocytoma, the most common glioma of the central nervous system, is subdivided into three grades (Grades II–IV) according to the parameters set by the World Health Organization.^[1] Isocitrate dehydrogenase 1 (IDH1) is a critical molecular marker in the diagnosis of glioma. Mutations in IDH1 occur in >70% of Grade II and Grade III astrocytomas but are much less frequent in Grade IV (glioblastoma [GBM]).^[2],[3] GBMs are subdivided into primary GBM and secondary GBM, the latter arising from Grade II or III cases. Thus, IDH1 mutations in GBM are used to identify secondary GBM.^[4],[5] The most common mutation of IDH1 is a single nucleotide change (CGT–CAT), resulting in arginine being replaced by histidine at amino acid 132, which is known as R132H.^[6] The mutation impairs the oxidative IDH1 activity of the enzyme but renders a new function to convert a-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG); the latter serves as a metabolic by-product.^{[7],[8],[9],[10]} Clinically, IDH1 mutation is closely related to the reduced invasion of glioma, and it is also associated with better prognosis.^[11],[12] However, the exact role of the IDH1 mutation in gliomas remains to be further studied.

The Hippo signaling pathway initially identified and named by screening mutant tumor suppressor in flies is involved in the progression of cancer.^{[13],[14],[15],[16]} The central components of the pathway consist of an upstream regulatory serine-threonine kinase module, including the mammalian sterile 20-like kinases serine/threonine kinases 1/2, the Salvador family WW domain-containing protein 1, the large tumor suppressor serine/threonine protein kinases 1/2 (LATS1/2), the MOB kinase activator 1A/B, and a downstream transcriptional module, which comprises transcriptional coactivator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP). YAP and TAZ are homologous proteins.^{[17],[18],[19],[20],[21]} When the regulators are stimulated, the mammalian Ser/Thr kinases LATS1/2 can phosphorylate and inactivate YAP and TAZ, which promotes either the cytoplasmic retention of YAP and TAZ by interaction with 14-3-3 protein or the degradation of YAP/TAZ, and ultimately, the Hippo signaling pathway inhibits the activity of YAP/TAZ via changing its protein level and subcellular distribution.^{[22],[23],[24],[25]}

Aberrant expression of TAZ has been observed in human gliomas, and its expression is significantly correlated with the malignancy and prognosis of the tumor, which suggests that TAZ may play an important role in the tumorigenesis and development of gliomas.^{[26],[27],[28]} Bhat et al.^[29] reported that TAZ regulates mesenchymal differentiation in malignant glioma, and the mesenchymal transition is tightly correlated with the aggressive proliferation of tumor. This study aimed to evaluate the correlation between TAZ expression and IDH1 mutation in astrocytoma at different tumor grades.

Materials and Methods

Human tissue specimens

Human astrocytoma samples were obtained from the Department of Pathology, Huashan Hospital affiliated to Fudan University, Shanghai, China, from June 2012 to June 2014 for this retrospective study. Two experienced neuropathologists made the diagnosis and graded the malignancy of the astrocytoma samples according to the criteria established by the World Health Organization (2016).^[30] The formalin-fixed paraffin-embedded tumor samples (90 cases) included diffuse astrocytoma (Grade II), IDH1 wild-type or mutant (15 cases each), anaplastic astrocytoma (Grade III), IDH1 wild-type or mutant (15 cases each), and GBMs (Grade IV), IDH1 wild-type or mutant (15 cases each). IDH1 gene status was verified by polymerase chain reaction (PCR) amplification over the entire exon 4 using a pair of primers derived from intron 3 and intron 4 (forward primer: 5′-TGA GCT CTA TAT GCC ATC ACT GC-3′ and reverse primer: 5′-CAA TTT CAT ACC TTG CTT AAT GGG-3′), followed by sequencing using the same pair of primers, as reported in the article of Xu et al.^[31] The procedures associated with the acquisition of samples from human subjects were approved by the Ethics Committee of Fudan University (approval No. 2016-Y013) on January 18, 2016, and performed in accordance with the ethical principles of the Declaration of Helsinki. Written informed consent was obtained from each participant.

Cell culture

Human GBM U87 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 2 mM L-glutamine in standard conditions. Human IDH1 wild-type and R132H-mutant plasmids were kindly provided by Prof. Xiong Yue (Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, North Carolina, USA).^[32] U87 cells were transfected using lipofectamine according to the manipulation protocol (Invitrogen, Carlsbad, CA, USA).

Immunohistochemistry

The formalin-fixed paraffin-embedded astrocytoma sections (thickness, 4 μm) were used for immunohistochemical staining using the labeled streptavidin-biotin method (Dako; Agilent Technologies, Inc, Santa Clara, CA, USA). The endogenous peroxidase activity was blocked in deparaffinized slides by incubating the sections in 3% hydrogen peroxide methanol solution at room temperature for 10 min, followed by antigen retrieval with 10 mM citrate buffer (pH 6.0) at 95–100°C for 10 min. Then, the slides were blocked with 10% goat serum (Abcam, Cambridge, UK) in phosphate-buffered saline (PBS) for 20 min at room temperature. Subsequently, the primary antibodies were incubated with the sections: anti-TAZ (1:200 dilution, BD Pharmingen, Franklin Lakes, NJ, USA) and anti-14-3-3e (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA). The sections were developed with the rabbit/mouse peroxidase/3,3′-diaminobenzidine EnVision™ Detection kit (Cat# GK500705; Dako; Agilent Technologies, Inc.) containing the secondary antibody and 3,3′-diaminobenzidine according to the manufacturer's protocol; the nuclei were counterstained with hematoxylin by Nikon (Nikon Instruments, Inc., Melville, NY, USA).

The intensity of TAZ staining was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong. The percentage scores were assigned, as follows: 1, ≤25%; 2, 26%–50%; 3, 51%–75%; and 4, >75%. These scores were multiplied to arrive at a final score ranging between 0 and 12, as we have shown before.^[33] Score 0 was labeled as 0, scores 1–4 were labeled as weak (Grade I), scores 5–8 were labeled as moderate (Grade II), and a score above 8 was labeled strong (Grade III).

Treating cells with 2R-Octyl-α-hydroxyglutarate

Cultured cells were treated with Octyl-2-hydroxyglutarate (Octyl-2-HG) (100 mM, Cayman, Ann Arbor, MI, USA) for 4 h, washed with PBS three times, and then collected for further analysis. Dimethyl sulfoxide (DMSO, Hengyu Chemical Co., Ltd., Changzhou, Jiangsu Province, China)-treated cells were used as a control.

Transcriptional coactivator with PDZ-binding motif transfection

The PRK7-N-flag-TAZ (kindly provided by Prof. Yue Xiong, Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, North Carolina, USA) is a eukaryotic expression plasmid, which containing full-length TAZ gene, with a flag marker. The TAZ vector or control vector was transfected into cells by Lipo2000 (Invitrogen) according to the manufacturer's instruction. The expression of TAZ was confirmed using real-time quantitative reverse transcription-PCR and western blot at 48 h after transfection.

Immunofluorescent staining

Glass coverslips plated cells were fixed in 4% formaldehyde, rinsed, and permeabilized in PBS containing 0.2% Triton X-100. Cells were then incubated in 10% goat serum for 30 min at room temperature. Samples were incubated with mouse anti-TAZ antibody at 37°C (1:200 at dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature and then cultured with the secondary antibody (fluorescein isothiocyanate-conjugated goat mouse-specific antibody (1:500 at dilution, Invitrogen). After rinsing with PBS, the slides were 4',6-diamzidino-2-phenylindole-containing for 5 min and then mounting with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). For fluorescence imaging, images were taken using a 40× objective lens on a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany).

Western blot analysis

Total protein from cultured cells was extracted in RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) that contained a protease inhibitor cocktail and phosphatase inhibitor. Protein samples (40 μg/sample) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck KGaA Co., Darmstadt, Germany). The membranes were incubated with primary antibodies overnight at 4°C after blocked with 5% non-fat milk and subsequently with HRP-conjugated anti-rabbit secondary antibodies (Santa Cruz Biotechnology). Primary antibodies were anti-TAZ (1:1000 dilution; Santa Cruz Biotechnology), the Hippo signaling antibody sampler kit, including anti-phospho-TAZ, anti-phospho-LAST1 (Thr35), anti-LATS1, anti-YAP, and anti-phospho-YAP (Ser127) (all derived from rabbit, 1:1000 dilution; Cell Signaling Technology), rabbit anti-14-3-3e (1:1000 dilution; Cell Signaling Technology), and rabbit anti-GAPDH (1:2000 dilution; Santa Cruz Biotechnology). Protein bands were visualized with the enhanced chemiluminescence kits (Pierce Chemical, Rockford, IL, USA). GAPDH was the loading control. In addition, the level of protein expression was calibrated to the band density of GAPDH by DRAFT-alpha view software (San Jose, CA, USA).

Quantitative real-time polymerase chain reaction

Total RNA was extracted from cells using TRIZOL reagent (Tiangen Biotech, Beijing, China). An equal amount of RNA was used for synthesis of the first-strand cDNA by reverse transcription using PrimeScript RT Master Mix (Takara, Beijing, China). Briefly, quantitative real-time PCR (qRT-PCR) (SYBR Green Assay, Roche Diagnostics GmbH, Mannheim, Germany) was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of TAZ were calculated and quantified using the 2^−ΔΔT methods.^[34] β-Actin served as the endogenous controls. The primer sequences were TAZ (forward primer: 5′-CTG AAG TTG ATG CGT TGG A-3′; reverse primer: 5′-GGC GGA CTG TTA GGA AGG-3′) and β-actin (forward primer: 5′-GGT GGC TTT TAG GAT GGC AAG-3′; reverse primer: 5′-ACT GGA ACG GTG AAG GTG ACA G-3′).

Cell viability assay

The viability of cells was assessed by Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan).^[35] Cells were cultured in 96-well plates (5 × 10³/well, 100 μL) for 24 h, the medium then was removed, and CCK-8 (0.5 mg/mL, 100 μL) was added to each well and incubated for 2 h. Dimethyl sulfoxide (100 μL) was added to dissolve the solid formazan, and the absorbance was measured with a microplate reader (Thermo Fisher Scientific) at 450 nm. To ensure the reliability of the experiment, we set up multiple subholes and repeated three times at least.

Transwell assay

Transwell assays were done as reported.^[36] Falcon^® inserts (BD Biosciences, San Jose, CA, USA) with 8 μm pore size coated with or without Matrigel (BD Biosciences) were used to measure the motility or invasiveness of cells exposed to different treatment. In the upper part of the Transwell unit, 50,000 cells suspended in culture medium without serum were seeded and keep for 24 h. The low layer of the Transwell unit was cultured with 500 μL DMEM with 10% FBS. After incubation, cells on the upper layer of the membrane were swapped away by a cotton swab. Cells on the bottom surface of the membrane were thought as invasive cells and then stained with crystal violet (Solarbio Technology Co., Ltd., Beijing, China). Cell numbers appearing in five randomly selected fields were counted. Assays were repeated three times.

Scratch wound-healing assay

The scratch wound-healing assay was used to detect the migratory properties of cells. Uniform wounds were scraped in monolayer cells with 80% confluent using a 10 μL pipette tip. The cells were incubated in DMEM and 10% FBS, and five visual fields were randomly chosen to observe the change of the scratched gap at a stage of 0, 6, 12, and 24 h under Ts2 Nikon optical microscopy (Nikon Instruments, Inc., Melville, NY, USA).

Cell cycle analysis

After fixed with ethanol (90%) 30 min at 4°C and washed with PBS, cells were re-suspended in propidium iodide (Thermo Fisher Scientific) solution (50 mg/mL containing ribonuclease A) at room temperature for 30 min in the dark. The distribution of cells with different DNA content was analyzed with Aria II flow cytometer (BD Biosciences).

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). The statistical methods included unpaired t-test or one-way analysis of variance followed by a Scheffe's test. Each presented experiment was performed at least three times, and data were presented as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

Results

Transcriptional coactivator with PDZ-binding motif expression decreased in isocitrate dehydrogenasegene-mutated astrocytoma

Consistent with literature reports,^[27],[29] TAZ immunoreactivity is enhanced with the increase in malignancy of glioma. TAZ immunoreactivity detected by immunohistochemical staining was significantly higher in IDH wild-type cases (IDH-wt) compared to IDH-mutation cases (IDH-mut) at the same tumor grade in human astrocytoma samples. Representative images of staining were shown in [Figure 1]A and [Figure 1]B. Statistic results are given in [Figure 1]C (P < 0.01).

Figure 1: The expression of TAZ is decreased in IDH1-mutated astrocytoma. (A and B) IHC staining of TAZ (arrows) in astrocytoma either IDH1 wild-type (A1–3) or IDH1- mutant cases (B1–3) of different malignant grades, each group contains 15 cases (A1 and B1: diffuse astrocytoma; A2 and B2: anaplastic astrocytoma; A3 and B3: glioblastoma). Scale bars: 50 μm. (C) The quantification of TAZ immunostaining. Statistical method was one-way analysis of variance followed by a Scheffe's test. (D) Western blot analysis of TAZ protein expression in U87 cells after transfected with IDH1^R132H mutation. (E) qRT-PCR analysis TAZ mRNA expression in IDH1-wt and IDH1^R132H U87 cells. qRT-PCR and western blot assay verified that the expression of TAZ mRNA (F) and protein (G) was decreased in IDH1-wt U87 cells after exogenous permeability Octyl-2-HG treated. All data are shown as the mean ± SD Results of western blot and qRT-PCR are representative of three independent experiments. **P < 0.01 (unpaired t-test). IHC: Immunohistochemical, TAZ: Transcriptional coactivator with PDZ-binding motif, IDH1: Isocitrate dehydrogenase 1, qRT-PCR: Quantitative real-time polymerase chain reaction, Octyl-2-HG: Octyl-2-hydroxyglutarate, SD: Standard deviation, wt: Wild type, mut: Mutation

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In vitro, we also verified that TAZ protein [Figure 1]D and mRNA were decreased in U87 cells after IDH1^R132H mutation [Figure 1]E. The 2-HG produced by IDH-mutation in gliomas is a structural analog of α-KG, which can inhibit the activity of α-KG-dependent enzymes.^[31],[37] To determine whether 2-HG affects TAZ activity, we used 10 mM Octyl-2-HG to treat IDH1- wild-type cells (which could produce equivalent to a 100-fold molar excess relative to normal 2-HG level in cell cytoplasm), we achieved strong inhibition of TAZ expression [Figure 1]F and [Figure 1]G, indicating that the decreased expression of TAZ may be caused by 2-HG (the oncogenic metabolites caused by the IDH1 mutation).

Isocitrate dehydrogenase 1 mutation activated the Hippo signaling pathway in cultured glioblastoma cells

Since the Hippo signaling pathway could negatively regulate the function of co-transcription factors YAP and TAZ,^[14] we examined the key signaling proteins of the pathway in IDH1-mutant glioma cells. We found that protein LAST1 was significantly activated in IDH1-mutated cells; as a consequence, the phosphorylation level of TAZ also increased significantly [Figure 2]A. As a potent transcriptional co-activator for TEAD/TEF transcription factors, TAZ needs to translocate from the cytoplasm to nuclei. If phosphorylated by the Hippo signaling pathway, TAZ and YAP would be retained in the cytoplasm by binding to the 14-3-3 protein for further proteasome degraded. We found that in IDH1-mutated cells, 14-3-3 protein expression also increased [Figure 2]B and [Figure 2]C.

Figure 2: The core components of the Hippo pathway was activated in IDH1-mutated U87. The core components of the Hippo pathway was activated in IDH1 mutated U87 cells and TAZ was extended in the cell cytoplasm. (A) Western blot analysis of the core components of the Hippo pathway. (B) 14-3-3e, which is the cytoplasm binding protein of phosphorylated TAZ, shows increasing expression after IDH1 mutated in U87 cells. (C) Quantification of the western blot bands grey density. (D and E) The location of TAZ in nuclear was significantly reduced in IDH1^R132H cells by immunofluorescence assay. TAZ labeling by FITC (green), nuclei labeling by PI (blue). Scale bars: 20 μm. All data are shown as the mean ± SD. Results are representative of three independent experiments. *P < 0.05 (unpaired t-test). wt: Wild type, LATS1: Large tumor suppressor serine/threonine protein kinases 1, YAP: Yes-associated protein, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, TAZ: Transcriptional coactivator with PDZ-binding motif, IDH1: Isocitrate dehydrogenase 1, SD: Standard deviation, PI: Propidium iodide, FITC: Fluorescein isothiocyanate

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Immunofluorescence staining for TAZ also confirmed that IDH1 gene status could affect the localization of TAZ in U87MG cells. As shown in [Figure 2]D, in IDH1 wild-type U87 cells, TAZ was mainly located in the nucleus, while in IDH1-mutant U87 cells, TAZ was mainly located in the cytoplasm and a small amount is located in the nucleus [Figure 2]E. This result indicated that IDH1 mutations can significantly downregulate TAZ expression and inhibit TAZs nuclear entry.

Isocitrate dehydrogenase 1-R132H mutation inhibited the proliferation, migration, and invasion of glioblastoma cells

Cancer progression indicates a cohort of cellular processes, including cell proliferation, migration, and invasion. Cell Counting Kit-8 assay showed that IDH1^R132H mutation was capable of inducing a significant reduction of viable cells [Figure 3]A. Meanwhile, by flow cytometry, we found cell-cycle arrest occurred in IDH1-mutated cells. From cytometric histograms on which the percentages of G1 and S + G2 cell-cycle stages were represented, we observed a significant increase of cells in G1 and decrease of cells in S + G2 cycle stages in IDH1-mutated cells when compared with controls [Figure 3]B, [Figure 3]C, [Figure 3]D.

Figure 3: IDH1^R132H could reduce cell proliferation, migration, and invasion of astrocytoma cells. (A) CCK-8 assay indicated that cell proliferation vitality was inhibited in U87 cells after IDH1^R132H mutated. (B, C) Flow cytometer showed that the number of cells in the S phase of IDH1^R132H cells (C) were significantly lower than IDH1- wild type cells (B). (D) Percentage of cells in S phase by flow cytometry. (E and F) Representative images at 0, 6, 12, and 24 h were showed the capacity of cell migration in U87 IDH1 wild-type (E1–4) and IDH1-mutated cells (F1–4). Scale bars: 100 μm. (G, H, J, K) Transwell assay without (G and H) or with (J and K) matrigel indicate the migration and invasive capacity of U87 cells (arrows) were decreased in IDH1-mutated cells compared with wild-type cells. Scale bars: 40 μm. Invasive (I) and migration property (L) was quantified, separately. All data are shown as the mean ± SD. Results are representative of three independent experiments. **P < 0.01 (unpaired t-test). OD: Optical density, IDH1: Isocitrate dehydrogenase 1, CCK-8: Cell Counting Kit-8, SD: Standard deviation

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In the cell scratch wound-healing assay, the rate of wound closure was different depending on IDH1 status. IDH1^R132H-mutated cells [Figure 3]F1–4 had a significantly wider wound width than IDH1 wild-type groups [Figure 3]E1–4.

Similar results were achieved in migration and invasive assay, IDH1 wild-type GBM cells exhibited strong migration and invasive capacity, while cells transfected with IDH1^R132H had dramatically decreased migration and invasiveness [Figure 3]G–L.

Taken together, these results revealed that the IDH1 gene mutation inhibited U87MG cells proliferation, migration, and invasion in vitro.

Transcriptional coactivator with PDZ-binding motif transfection rescued the invasive proliferation of glioma cells with isocitrate dehydrogenase 1-R132H mutation

Using IDH1^R132H GBM cells and lentivirus, we induced the stable transfection of TAZ. qRT-PCR and western blot assay were used to confirm transfection efficiency [P < 0.01, [Figure 4]A and [Figure 4]B. The overexpression of TAZ in IDH1^R132H-mutant cell could promote the cell viability [Figure 4]C and the proportion of S phase in cell cycle than those of control [P < 0.01, [Figure 4]D and [Figure 4]E. Overexpressed TAZ in IDH1^R132H-mutant cells were also scratched, and the wound width was measured. The cells had a significantly less wound width (P ≤ 0.001) than IDH1^R132H cells. At 24 h, wounds in the TAZ overexpressed group were almost healed, while in the control cells, the gaps were still open [Figure 4]F and [Figure 4]G. The cells migration and invasion capacity were also improved significantly [P < 0.05, [Figure 4]H–M after TAZ was transfected into IDH1-mutated cells. Given the above results, overexpression of TAZ could rescue the active proliferation and aggressive invasion phenotype of U87 cells with IDH1^R132H mutation.

Figure 4: TAZ rescue the invasive proliferation phenotype of IDH1^R132H-mutant astrocytoma cells. (A and B) qRT-PCR (A) and western blot assay (B) verified the successful transfection of TAZ in IDH1^R132H U87 cells. (C) CCK-8 assay indicated that cell viability was increased after transfected with TAZ in IDH1^R132H cells. (D) Flow cytometry showed that the number of cells in S phase of IDH1^R132H cells after transfected with TAZ was significantly higher compared with the vector control group. (E) Percentage of cells in S phase by flow cytometry. (F and G) Representative images at 0, 6, 12, and 24 h showed the capacity of cell migration by scratch assay in U87 IDH1^R132H-mutant cells before (F1–4) and after (G1–4) TAZ transfected. Scale bars: 100 μm. (H, I, K, L) Transwell assay without (H and I) or with (K and L) matrigel indicate the migration and invasive capacity of U87 IDH1-mutated cells (arrows) increased after TAZ transfected. Scale bars: 40 μm. (J and M) Quantifications of Transwell assay. All data are shown as the mean ± SD. Results are representative of three independent experiments. **P < 0.01 (unpaired t-test). TAZ: Transcriptional coactivator with PDZ-binding motif, IDH1: Isocitrate dehydrogenase 1, qRT-PCR: Quantitative real-time polymerase chain reaction, CCK-8: Cell Counting Kit-8, SD: Standard deviation, mut: Mutation, wt: Wild type, EV: Ev virus vector

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Discussion

Transcriptional coactivator with PDZ-binding motif inhibition may correlate with a good prognosis in glioma

Diffuse astrocytomas are very infiltrative, and it is generally accepted that IDH1-mutated glioma is less invasive compared with wild-type glioma at the same grade,^[38] but the mechanism why this is the case has not been elucidated.

TAZ is one of the key downstream effectors of the Hippo signaling pathway, which plays an important role in cell proliferation and organ size control. Recent work indicates that YAP/TAZ is essential for cancer initiation and growth in most solid tumors. Their activation induces proliferation, chemoresistance, and metastasis in cancer cells.^[39] Verhaak et al.^[40] reported that the TAZ expression was lower in primary GBMs, which correlated with CpG island hypermethylation of the TAZ promoter compared with secondary GBMs. Since results from The Cancer Genome Atlas ^{More Details} consortium revealed that the IDH1 mutation was a defining feature of different grades and subtypes of glioma,^[40] we evaluated the correlation between IDH1 mutational status and TAZ expression and showed a correlation.

It has been established that the IDH1 mutation causes two things to happen: reduces the ability of NADP+ dependent isocitrate catalytic conversion to α-KG and the mutation causes a new catalytic function, promoting α-KG to D-2-hydroxyglutarate (D-2-HG), accompanied by the oxidation of NADPH. The final result is low α-KG, high D-2-HG levels in IDH1-mutated cells compared with wild-type cells.^[41],[42] The D-2-HG then competitively inhibits α-KG dependent enzymes due to its similar structure and causes metabolism and epigenetic changes.^[43] We used Octyl-2-HG, a cytomembrane permeable analog of 2-HG, to see if it could inhibit TAZ levels in cultured IDH1 wild-type GBM cells, and show that this oncological metabolite can inhibit TAZ expression.

The Hippo signaling pathway was first discovered in Drosophila, where it was shown to regulate organ size. The Hippo signaling pathway can respond to a variety of upstream stimuli, such as exogenous signal input, intercellular contact, cell pressure, mechanical conductance, and cell polarity. One member of this signaling pathway, LATS1/2 kinase, can directly phosphorylate multiple serine residues of YAP/TAZ. After being phosphorylated, YAP/TAZ remain in the cytoplasm by binding the 14-3-3 protein, resulting in cytoplasmic retention and further degradation through the proteasome pathway.^[19],[25] In this study, we found that accompanying the IDH1 mutation, the subcellular localization of TAZ in the cell was changed from nuclear to cytoplasmic, and the expression of 14-3-3 protein was elevated. Our studies in glioma samples and cells consistently show that IDH1 mutations can significantly downregulate TAZ expression and inhibit TAZ nuclear entry.

Our study showed the proliferation and invasive capacity of GBM cells was significantly inhibited by the IDH1 mutation. Overexpression of TAZ could rescue the invasive phenotype of GBM cells harboring the IDH1^R132H mutation.

Conclusion

We found that the IDH1^R132H mutation could not only decrease TAZ protein expression at the transcriptional level but also inhibit TAZ activity by activating the Hippo signaling pathway, which may further inhibit the proliferation and invasive capacity of glioma cells. Our study provides a molecular mechanism for the better prognosis of IDH1^R132H gliomas.

Acknowledgments

We thank Prof. Feng Tang, Department of Pathology, Huashan Hospital, Fudan University, for his generous help in providing all human astrocytoma samples. We thank Prof. Xiong Yue, Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, North Carolina, USA, for providing the human IDH1 wild-type and R132H-mutant plasmids. We thank Prof. Qunying Lei, Department of Biochemistry, Fudan University, China, for providing the PRK7-N-flag-TAZ plasmid.

Financial support and sponsorship

This study was supported by the National Natural Science Foundation of China (No. 81272796).

Institutional review board statement

The study was approved by the Ethics Committee of Fudan University (approval No. 2016-Y013) on January 18, 2016.

Conflicts of interest

There are no conflicts of interest.

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