Methylation dependent down-regulation of G0S2 leads to suppression of invasion and improved prognosis of IDH1-mutant glioma

Isocitrate dehydrogenase (IDH) mutations are a prognostic factor in diffuse glioma. However, the mechanism by which these mutations improve prognosis are not clear. In a subset of IDH-mutant glioma, remodeling of the methylome results in the glioma-CpG island methylator phenotype (G-CIMP) and transcriptional reorganization. In this study, we focus on G0/G1 switch 2 (G0S2), which is highly downregulated in G-CIMP glioma. We found that G0S2 expression tended to increase as the WHO grade increased, and G0S2 knockdown inhibited glioma invasion. Additionally, we revealed that the overexpression of the DNA demethylase Ten-eleven translocation 2 (TET2) in IDH1-plasmid transfected glioblastoma multiforme (GBM) cells restored G0S2 expression. These results indicate that G0S2 is epigenetically silenced in IDH1-mutant glioma. In addition, the stereotactic delivery of glioma cells with decreased G0S2 expression in the mouse brain resulted in prolonged survival. The Cancer Genome Atlas (TCGA) analysis also indicated that survival is longer in the lower G0S2 expression group than in the higher G0S2 expression group. Moreover, G0S2 expression was higher in recurrent tumor specimens than at the initial diagnosis in the same patient. These results provide one explanation for the improved survival in IDH1-mutant glioma as well as a new epigenetic target for glioma treatment.


Introduction
Glioma, the most common primary tumor in the central nervous system, is classified into four grades according to its malignancy based on histopathological and molecular features established by the World Health Organization (WHO). WHO grade IV glioblastoma multiforme (GBM) is the most malignant and has the worst prognosis among all glioma. Although the current standard treatment includes the maximal removal of the tumor, followed by adjuvant chemotherapy and radiation therapy, the median overall survival is only 16.8 months [1]. Treatment approaches and survival have not improved for decades. PLOS  Moreover, as glioma cells invade eloquent areas and migrate undetectably toward normal outer brain tissues, it is difficult to resect the entire glioma by surgery. Accordingly, most gliomas recur and progress to more malignant WHO grades than that at primary diagnosis. Unfortunately, there is no standard therapy for recurrent glioma. Thus, new treatment targets are urgently needed.
Most GBM (~90%) occur in elderly people and develop rapidly (primary GBM or de novo GBM). However, a subset of GBM that progresses from a lower grade (WHO grade II/III) glioma (termed secondary GBM) occurs in younger people and has a significantly better prognosis than primary GBM [2,3,4].
In addition, the updated fourth edition of the WHO Classification of CNS Tumors published in 2016 includes well-established molecular genetic parameters for subclassification [12]. In this classification, IDH1 mutations, the codeletion status of chromosome arms 1p and 19q, and the histone 3 mutational status can be used to distinguish between biologically distinct glioma [13,14]. Moreover, it has been reported that the genomic status better predicts outcome than histologic grade [15].
Patients with WHO grade III anaplastic astrocytoma and GBM carrying IDH1 mutations have a significantly longer median overall survival than patients with wild-type IDH1 [6]. However, the mechanism underlying the improved prognosis in patients with glioma carrying IDH1 mutations is not fully understood.
Concurrent genome-wide DNA methylation and DNA promoter CpG island hypermethylation leads to transcriptional silencing in cancer [16]. The frequency of promoter methylation is higher in secondary GBM than in primary GBM [17]. A distinct subset of glioma displaying DNA hypermethylation, i.e., glioma-CpG island methylator phenotype (G-CIMP), has a characteristic profile; it is more prevalent among lower grade glioma, strongly associated with IDH1 mutation, diagnosed at a younger age, and has a significantly better prognosis [18]. However, the functional importance of this altered epigenetic state remains unclear. In comparisons between G-CIMP-positive tumors and G-CIMP-negative tumors, G0/G1 switch 2 (G0S2) exhibits the strongest differential expression [18], yet the mechanism and function of G0S2 in glioma are unclear. These observations prompted us to hypothesize that the epigenetic silencing of G0S2 in G-CIMP may improve prognosis in patients with IDH1 mutations. In this study, we revealed the functional role of G0S2 in glioma and elucidated one explanation for the increased survival in glioma with IDH1 mutation.
Gibco (N7805-100, Gaithersburg, MD, USA) and cultured in complete astrocyte medium according to the manufacturer's instructions.

Analysis of G0S2 expression in tumor samples
Either signed consent forms or exemptions were obtained for all human samples. The study of human samples was approved by the Osaka University Institutional Review Board (17022-3). Tumor specimens were obtained during surgery and classified according to the histological grade of nervous system tumors published by the WHO in 2007. All tissue samples used in this study were acquired with informed consent, and all experiments were performed in accordance with relevant guidelines and regulations of the Graduate School of Medicine of Osaka University. Human astrocytes were used as normal controls. Gene expression levels were analyzed by qRT-PCR, as described above.
On day 0, U251 cells were plated in a 60-mm dish and cultured at 37˚C. On day 1, cells were 70-90% confluent, and siRNA/plasmid transfection was performed. On day 3, cells were extracted.

Rescue experiment for G0S2 siRNA
Using the above G0S2 overexpression plasmid as a template, a siRNA-resistant form of G0S2 was generated by changing the targeted sequence of the siRNA to 5 0 -AAATGGTCAAACTG TATGT-3 0 (4 mutant nucleotides are underlined) using the KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan). The plasmid was confirmed by DNA sequencing.

Production of a lentiviral vector expressing G0S2 shRNA
The shRNA sequence was made using G0S2 siRNA sense and antisense sequences with one nucleotide exchanged at the BglII site (GATCT -> GATCC) and XbaI site (G0S2 shRNA: 5 0 -GATCCCCAGATGGTGAAGCTGTACGTACGTGTGCTGTCCGTACGTACAGCTTCACCATC TTTTTTGGAAAT, control shRNA: 5 0 -GATCCCCACTACCGTTGTTAAGGTGACGTG TGCTGTCCGTCACCTTAACAACGGTAGTTTTTTGGAAAT) and their complementary strands were synthesized (Fasmac) and annealed in TE buffer by heating to 100˚C for 2-3 min followed by cooling to room temperature. The resulting double-stranded oligo-DNA was subcloned to the pENTR4-H1 entry vector at the BglII and XbaI sites. The insert was confirmed by both restriction enzyme digestion and DNA sequencing. Next, pENTR4-H1-shRNA was cloned into the lentiviral vector (CS-RfA-CG) using Gateway LR Clonase II Enzyme Mix (Invitrogen) according to the manufacturer's instructions. The insert was confirmed by both restriction enzyme digestion and DNA sequencing. These control or G0S2 shRNA-expressing lentiviral vector stocks were generated by the transfection of 293T cells. The 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum. 293T cells were plated in 100-cm 2 dishes in 10 ml of the medium and transfected the following day with 5 μg of the pCAG-HIV plasmid, 5 μg of the pCMV-VSV-G-RSV-Rev plasmid, and 10 μg of each lentiviral vector plasmid. Lentiviral vectors were collected on day 5 post-transfection and filtered through a 0.45-μm pore size filter and concentrated by ultracentrifugation at 4˚C for 2 h at 48,820 × g.
U87 cells were plated on 100-cm 2 dishes at a density of 1 × 10 6 cells in 9 ml of medium per dish. Transductions were carried out in the presence of 4 μg/ml Polybrene for Lenti-control or G0S2 shRNA. After incubation for 12 h, the transduction medium was replaced with fresh DMEM.

Transwell invasion assay
Transwell inserts (6.5 mm, 8-μm pore size membrane) were coated with vitronectin (10 μg/mL in PBS). The assay was largely performed as previously reported [19]. At 2 days after siRNA transfection, U251 cells were harvested with Trypsin-EDTA (0.25%), washed with PBS, and resuspended in invasion assay buffer at 4 × 10 5 cells/ml. Cell aliquots of 100 μl were plated on the upper filter surface and incubated at 37˚C for 3 h. Filters were then washed, and cells on the upper surface were removed using cotton swabs. Cells on the lower filter surface were fixed in 4% paraformaldehyde for 30 min, blocked in PBS with 0.1% Triton-X and 5% BSA for 1 h, and stained with DAPI. Five random selected fields were evaluated with a 100× oil immersion lens; the number of invaded cells was compared with cells counts in the control group. An average of five fields at 100× total magnification was used for the quantitative analysis.

In vivo tumorigenicity assay
All experiments were performed with the approval of the Animal Studies Ethics Committee of the Osaka University Hospital (J4822), and all experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Graduate School of Medicine of Osaka University. Five-week-old NOD-SCID mice (Charles River) were randomly divided into two groups (three mice per group). Under anesthesia, a 1-mm burr hole was made (2 mm to the midline and 2 mm posterior to the bregma) using a microskull drill. A total of 2 × 10 5 cells (U87 transfected with Lenti-control or G0S2 shRNA) suspended in 1 μl of PBS were slowly and smoothly injected to a depth of 3 mm via the skull hole using a 10-μl Hamilton syringe with a 30-gauge needle at a rate of 0.5 μl/min into the subcortex of the mouse brain. The needle was retained in place for 2 min. The scalp was sutured. Survival was recorded every 2 days over the course of the study.

Immunohistochemistry for G0S2
Immunohistochemical analyses of G0S2 were performed using paraffin blocks of specimens. The sections were deparaffinized in xylene and rehydrated in a descending ethanol-to-water gradient series. Endogenous peroxidase was blocked by exposure to 1% H 2 O 2 at room temperature for 1 h. Sections were soaked for antigen retrieval in pH 6.0 citrate buffer at 125˚C for 5 min. After cooling to room temperature, sections were blocked with 10% goat serum in PBS for 30 min at room temperature. Sections were incubated for 2 h at room temperature with a primary antibody against G0S2 (1:200; Novus, St. Louis, MO, USA) followed by incubation for 30 min at room temperature with Anti-rabbit IgG labeled with biotin (1:1000). The sections were prepared for the chromogen reaction with 0.05% diaminobenzidine and 0.01% H 2 O 2 , and were counterstained with hematoxylin.

Analysis of the cancer genome Atlas dataset
For the WHO grade II-IV glioma analysis, G0S2 gene expression based on RNASeq data (TOIL RSEM log2(normalized_count+1)), DNA methylation β values (the ratio of methylation-specific and demethylation-specific fluorophores), average Methylation450K data, IDH1 binary non-silent mutation (broad), primary tumor (sample_type) for brain lower grade glioma (LrGG, WHO grade II/III), and glioblastoma multiforme (GBM, WHO grade IV) samples were extracted from the TCGA Pan-Cancer (PANCAN) dataset using the University of California Santa Cruz Xena Browser (http://xena.ucsc.edu/). Excluding null data, 554 matching samples were available (509 lower grade glioma and 45 primary GBM, 157 wt IDH1 and 397 mut IDH1). For the lower grade (WHO grade II/III) glioma analysis, G0S2 gene expression based on RNASeq (polyA+ IlluminaHi-Seq) data, DNA methylation β values, average Methylation450K data, IDH1 mutation found (0 = No, 1 = Yes), WHO grade II/III (neoplasm histologic grade), and primary tumor (sample_ type) samples were extracted. Excluding null data, 125 matching samples were obtained (57 WHO grade II and 68 WHO grade III), dividing into two groups according to the G0S2 expression level ("G0S2 low" and "G0S2 high"). The survival ratio was compared with respect to G0S2 expression within the same WHO grade. For the GBM analysis, G0S2 gene expression based on RNASeq (polyA+ IlluminaHiSeq) data and primary tumor (sample_type) samples were extracted from the TCGA Glioblastoma (GBM) dataset. Excluding null data, 154 matching samples were obtained, the survival ratio was compared with respect to G0S2 expression among the GBM samples.
Additionally, the G0S2 mutation status was extracted from the glioma dataset {Merged Cohort of LGG and GBM (TCGA, Cell 2016)} using cBioPortal for Cancer Genomics (www. cbioportal.org), and 1102 samples were available.

Statistical analysis
Data are presented as means ± SEM (standard error of the mean). Comparisons between groups were performed using Student's t-tests and one-way ANOVA followed by Tukey-Kramer tests. Statistical significance was set at p < 0.05. JMP statistical package was used for the statistical analyses.
The statistical analyses of TCGA datasets using Welch's t-test, Pearson's and Spearman's rank correlation were collected from the University of California Santa Cruz Xena Browser (http://xena.ucsc.edu/)

Glioma with low G0S2 expression is associated with a longer overall survival in the TCGA dataset
Using the Cancer Genome Atlas (TCGA) database, we found that G0S2 expression elevated as the WHO grade increased among WHO grade II-IV gliomas. Relative G0S2 expression was higher in GBM than in lower grade gliomas (7.57 vs. 3.59, p = 1269e-16), and was higher in WHO grade III glioma than in WHO grade II glioma (3.12 vs. 2.49, p = 0.006330) among the lower grade gliomas (Fig 1A). Mutant IDH1 was statistically significantly related to both higher G0S2 methylation (0.749 vs. 0.366, p = 9.439e-53) and lower G0S2 expression (3.1 vs. 7.03, p = 0.000) compared to wild-type IDH1 among WHO grade II-IV gliomas (Fig 1B and 1C). G0S2 gene expression was negatively associated with G0S2 methylation (Pearson's r = -0.7719, Spearman's rho = -0.6932) within WHO grade II-IV gliomas (Fig 1D). Interestingly, G0S2 mutations were not reported in 1102 gliomas among TCGA WHO grade II-IV gliomas (Fig 1D), supporting the notion that G0S2 is epigenetically silenced in gliomas harboring IDH1 mutations. In addition, the lower G0S2 expression group clearly showed better over survival than the higher G0S2 expression group for all WHO grade II-IV gliomas (p = 1.181e-8) and for WHO grade II/III lower grade gliomas (p = 0.01371) (Fig 1E). Furthermore, the lower G0S2 expression group tended to exhibit a better prognosis than the higher G0S2 expression group among the same WHO grade (Fig 1F). These observations suggest that G0S2 is a new potential diagnostic marker for glioma.

Decreased G0S2 expression in glioma with IDH1 mutation
To verify the role of G0S2 in glioma malignancy, we measured G0S2 gene expression in WHO grade II diffuse astrocytoma, grade III anaplastic astrocytoma, and grade IV primary GBM tissue samples (Fig 2A). G0S2 levels were significantly higher for wild-type IDH1 than for mutant IDH1 among WHO grade III/IV gliomas. Although the correlation was not analyzed owing to the limited number of samples (n = 2, wild-type IDH1 WHO grade II specimens), G0S2 mRNA levels tended to be elevated as the WHO grade increased. These findings suggest that reduced expression of G0S2 contributes to better pathological features in glioma with IDH1 mutation.
Further, mRNA expression of G0S2 was higher in recurrent malignant glioma, diagnosed as WHO grade IV secondary GBM, than in initial glioma, diagnosed as WHO grade II diffuse astrocytoma, within the same patient (Fig 2B). G0S2 mRNA expression was higher in the same human high-grade glioma tissues (secondary GBM) than in low-grade glioma tissues (diffuse astrocytoma; DA) ( Fig 2C). These results suggest that increased G0S2 gene expression is associated with the malignancy of glioma.

Knockdown of G0S2 reduces the invasion of glioma cells
Next, we analyzed the role of G0S2 in glioma invasion by a transwell assay. To perform loss-offunction experiments using small interfering RNA (siRNA), we examined the knockdown efficacy of G0S2 siRNA. Efficient G0S2 mRNA downregulation was observed in G0S2 siRNA #1-, 2-, and 3-transfected cells, but not in the control, non-target siRNA-transfected cells ( Fig  3A). siRNA-mediated knockdown of G0S2 in the U251 malignant glioma cell line significantly suppressed cellular invasion (Fig 3B and 3C). To determine the specificity, the cells were transfected with both G0S2 siRNA and the RNA interference (RNAi)-resistant G0S2 rescue plasmid, and G0S2 expression was recovered to the control level (Fig 3D). Reconstituting G0S2 expression with the G0S2 rescue plasmid restored the cellular invasion ability (Fig 3B and 3C). These results indicate that the inhibition of G0S2 expression suppresses the invasion ability of glioma cells.

Antitumor effect of G0S2 knockdown in U87 glioma xenograft-bearing mice
Our in vitro experiments indicated that the downregulation of G0S2 expression can efficiently attenuate the invasion of glioma cells. Therefore, we further examined the antitumor effect of G0S2 in vivo using a xenograft model. We first transplanted both U251 and U87 cells into mice brains, and found that U87 cells, but not U251 cells, tended to survive in the brain. We examined the knockdown efficacy of Lenti-G0S2 shRNA in U87 cells (Fig 4A). U87 GBM cells infected with the lentivirus encoding G0S2 shRNA in mouse brains exhibited reduced cell invasion toward surrounding normal brain tissues ( Fig 4B). Importantly, mice in which G0S2silenced GBM cells were injected into the brain exhibited prolonged overall survival (Fig 4C).

IDH1 mutation epigenetically represses the transcription of G0S2
Since 2-HG, which is increased in IDH-mutant glioma, directly inhibits the activity of TET DNA demethylase, many genes in cancer are epigenetically silenced by promoter hypermethylation [8,20]. Of note, the G0S2 promoter contains a CpG island [21], suggesting that G0S2 down-regulation in the mutant IDH1 background results from epigenetic silencing. To test this hypothesis, we examined whether GBM cells transfected with a mutant IDH1 plasmid (pcDNA3-Flag-IDH1-R132H, a gift from Yue Xiong, #62907; Addgene, Cambridge, MA, USA) decreased the G0S2 mRNA level, and DNA hypomethylation by TET2 overexpression could restore the G0S2 mRNA level. It was reported that expression of IDH1 mutation lead to decrease in 5hmC [22], and that 5hmC decrease was associated with TET2 gene expression . The data were normalized by β-actin, used as the internal control, followed by normalization by cultured human astrocyte. G0S2 mRNA was significantly higher in gliomas with wild-type IDH1 than with mutant IDH1 for WHO grade III/IV glioma. Although there was no significant difference owing to the limited specimen number, G0S2 mRNA tended to increase as the WHO grade increased. Wild-type (wt) IDH1 diffuse astrocytoma, n = 2; mutant (mut) IDH1 diffuse astrocytoma, n = 3; wt IDH1 anaplastic astrocytoma, n = 3; mut IDH1 anaplastic astrocytoma, n = 5; wt IDH1 primary GBM, n = 10; mut IDH1 primary GBM, n = 3. � p < 0.05, Student's t-test. (b) G0S2 expression was higher at the recurrent diagnosis than at the initial diagnosis in the same patient. Relative G0S2 expression was quantified by RT-PCR using normal human astrocytes as a control. G0S2 expression was elevated in recurrent glioma, diagnosed as WHO grade IV secondary GBM, compared to initial glioma, diagnosed as WHO grade II diffuse astrocytoma, in the same patient. β-Actin was used as an internal control. n = 1. (c) Representative images of the sections of diffuse astrocytoma (DA) and secondary GBM (sGBM) immunostained with an anti-G0S2 antibody. The specimens were prepared from the same case. G0S2 expression was higher in sGBM than in DA. Scale bar: 200 μm (low-magnification, upper panel), 50 μm (high-magnification, lower panel).

Discussion
Mutations and the overexpression of several oncogenes have been identified in glioma [24,25,26]. The IDH R132H mutation, in particular, is thought to play a key role in gliomagenesis. This point mutation results not only in a loss of function, i.e., an inability to catalyze the conversion of isocitrate to α-ketoglutarate, but also in a gain of function, i.e., the ability to catalyze the NADPH-dependent reduction of α -ketoglutarate to 2-hydroxyglutarate (2-HG). Excess 2-HG contributes to the progression of epigenetic alterations, including DNA methylation at promoter CpG islands, and finally increases the risk of developing glioma [7]. Chao et al.  reported that 2-HG accumulation can inhibit H3K9 demethylase KDM4C (also known as JMJD2C) and that the inhibition of histone demethylation impairs the differentiation of nontransformed cells into terminally differentiated cells [27]. In addition, Flavahan et al. found that IDH mutations promote gliomagenesis by disrupting the chromosomal topology and allowing aberrant regulatory interactions that induce PDGFRA expression [28]. Hypermethylation of the O 6 -methylguanine-DNA methyltransferase (MGMT) promoter-associated CpG island has been reported as a favorable prognostic indicator. MGMT encodes an O 6 -methylguanine methyltransferase that removes alkyl groups from the O-6 position of guanine. Patients with MGMT hypermethylation show sensitivity to alkylating agents, such as temozolomide, with improved outcomes [29]. Rivera et al. reported that the methylation status of the MGMT promoter also predicts the response to radiotherapy in the absence of adjuvant alkylating agents in patients with newly diagnosed GBM [30].
IDH mutations are associated with improved survival in patients with glioma [6], and are a useful prognostic marker in clinical settings. However, the mechanism underlying the improved prognosis in patients with IDH mutations has not been determined. In this study, we elucidated the role of G0S2 in glioma cell invasion and identified a mechanism by which patients with glioma carrying IDH mutations and G-CIMP show better prognosis.
At present, only histopathological findings can be used to distinguish between WHO grade II and WHO grade III gliomas. According to our analysis, survival was longer for the lower G0S2 expression group than for the higher G0S2 expression group among the patients with WHO grade II-IV gliomas and WHO grade II/III gliomas in the TCGA dataset. Moreover, G0S2 expression increased as the WHO grade increased. We also demonstrated that the expression of G0S2 was higher in post-operative recurrence diagnosed as WHO grade IV secondary GBM compared with initial glioma diagnosed as WHO grade II diffuse astrocytoma in the same patient. It should be noted that there may be a contamination of normal brain parenchyma when using surgical material from low grade gliomas, resulting in lower relative G0S2 expression levels in the samples we examined. These data suggest that G0S2 is correlated with malignant transformation and can be a new marker when glioma progresses to a higher WHO grade or recurrence. It is necessary to evaluate a larger cohort to validate the correlation between G0S2 and WHO grade diagnosis. It is difficult to evaluate the mechanism underlying glioma malignant transformation, such as progression from WHO grade II/III to WHO grade IV, in vitro or in vivo; accordingly, an effective therapy for glioma recurrence is lacking. If a patient is diagnosed WHO grade II/III low-grade glioma, specifically suppressing G0S2 methylation has the potential to prevent malignant transformation.
The inhibition of 2-HG has been proposed as a possible treatment strategy for IDH-mutant glioma [31]. Although DNA methyltransferase inhibitors and histone deacetylase inhibitors are in clinical trials and show promise for the treatment of hematopoietic malignancies [32,33,34], treatment with vorinostat, a histone deacetylase (HDAC) inhibitor, combined with standard chemoradiation in newly diagnosed GBM in a phase II cohort did not meet efficacy objectives [35,36]. With respect to the molecular landscape, it is thought that focusing on specific alterations is more advantageous than conventional treatment strategies [37]. Our results further suggest that G0S2 methylation may be a potential therapeutic target.
In other cancers, G0S2 is correlated with cancer invasion [38], apotosis [39], and metabolism [40]. In a previous study of the genome-wide DNA methylation status of invasive cancer cell lines derived from breast, liver, and prostate cancers, G0S2 mRNA was significantly upregulated compared to expression in non-invasive cell lines, and its depletion decreased cell invasion in these three invasive cell lines [38]. Previous study reported that U87 cells were used for analyzing invasion ability [41]. We demonstrated that glioma harboring mutant IDH1 epigenetically represses G0S2 expression, thereby suppressing surrounding cell invasion and resulting in a better prognosis. Consistent with our results, G0S2 expression was down-regulated epigenetically by DNA hypermethylation in non-small cell lung cancer cell lines [38,39]. G0S2 is induced by tumor necrosis factor alpha (TNFA), whose activation also requires NFkB. G0S2 promotes apoptosis by interacting with BCL2 and by preventing the formation of protective BCL2 and BAX heterodimers [39]. Kim et al. have demonstrated that the G0S2 gene is significantly upregulated in BRAF V600E cells compared to wild-type BRAF human thyroid cells, and this expression change caused by the BRAF V600E mutation may have an important role in thyroid cancer development [42]. These observations are consistent with our results, demonstrating that G0S2 downregulation is related to the suppression of glioma cell invasion (Fig 3).
Clearly, as the characteristics of glioma with IDH mutations are related to both the inactivation of several tumor suppressor genes and the overexpression of other genes, although it should be noted that G0S2 is not an only marker for the better prognosis of IDH mutation glioma, the epigenetic down-regulation of the G0S2 is not the only factor determining the biological characteristics of glioma, however, our results showed that epigenetic G0S2 downregulation following IDH1 mutation effectively inhibits the invasion of GBM cells both in vitro and in vivo. Unlike genetic mutations, epigenetic changes, such as promoter methylation, are reversible and can be targeted by drugs. Future studies should elucidate the downstream pathways involved in the suppression of invasion by G0S2.
In conclusion, we identified one mechanism by which IDH mutations improve prognosis in glioma. Continued investigations are necessary to shed light on the roles of IDH mutations.