|Year : 2015 | Volume
| Issue : 4 | Page : 123-130
Promoter Methylated Tumor Suppressor Genes in Glioma
Yingduan Cheng1, Yanyang Tu2, Pei Liang3
1 Cipher Ground, North Brunswick, NJ, USA
2 Department of Experimental Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China
3 Department of Urology, School of Medicine, University of California-Los Angeles, Los Angeles, CA, USA
|Date of Submission||02-Aug-2015|
|Date of Acceptance||10-Aug-2015|
|Date of Web Publication||27-Aug-2015|
Dr. Yingduan Cheng
Cipher Ground, 675 Rt. 1 South, North Brunswick, NJ 08902
Source of Support: None, Conflict of Interest: None
Epigenetic silencing of tumor suppressor genes (TSGs) is critical for glioma initiation and progression. Emerging reports suggest that increased numbers of TSGs, which are critical to cell cycle, proliferation, apoptosis, migration, invasion, DNA repair, and signaling pathways, were silenced in gliomas. Tumor-specific methylation of TSGs in glioma indicates that they could be used as epigenetic biomarkers for molecular diagnosis and therapeutics. This review summarizes the recent discoveries of epigenetically silenced TSGs in human gliomas, providing better understanding of disrupted epigenetic regulation in glioma progression.
Keywords: DNA methylation, glioma, tumor suppressor gene
|How to cite this article:|
Cheng Y, Tu Y, Liang P. Promoter Methylated Tumor Suppressor Genes in Glioma. Cancer Transl Med 2015;1:123-30
| Introduction|| |
In adults, glioma is the most common malignancy of the central nervous system.  Based on the specific type of cells with which they share histological features, gliomas have several main types: ependymomas (ependymal cells), astrocytomas (astrocytes), and oligodendrogliomas (oligodendrocytes).  According to WHO classification of brain tumors, gliomas are further classified into low-grade (Grades I and II), and high-grade (Grades III and IV) tumors.  Grade IV glioblastoma (GBM) is the most devastating malignant form of glioma with a median overall survival of 14.6 months despite the continuous progress in therapeutic technologies, including surgery, radiotherapy, photodynamic therapy, and chemotherapy. , Around 60% of the high-grade gliomas are GBMs, the incidence rate of which is approximately 3/100,000.  Understanding of the molecular mechanisms, and early detection of this malignant tumor are critical for a successful therapy.
The development of a tumor from a normal cell is a complex and multi-step process. A large number of oncogenes, tumor suppressor genes (TSGs), and signaling transduction pathways are involved in this process. , It is well accepted that carcinogenesis is driven by the accumulation of genetic and epigenetic changes, which result in the uncontrollable balance between cell proliferation and cell death.  Aberrant epigenetic mechanisms, such as DNA methylation, histone modification, nucleosome positioning, noncoding RNA, and microRNAs are recognized as key events in cancer development. Currently, it is well accepted that epigenetic alterations even precede the genetic changes during tumorigenesis.  Many epigenetic silenced functional TSGs are identified in multiple tumors, and these genes could function as biomarkers for cancer diagnosis. ,,,,, Hypermethylated TSGs in cancer cells are the most well-defined epigenetic hallmarks in gliomas.  Lots of functional TSGs were identified in gliomas including p16 INK4a, p14 ARF, MLH1, O 6 -methylguanine-DNA methyltransferase (MGMT), NDRG2, SLIT2, etc. This review will summarize previously identified candidate epigenetic biomarkers in gliomas based on their biological functions, such as cell cycle, apoptosis, invasion, DNA repair, and pathway related genes [Table 1]. Some representative genes are described in details below. Furthermore, these genes could be potential diagnosis markers and therapeutic targets for gliomas.
| Cell Cycle and Proliferation|| |
In normal cells, cell cycle and proliferation are controlled by a series of factors. Defects of these have been implicated in cancer initiation and progression. Both p16 INK4a and p14 ARF are alternative splicing isoforms of CDKN2A, located on human chromosome 9p21. , As an inhibitor of cyclin-dependent kinase 4 (CDK4) and 6 (CDK6), p16 maintains the unphosphorylated state of tumor suppressor Rb thus inhibiting cell cycle progression. Epigenetic inactivation of p16 by DNA methylation is frequently found in glioma, leading to uncontrollable cell cycle progression, and proliferation in glioma cells. ,,, Meanwhile, p14 inhibits MDM2-mediated degradation of p53 protein through inducing conformational changes of MDM2. Epigenetic inactivation of p14 ARF increases the degradation of the p53 protein, which results in the downregulation of p53 induced apoptosis. , Methylation of p16 INKa and p14 ARF in gliomas was also reported in many papers. ,,,
Besides these well-known cell cycle regulators, other DNA methylated TSGs have the ability to regulate cell cycle and proliferation in gliomas. NSD1 gene encodes a histone methyltransferase involved in chromatin regulation. It contains several functional domains including a SET domain, plant homeodomain, and proline-tryptophan-tryptophan-proline domain.  NSD1 function is abrogated in human neuroblastoma and glioma cells by CpG island-promoter hypermethylation. Epigenetic inactivation of NSD1 in transformed cells leads to specifically diminished methylation of the histone lysine residues H4-K20 and H3-K36. NSD1 is a functional TSG, which shows tumor suppressor-like features such as reducing colony formation density and inhibiting cellular growth. NSD1 CpG island hypermethylation was frequently detected in neuroblastomas and gliomas. Most importantly, NSD1 hypermethylation is a predicting factor of poor outcome in high-risk neuroblastoma. These findings suggest that inactivation of NSD1 leads to a disrupted histone methylation landscape, and it has a translational value as a prognostic marker. 
Receptor protein tyrosine phosphatase delta (PTPRD) is a member of the highly conserved family of receptor PTPs. PTPRD gene encodes a transmembrane protein with a cytoplasmic tyrosine phosphatase domain. This gene is frequently silenced via promoter CpG island hypermethylation. Its inactivation occurs in more than 50% of GBM tumors, and this inactivation also predicts poor prognosis in glioma patients. Functional studies suggest that wild-type PTPRD inhibits the growth of GBM and other tumor cells through dephosphorylating oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor silenced during the development of GBMs. 
Another cell cycle regulator, Krüppel-like factor 4 (KLF4), is also downregulted in glioma. KLF4 is a member of the KLF zinc-finger-containing transcription factor family. It contains three highly conserved zinc fingers of the C2H2 type along with a transactivating domain at the N-terminus.  Loss in physiological KLF4 expression at the RNA and protein levels was found in more than 40% of primary medulloblastomas. Re-expression of KLF4 in D283 medulloblastoma cell line results in significant growth suppression, both in vitro and in vivo.  All these data suggests that KLF4 functions as a TSG in the pathogenesis of medulloblastoma.
NDRG2, N-Myc downstream-regulated gene 2, is located at chromosome 14q11.2. The mRNA and protein levels, associated with this gene, were significantly lower in glioma tissues than in adjacent normal tissues. The downregulation of NDRG2, caused by promoter methylation, negatively correlates with the higher glioma grade and poor patient outcome. Overexpression of NDGR2 in human GBM U373 and U138 cells markedly reduced cell proliferation. Studies suggest that NDRG2 can regulate the level of histone acetylation to control glioma cell growth. All these data suggested that NDRG2 plays an important role in GBM carcinogenesis. ,,
| Apoptosis|| |
Apoptosis plays a critical role in many normal processes like fetal development and tissue homeostasis. A dysregulation in this process contributes to the development of many diseases, including cancer.  Some proapoptotic genes are also silenced by DNA methylation in gliomas. RANK/TNFRSF11A gene encodes a type 1 membrane protein, which binds to its ligand RANKL, activates signaling pathways such as nuclear factor kappa B (NF-kB), JNK, ERK, p38, and AKT/PKB, through phosphorylation of tumor necrosis factor receptor-associated factors protein.  Promoter methylation of RANK/TNFRSF11A is frequently detected in GBM tissues and glioma cell lines. Restoration of RANK/TNFRSF11A in GBM cell lines leads to a significant reduction in colony formation and increased apoptosis. This gene regulates apoptosis through NF-kB signaling and other distinct pathways such as cyclic adenosine monophosphate (cAMP)/protein kinase alpha (cAMP response element [CRE]), hypoxia inducible factor, Oct4, and Wnt (T-cell factor/lymphoid enhancer factor) pathway. 
Neogenin is a homologue of deleted in colorectal cancer. This gene plays fundamental roles in apoptosis, cellular differentiation, and axon guidance. Studies suggest that Neogenin was frequently downregulated in gliomas by promoter hypermethylation. The downregulation of Neogenin negatively correlates not only with glioma malignancy but also with glioma recurrence. Overexpression of Neogenin elevated apoptotic rate in SHG-44 cells. Studies suggest that Neogenin induces apoptosis and its downregulation, through promoter methylation, is a selective advantage for glioma genesis. 
Nonsteroidal antiinflammatory drug-activated gene, NAG-1, is a transforming growth factor-β member. Studies suggest that NAG-1 was frequently methylated in both gliomas and glioma cell lines. NAG-1 basal expression level inversely correlates with tumor grade in glioma. Its expression could be restored by pharmacological demethylation in methylated glioma cells.  NAG-1 functions as a tumor suppressor, that acts by inducing apoptosis in glioma cells. PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced GBM cell apoptosis. 
| Invasion and Migration|| |
The ability of a cancer cell to undergo migration and invasion allows it to change its position from the primary tumor into surrounding tissue.  Cell-cell and cell-matrix interactions are crucially involved in metastasis. Disruption of cell adhesion leads to a loss of contact growth inhibition, which is the early step of the neoplastic process.  With no lysine (K) 2 (WNK2), a serine/threonine kinase is a member of the WNK protein kinase subfamily. WNK2 is a TSG silenced by promoter methylation in gliomas. Patients without WNK2 exhibited poor prognosis. Studies found that WNK2 downregulation was associated with increased glioma cell invasion, due to negatively regulated MMP2 expression and activity, through a mechanism involving inactivation of JNK. ,
Adherens junctional associated protein 1 (AJAP1) is a transmembrane protein found in adhesion junctions and functions to inhibit glioma cell adhesion and migration. AJAP1 may be translocated to the nucleus and regulate gene expression via its interaction with β-catenin complexes. Promoter methylation of AJAP1 is frequently detected in oligodendrogliomas and correlates with low levels of AJAP1 expression. Low AJAP1 gene expression is associated with decreased patient survival rate. ,
Gliomas invasiveness is also regulated by the interplay between secreted proteases (e.g., cathepsins) and endogenous inhibitors (cystatins). Cystatin E/M (CST6) is a potent inhibitor of cathepsin B, which is frequently overexpressed in gliomas. Research suggests that CST6 is frequently downregulated in glioma samples and this reduced expression correlates with CST6 promoter hypermethylation. Ectopic expression of cystatin E/M in glioma cell lines results in reduced cell motility and invasion. ,
SLIT2 is a large extracellular matrix-secreted, and membrane-associated glycoprotein.  SLIT2/ROBO1 is a conserved ligand-receptor system. Their interactions mediate the repulsive cues on axons and growth cones during neural development. SLIT2 promoter was frequently methylated in glioma cell lines and tumor tissues, which correlates with the downregulated gene expression. The treatment with demethylating agent 5-aza-2'- deoxycytidine in SLIT2 methylated cells will restore SLIT2 gene expression. SLIT2 inhibits glioma migration and invasion by inactivating CDC42-GTP through SLIT2/ROBO1 pathway. ,,
| DNA Repair|| |
DNA repair genes could repair spontaneous errors that normally occur at microsatellite sequences during DNA replication.  Defects in these DNA repair genes will end up in a particular type of genetic instability. MLH1, a mismatch repair enzyme, is activated in response to DNA damage. Methylation analysis of the CpG sites in MLH1 promoter revealed hypermethylation in recurrent gliomas. This suggests that MLH1 promoter hypermethylation is an early event in the development and progression and the clonal evolution of gliomas. 
The product of DNA repair enzyme MGMT gene antagonizes the genotoxic effects of alkylating agents. MGMT promoter methylation is a frequent event and a key mechanism of MGMT inactivation in GBM patients. Its epigenetic silencing pattern predicts a favorable outcome in patients with GBM who are exposed to alkylating agent chemotherapy. ,,,
| RAS Pathway|| |
Some pathway related genes are downregulated by DNA methylation in gliomas. The human Ras association domain family 1A (RASSF1A) gene is frequently inactivated by promoter hypermethylation in primary brain tumors and glioma cell lines. RASSF1A was re-expressed in all methylated cell lines after treatment with the demethylating agent 5-aza-2'- deoxycytidine. It could induce apoptosis through MST1 or MOAP-1 proteins. Methylation of promoter CpG islands of RASSF1A may play an important role in the pathogenesis of glioma and medulloblastoma. ,,,
Another N-terminal RAS association domain family of genes, RASSF10, was consistently methylated in astrocytic gliomas. Specifically, RASSF10 was constantly methylated in WHO Grades II, and III astrocytomas and WHO Grade IV primary GBMs (67.5%), but unmethylated in Grade I astrocytomas and in DNA from age-matched control brain samples. Pharmacological demethylation by 5-aza-2'- deoxycytidine in methylated glioma cell lines restored RASSF10 expression. In secondary GBMs, RASSF10 methylation was an independent prognostic factor associated with worst progression-free survival, overall survival, and occurred at an early stage in their development. Overexpression of RASSF10 inhibited colony forming ability in two RASSF10-methylated glioma cell lines, and knockdown of RASSF10 increased cell proliferation in U87 glioma cells. 
| STAT Pathway|| |
JAK/STAT pathway is involved in initiation and progression of several types of cancer. , Suppressor of cytokine signaling 3 (SOCS3) is a functional tumor suppressor that inhibits the JAK/STAT signaling pathway. Reports found that SOCS3 promoter hypermethylation was frequently detected in primary GBMs, which was characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. SOCS3-depletion strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation. , Hypermethylation of SOCS1 was also reported in gliomas. , Reintroduction of SOCS1 into glioma cells sensitizes them to radiation-induced destruction.  These genes are critical inhibitors of STAT pathway in gliomas.
| Wnt Pathway|| |
Aberrant activation of wingless (Wnt) signaling is involved in the pathogenesis of various cancers including gliomas.  A study on hypermethylation investigation of Wnt pathway inhibitors suggested that some Wnt pathway inhibitors are frequently silenced by DNA methylation in gliomas. Hypermethylation of SFRP1, SFRP2, and NKD2 occurred in more than 40% of the primary GBMs, while DKK1 hypermethylation was found in 50% of secondary GBMs. SFRP1−, SFRP5−, DKK1−, DKK3−, NKD1−, and NKD2 expression increased in demethylase drug treatment in their hypermethylated U87-MG GBM cells.  Silencing Wnt inhibitors in glioma result in activation of Wnt pathway, leading to neoplastic progression.
LATS2 is a tumor suppressor which inhibits oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction.  LATS2 is frequently methylated in astrocytoma, but not in normal brain tissues. The mRNA level of LATS2 in astrocytomas with hypermethylation was significantly lower than those without methylation. LATS2 methylation was detectable in U251 and SHG-44 cell lines, and 5-aza-deoxycytidine restored its expression.  The methylation status of LATS2 may provide useful clues to the development of the diagnostic assays for astrocytoma.
| Clinical Application|| |
Gliomas are the most common type of primary brain tumor. Around 200,000 patients are diagnosed with a glioma around the world, each year.  Even with the available clinical treatments such as surgery, radiotherapy, and chemotherapy the median overall survival is only 14.6 months for GBM patients with a 5-year survival rate in < 5%. ,,, Understanding molecular mechanisms in gliomas are critical for the development of new treatment strategies. Identifying useful biomarkers will greatly benefit the early diagnosis of the condition and improve therapeutic treatment in glioma patients. Many methylated TSGs in gliomas have proven that their methylation pattern correlated with poor prognosis in patients [Table 1].
There are three ways for the application usage of aberrant DNA methylation in cancer diagnosis: (1) as a marker to detect cancer cells or cancer derived DNA; (2) as a marker to predict prognosis; and (3) as a biomarker for the assessment of therapeutic response.  There are several advantages for promoter methylated biomarkers. Promoter methylation testing in DNA samples provides more stability data than RNA samples. Frequently, DNA samples from cancer cells is only a small fraction of the total DNA in the clinical samples, which are composed of bodily fluids, such as blood, urine, sputum, saliva, and stools. Due to the development in high sensitivity DNA methylation detection technology, epigenetic biomarkers could be easily detected in these samples, even with the small fraction of cancer cell DNA.  What is more, the methylation of MGMT and p16 INK4a are already tested and works well in serum samples of glioma patients. ,,
The methylated promoters of TSGs can modulate the sensitivity of GBMs to drugs and radiotherapy.  For example, reactivated SOCS1, which is silenced by hypermethylation in glioma, increases the sensitivity of glioma to radiation via inactivation of MAPK.  Viral-mediated re-expression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis.  The methylation status also provides insight for patient specific therapy. The well-known example is MGMT promoter methylation and the resultant response to DNA alkylating agents. The mechanism of DNA alkylating agents is to form cross-links between adjacent stands of DNA, thus inhibit DNA replication to kill cells. The MGMT could directly and specifically remove the cytotoxic alkyl adducts formed at the O 6 position of the guanine by these alkylating agents. The active form of MGMT will inhibit the efficiency alkylating agents.  Because MGMT gene is seldom deleted or mutated in cancers, epigenetically silenced MGMT is a predictive biomarker for the response of clinical treatment with radiation and alkylating agents.  The predictive value of hypermethylated MGMT promoter is confirmed in several clinical investigations, and MGMT methylation is associated with significantly longer survival in GBMs when treated with radiation and alkylating agents.
In conclusion, both genetic changes and aberrant epigenetic changes contribute to glioma pathogenesis. Epigenetically silenced TSGs fail to suppress the initiation and development of gliomas. The studies on the development of methylated promoters of TSGs as biomarkers, in gliomas, will greatly benefit the diagnosis, prognosis, and therapy of such a devastating malignant tumor. We summarized the recently identified epigenetic silenced TSGs in this review. Continued efforts are needed to investigate TSGs' molecular mechanism, which will expand the knowledge of these markers and lead to the clinical translation of these makers.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Tu Y, Gao X, Li G, Fu H, Cui D, Liu H, Jin W, Zhang Y. MicroRNA-218 inhibits glioma invasion, migration, proliferation, and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1. Cancer Res
2013; 73 (19): 6046-55.
Kaba SE, Kyritsis AP. Recognition and management of gliomas. Drugs
1997; 53 (2): 235-44.
Louis DN. Molecular pathology of malignant gliomas. Annu Rev Pathol
2006; 1: 97-
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med
2005; 352 (10): 987-96.
Wang J, Su HK, Zhao HF, Chen ZP, To ST. Progress in the application of molecular biomarkers in gliomas. Biochem Biophys Res Commun
2 doi: 10.1016/j.bbrc. 207.
Cheng Y, Geng H, Cheng SH, Liang P, Bai Y, Li J, Srivastava G, Ng MH, Fukagawa T, Wu X, Chan AT, Tao Q. KRAB zinc finger protein ZNF382 is a proapoptotic tumor suppressor that represses multiple oncogenes and is commonly silenced in multiple carcinomas. Cancer Res
2010; 70 (16): 6516-26.
Coleman WB, Tsongalis GJ. Molecular mechanisms of human carcinogenesis. EXS
2006; (96): 321-49.
Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet
2002; 3 (6): 415-28.
Shu XS, Li L, Ji M, Cheng Y, Ying J, Fan Y, Zhong L, Liu X, Tsao SW, Chan AT, Tao Q. FEZF2, a novel 3p14 tumor suppressor gene, represses oncogene EZH2 and MDM2 expression and is frequently methylated in nasopharyngeal carcinoma. Carcinogenesis
2013; 34 (9): 1984-93.
Cheng Y, Liang P, Geng H, Wang Z, Li L, Cheng SH, Ying J, Su X, Ng KM, Ng MH, Mok TS, Chan AT, Tao Q. A novel 19q13 nucleolar zinc finger protein suppresses tumor cell growth through inhibiting ribosome biogenesis and inducing apoptosis but is frequently silenced in multiple carcinomas. Mol Cancer Res
2012; 10 (7): 925-36.
Wang S, Cheng Y, Du W, Lu L, Zhou L, Wang H, Kang W, Li X, Tao Q, Sung JJ, Yu J. Zinc-finger protein 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA transcription in gastric cancer. Gut
2013; 62 (6): 833-41.
Fu L, Dong SS, Xie YW, Tai LS, Chen L, Kong KL, Man K, Xie D, Li Y, Cheng Y, Tao Q, Guan XY. Down-regulation of tyrosine aminotransferase at a frequently deleted region 16q22 contributes to the pathogenesis of hepatocellular carcinoma. Hepatology
2010; 51 (5): 1624-4.
Yu J, Liang QY, Wang J, Cheng Y, Wang S, Poon TC, Go MY, Tao Q, Chang Z, Sung JJ. Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer. Oncogene
2013; 32 (3): 307-17.
He Z, Li B. Recent progress in genetic and epigenetic profile of diffuse gastric cancer. Cancer Transl Med
2015; 1 (3): 80-93.
Qu Y, Dang S, Hou P. Gene methylation in gastric cancer. Clin Chim Acta
2013; 424: 53-65.
Fueyo J, Gomez-Manzano C, Bruner JM, Saito Y, Zhang B, Zhang W, Levin VA, Yung WK, Kyritsis AP. Hypermethylation of the CpG island of p16/CDKN2 correlates with gene inactivation in gliomas. Oncogene
1996; 13 (8): 1615-9.
Costello JF, Berger MS, Huang HS, Cavenee WK. Silencing of p16/CDKN2 expression in human gliomas by methylation and chromatin condensation. Cancer Res
1996; 56 (10): 2405-10.
Little M, Wainwright B. Methylation and p16: suppressing the suppressor. Nat Med
1995; 1 (7): 633-4.
Sibin MK, Bhat DI, Narasingarao KV, Lavanya C, Chetan GK. CDKN2A (p16) mRNA decreased expression is a marker of poor prognosis in malignant high-grade glioma. Tumour Biol
2 doi: 10.1007/s13277-015-3480-5.
Arifin MT, Hama S, Kajiwara Y, Sugiyama K, Saito T, Matsuura S, Yamasaki F, Arita K, Kurisu K. Cytoplasmic, but not nuclear, p16 expression may signal poor prognosis in high-grade astrocytomas. J Neurooncol
2006; 77 (3): 273-7.
He J, Qiao JB, Zhu H. p14ARF promoter region methylation as a marker for gliomas diagnosis. Med Oncol
2011; 28 (4): 1218-24.
Carr J, Bell E, Pearson AD, Kees UR, Beris H, Lunec J, Tweddle DA. Increased frequency of aberrations in the p53/MDM2/p14(ARF) pathway in neuroblastoma cell lines established at relapse. Cancer Res
2006; 66 (4): 2138-45.
Veeriah S, Brennan C, Meng S, Singh B, Fagin JA, Solit DB, Paty PB, Rohle D, Vivanco I, Chmielecki J, Pao W, Ladanyi M, Gerald WL, Liau L, Cloughesy TC, Mischel PS, Sander C, Taylor B, Schultz N, Major J, Heguy A, Fang F, Mellinghoff IK, Chan TA. The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers. Proc Natl Acad Sci U S A
2009; 106 (23): 9435-40.
Nakahara Y, Northcott PA, Li M, Kongkham PN, Smith C, Yan H, Croul S, Ra YS, Eberhart C, Huang A, Bigner D, Grajkowska W, Van Meter T, Rutka JT, Taylor MD. Genetic and epigenetic inactivation of Kruppel-like factor 4 in medulloblastoma. Neoplasia
2010; 12 (1): 20-7.
Zhou B, Tang Z, Deng Y, Hou S, Liu N, Lin W, Liu X, Yao L. Tumor suppressor candidate gene, NDRG2 is frequently inactivated in human glioblastoma multiforme. Mol Med Rep
2014; 10 (2): 891-6.
Deng Y, Yao L, Chau L, Ng SS, Peng Y, Liu X, Au WS, Wang J, Li F, Ji S, Han H, Nie X, Li Q, Kung HF, Leung SY, Lin MC. N-Myc downstream-regulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation. Int J Cancer
2003; 106 (3): 342-7.
Waha A, Felsberg J, Hartmann W, von dem Knesebeck A, Mikeska T, Joos S, Wolter M, Koch A, Yan PS, Endl E, Wiestler OD, Reifenberger G, Pietsch T, Waha A. Epigenetic downregulation of mitogen-activated protein kinase phosphatase MKP-2 relieves its growth suppressive activity in glioma cells. Cancer Res
2010; 70 (4): 1689-99.
Berdasco M, Ropero S, Setien F, Fraga MF, Lapunzina P, Losson R, Alaminos M, Cheung NK, Rahman N, Esteller M. Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma. Proc Natl Acad Sci U S A
2009; 106 (51): 21830-5.
Yang Y, Huang JQ, Zhang X, Shen LF. MiR-129-2 functions as a tumor suppressor in glioma cells by targeting HMGB1 and is down-regulated by DNA methylation. Mol Cell Biochem
2015; 404 (1-2): 229-39.
Dong X, Deng Q, Nie X, Zhang M, Jia W, Chen C, Xu C, Xu R. Downregulation of HTATIP2 expression is associated with promoter methylation and poor prognosis in glioma. Exp Mol Pathol
2015; 98 (2): 192-9.
Chu SH, Feng DF, Ma YB, Zhang H, Zhu ZA, Li ZQ, Jiang PC. Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma. J Transl Med
Chu SH, Ma YB, Feng DF, Zhang H, Qiu JH, Zhu ZA. Effect of 5-Aza-2'- deoxycytidine on SLC22A18 in glioma U251 cells. Mol Med Rep
2012; 5 (1): 138-41.
Alaminos M, Dávalos V, Ropero S, Setién F, Paz MF, Herranz M, Fraga MF, Mora J, Cheung NK, Gerald WL, Esteller M. EMP3, a myelin-related gene located in the critical 19q13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. Cancer Res
2005; 65 (7): 2565-71.
Kunitz A, Wolter M, van den Boom J, Felsberg J, Tews B, Hahn M, Benner A, Sabel M, Lichter P, Reifenberger G, von Deimling A, Hartmann C. DNA hypermethylation and aberrant expression of the EMP3 gene at 19q13.3 in Human Gliomas. Brain Pathol
2007; 17 (4): 363-70.
Watanabe T, Huang H, Nakamura M, Wischhusen J, Weller M, Kleihues P, Ohgaki H. Methylation of the p73 gene in gliomas. Acta Neuropathol
2002; 104 (4): 357-62.
Jancalek R. The role of the TP73 gene and its transcripts in neuro-oncology. Br J Neurosurg
2014; 28 (5): 598-
von dem Knesebeck A, Felsberg J, Waha A, Hartmann W, Scheffler B, Glas M, Hammes J, Mikeska T, Yan PS, Endl E, Simon M, Reifenberger G, Pietsch T, Waha A. RANK (TNFRSF11A) is epigenetically inactivated and induces apoptosis in gliomas. Neoplasia
2012; 14 (6): 526-34.
Wu X, Li Y, Wan X, Kayira TM, Cao R, Ju X, Zhu X, Zhao G. Down-regulation of neogenin accelerated glioma progression through promoter Methylation and its overexpression in SHG-44 induced apoptosis. PLoS One
2012; 7 (5): e38
Kadowaki M, Yoshioka H, Kamitani H, Watanabe T, Wade PA, Eling TE. DNA methylation-mediated silencing of nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) in glioma cell lines. Int J Cancer
2012; 130 (2): 267-77.
Zhang Z, Wu L, Wang J, Li G, Feng D, Zhang B, Li L, Yang J, Ma L, Qin H. Opposing effects of PI3K/Akt and Smad-dependent signaling pathways in NAG-1-induced glioblastoma cell apoptosis. PLoS One
2014; 9 (4): e96
Chilukamarri L, Hancock AL, Malik S, Zabkiewicz J, Baker JA, Greenhough A, Dallosso AR, Huang TH, Royer-Pokora B, Brown KW, Malik K. Hypomethylation and aberrant expression of the glioma pathogenesis-related 1 gene in Wilms tumors. Neoplasia
2007; 9 (11): 970-8.
Li L, Abdel Fattah E, Cao G, Ren C, Yang G, Goltsov AA, Chinault AC, Cai WW, Timme TL, Thompson TC. Glioma pathogenesis-related protein 1 exerts tumor suppressor activities through proapoptotic reactive oxygen species-c-Jun-NH2 kinase signaling. Cancer Res
2008; 68 (2): 434-43.
Mueller W, Nutt CL, Ehrich M, Riemenschneider MJ, von Deimling A, van den Boom D, Louis DN. Downregulation of RUNX3 and TES by hypermethylation in glioblastoma. Oncogene
2007; 26 (4): 583-93.
Bai Y, Zhang QG, Wang XH. Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome. Eur J Med Res
2014; 19: 66.
Foltz G, Ryu GY, Yoon JG, Nelson T, Fahey J, Frakes A, Lee H, Field L, Zander K, Sibenaller Z, Ryken TC, Vibhakar R, Hood L, Madan A. Genome-wide analysis of epigenetic silencing identifies BEX1 and BEX2 as candidate tumor suppressor genes in malignant glioma. Cancer Res
2006; 66 (13): 6665-74.
Karakoula K, Jacques TS, Phipps KP, Harkness W, Thompson D, Harding BN, Darling JL, Warr TJ. Epigenetic genome-wide analysis identifies BEX1 as a candidate tumour suppressor gene in paediatric intracranial ependymoma. Cancer Lett
2014; 346 (1): 34-44.
Zhou X, Meng Q, Xu X, Zhi T, Shi Q, Wang Y, Yu R. Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway. Biochem Biophys Res Commun
2012; 427 (3): 574-80.
Zhou X, Xu X, Meng Q, Hu J, Zhi T, Shi Q, Yu R. Bex2 is critical for migration and invasion in malignant glioma cells. J Mol Neurosci
2013; 50 (1): 78-87.
Costa AM, Pinto F, Martinho O, Oliveira MJ, Jordan P, Reis RM. Silencing of the tumor suppressor gene WNK2 is associated with upregulation of MMP2 and JNK in gliomas. Oncotarget
2015; 6 (3): 1422-34.
Moniz S, Martinho O, Pinto F, Sousa B, Loureiro C, Oliveira MJ, Moita LF, Honavar M, Pinheiro C, Pires M, Lopes JM, Jones C, Costello JF, Paredes J, Reis RM, Jordan P. Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness. Hum Mol Genet
2013; 22 (1): 84-95.
Cogdell D, Chung W, Liu Y, McDonald JM, Aldape K, Issa JP, Fuller GN, Zhang W. Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma. Chin J Cancer
2011; 30 (4): 247-53.
Zeng L, Fee BE, Rivas MV, Lin J, Adamson DC. Adherens junctional associated protein-1: a novel 1p36 tumor suppressor candidate in gliomas (Review). Int J Oncol
2014; 45 (1): 13-7.
Qiu J, Ai L, Ramachandran C, Yao B, Gopalakrishnan S, Fields CR, Delmas AL, Dyer LM, Melnick SJ, Yachnis AT, Schwartz PH, Fine HA, Brown KD, Robertson KD. Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas. Lab Invest
2008; 88 (9): 910-25.
Kim TY, Zhong S, Fields CR, Kim JH, Robertson KD. Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. Cancer Res
2006; 66 (15): 7490-
Dallol A, Krex D, Hesson L, Eng C, Maher ER, Latif F. Frequent epigenetic inactivation of the SLIT2 gene in gliomas. Oncogene
2003; 22 (29): 4611-6.
Xu Y, Li WL, Fu L, Gu F, Ma YJ. Slit2/Robo1 signaling in glioma migration and invasion. Neurosci Bull
2010; 26 (6): 474-8.
Yiin JJ, Hu B, Jarzynka MJ, Feng H, Liu KW, Wu JY, Ma HI, Cheng SY. Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity. Neuro Oncol
2009; 11 (6): 77-89.
Tivnan A, Zhao J, Johns TG, Day BW, Stringer BW, Boyd AW, Tiwari S, Giles KM, Teo C, McDonald KL. The tumor suppressor microRNA, miR-124a, is regulated by epigenetic silencing and by the transcriptional factor, REST in glioblastoma. Tumour Biol
2014; 35 (2): 1459-65.
Fowler A, Thomson D, Giles K, Maleki S, Mreich E, Wheeler H, Leedman P, Biggs M, Cook R, Little N, Robinson B, McDonald K. miR-124a is frequently down-regulated in glioblastoma and is involved in migration and invasion. Eur J Cancer
2011; 47 (6): 953-63.
Lu SH, Jiang XJ, Xiao GL, Liu DY, Yuan XR. miR-124a restoration inhibits glioma cell proliferation and invasion by suppressing IQGAP1 and β-catenin. Oncol Rep
2014; 32 (5): 2104-10.
Vaitkienë P, Skiriutë D, Skauminas K, Tamaðauskas A. Associations between TFPI-2 methylation and poor prognosis in glioblastomas. Medicina (Kaunas)
2012; 48 (7): 345-9.
Gessler F, Voss V, Seifert V, Gerlach R, Kögel D. Knockdown of TFPI-2 promotes migration and invasion of glioma cells. Neurosci Lett
2011; 497 (1): 49-54.
George J, Gondi CS, Dinh DH, Gujrati M, Rao JS. Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis. Clin Cancer Res
2007; 13 (12): 3507-17.
Bertrand KC, Mack SC, Northcott PA, Garzia L, Dubuc A, Pfister SM, Rutka JT, Weiss WA, Taylor MD. PCDH10 is a candidate tumour suppressor gene in medulloblastoma. Childs Nerv Syst
2011; 27 (8): 1243-9.
Echizen K, Nakada M, Hayashi T, Sabit H, Furuta T, Nakai M, Koyama-Nasu R, Nishimura Y, Taniue K, Morishita Y, Hirano S, Terai K, Todo T, Ino Y, Mukasa A, Takayanagi S, Ohtani R, Saito N, Akiyama T. PCDH10 is required for the tumorigenicity of glioblastoma cells. Biochem Biophys Res Commun
2014; 444 (1): 13-8.
Mei PJ, Bai J, Liu H, Li C, Wu YP, Yu ZQ, Zheng JN. RUNX3 expression is lost in glioma and its restoration causes drastic suppression of tumor invasion and migration. J Cancer Res Clin Oncol
2011; 137 (12): 1823-30.
Berghoff AS, Hainfellner JA, Marosi C, Preusser M. Assessing MGMT methylation status and its current impact on treatment in glioblastoma. CNS Oncol
2015; 4 (1): 47-52.
Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W, Hegi ME. MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol
2010; 6 (1): 39-51.
Kanemoto M, Shirahata M, Nakauma A, Nakanishi K, Taniguchi K, Kukita Y, Arakawa Y, Miyamoto S, Kato K. Prognostic prediction of glioblastoma by quantitative assessment of the methylation status of the entire MGMT promoter region. BMC Cancer
Weller M. Assessing the MGMT status in glioblastoma: one step forward, two steps back? Neuro Oncol
2013; 15 (3): 253-4.
Gömöri E, Pál J, Mészáros I, Dóczi T, Matolcsy A. Epigenetic inactivation of the hMLH1 gene in progression of gliomas. Diagn Mol Pathol
2007; 16 (2): 104-7.
Martini M, Pallini R, Luongo G, Cenci T, Lucantoni C, Larocca LM. Prognostic relevance of SOCS3 hypermethylation in patients with glioblastoma multiforme. Int J Cancer
2008; 123 (12): 2955-60.
Zhou H, Miki R, Eeva M, Fike FM, Seligson D, Yang L, Yoshimura A, Teitell MA, Jamieson CA, Cacalano NA. Reciprocal regulation of SOCS 1 and SOCS3 enhances resistance to ionizing radiation in glioblastoma multiforme. Clin Cancer Res
2007; 13 (8): 2344-53.
Lindemann C, Hackmann O, Delic S, Schmidt N, Reifenberger G, Riemenschneider MJ. SOCS3 promoter methylation is mutually exclusive to EGFR amplification in gliomas and promotes glioma cell invasion through STAT3 and FAK activation. Acta Neuropathol
2011; 122 (2): 241-51.
Horiguchi K, Tomizawa Y, Tosaka M, Ishiuchi S, Kurihara H, Mori M, Saito N. Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors. Oncogene
2003; 22 (49): 7862-5.
Gao Y, Guan M, Su B, Liu W, Xu M, Lu Y. Hypermethylation of the RASSF1A gene in gliomas. Clin Chim Acta
2004; 349 (1-2): 173-9.
Levesley J, Lusher ME, Lindsey JC, Clifford SC, Grundy R, Coyle B. RASSF1A and the BH3-only mimetic ABT-737 promote apoptosis in pediatric medulloblastoma cell lines. Neuro Oncol
2011; 13 (12): 1265-76.
Hill VK, Underhill-Day N, Krex D, Robel K, Sangan CB, Summersgill HR, Morris M, Gentle D, Chalmers AD, Maher ER, Latif F. Epigenetic inactivation of the RASSF10 candidate tumor suppressor gene is a frequent and an early event in gliomagenesis. Oncogene
2011; 30 (8): 978-89.
Götze S, Wolter M, Reifenberger G, Müller O, Sievers S. Frequent promoter hypermethylation of Wnt pathway inhibitor genes in malignant astrocytic gliomas. Int J Cancer
2010; 126 (11): 2584-93.
Schiefer L, Visweswaran M, Perumal V, Arfuso F, Groth D, Newsholme P, Warrier S, Dharmarajan A. Epigenetic regulation of the secreted frizzled-related protein family in human glioblastoma multiforme. Cancer Gene Ther
2014; 21 (7): 297-
Kongkham PN, Northcott PA, Croul SE, Smith CA, Taylor MD, Rutka JT. The SFRP family of WNT inhibitors function as novel tumor suppressor genes epigenetically silenced in medulloblastoma. Oncogene
2010; 29 (20): 3017-24.
Vibhakar R, Foltz G, Yoon JG, Field L, Lee H, Ryu GY, Pierson J, Davidson B, Madan A. Dickkopf-1 is an epigenetically silenced candidate tumor suppressor gene in medulloblastoma. Neuro Oncol
2007; 9 (2): 135-44.
Mizobuchi Y, Matsuzaki K, Kuwayama K, Kitazato K, Mure H, Kageji T, Nagahiro S. REIC/Dkk-3 induces cell death in human malignant glioma. Neuro Oncol
2008; 10 (3): 244-53.
Jiang Z, Li X, Hu J, Zhou W, Jiang Y, Li G, Lu D. Promoter hypermethylation-mediated down-regulation of LATS1 and LATS2 in human astrocytoma. Neurosci Res
2006; 56 (4): 450-8.
Watanabe T, Katayama Y, Yoshino A, Yachi K, Ohta T, Ogino A, Komine C, Fukushima T. Aberrant hypermethylation of p14ARF and O6-methylguanine-DNA methyltransferase genes in astrocytoma progression. Brain Pathol
2007; 17 (1): 5-10.
Chin L, Pomerantz J, DePinho RA. The INK4a/ARF tumor suppressor: one gene-two products-two pathways. Trends Biochem Sci
1998; 23 (8): 291-6.
Gonzalez-Gomez P, Bello MJ, Arjona D, Lomas J, Alonso ME, De Campos JM, Vaquero J, Isla A, Gutierrez M, Rey JA. Promoter hypermethylation of multiple genes in astrocytic gliomas. Int J Oncol
2003; 22 (3): 601-8.
Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC, Wu TT, Huang S, Xie K. Drastic down-regulation of Krüppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res
2005; 65 (7): 2746-54.
Li L, Qin X, Shi M, Miao R, Wang L, Liu X, Yao L, Deng Y. Regulation of histone acetylation by NDRG2 in glioma cells. J Neurooncol
2012; 106 (3): 485-92.
Vermeulen K, Van Bockstaele DR, Berneman ZN. Apoptosis: mechanisms and relevance in cancer. Ann Hematol
2005; 84 (10): 627-39.
Darnay BG, Besse A, Poblenz AT, Lamothe B, Jacoby JJ. TRAFs in RANK signaling. Adv Exp Med Biol
2007; 597: 152-9.
Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer
2003; 3 (5): 362-74.
Duval A, Hamelin R. Genetic instability in human mismatch repair deficient cancers. Ann Genet
2002; 45 (2): 71-5.
Hesson L, Bièche I, Krex D, Criniere E, Hoang-Xuan K, Maher ER, Latif F. Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas. Oncogene
2004; 23 (13): 2408-19.
Hesson LB, Cooper WN, Latif F. The role of RASSF1A methylation in cancer. Dis Markers
2007; 23 (1-2): 73-87.
Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE Jr. Stat3 as an oncogene. Cell
1999; 98 (3): 295-
Wen Z, Zhong Z, Darnell JE Jr. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell
1995; 82 (2): 241-50.
Zhang K, Zhang J, Han L, Pu P, Kang C. Wnt/beta-catenin signaling in glioma. J Neuroimmune Pharmacol
2012; 7 (4): 740-9.
Li J, Chen X, Ding X, Cheng Y, Zhao B, Lai ZC, Al Hezaimi K, Hakem R, Guan KL, Wang CY. LATS2 suppresses oncogenic Wnt signaling by disrupting β-catenin/BCL9 interaction. Cell Rep
2013; 5 (6): 1650-63.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2 CA Cancer J Clin
2005; 55 (2): 74-
Kondo Y, Katsushima K, Ohka F, Natsume A, Shinjo K. Epigenetic dysregulation in glioma. Cancer Sci
2014; 105 (4): 363-9.
Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med
2008; 359 (5): 492-
Hu XT, He C. Recent progress in the study of methylated tumor suppressor genes in gastric cancer. Chin J Cancer
2013; 32 (1): 31-41.
Balaña C, Carrato C, Ramírez JL, Cardona AF, Berdiel M, Sánchez JJ, Tarón M, Hostalot C, Musulen E, Ariza A, Rosell R. Tumour and serum MGMT promoter methylation and protein expression in glioblastoma patients. Clin Transl Oncol
2011; 13 (9): 677-85.
Liu BL, Cheng JX, Zhang W, Zhang X, Wang R, Lin H, Huo JL, Cheng H. Quantitative detection of multiple gene promoter hypermethylation in tumor tissue, serum, and cerebrospinal fluid predicts prognosis of malignant gliomas. Neuro Oncol
2010; 12 (6): 540-8.
Lavon I, Refael M, Zelikovitch B, Shalom E, Siegal T. Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades. Neuro Oncol
2010; 12 (2): 173-80.
Natsume A, Kondo Y, Ito M, Motomura K, Wakabayashi T, Yoshida J. Epigenetic aberrations and therapeutic implications in gliomas. Cancer Sci
2010; 101 (6): 1331-6.
Day RS 3 rd
, Ziolkowski CH, Scudiero DA, Meyer SA, Lubiniecki AS, Girardi AJ, Galloway SM, Bynum GD. Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains. Nature
1980; 288 (5792): 724-7.
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