Identification of Promoter Region Methylation Patterns of MGMT,CDKN2A,GSTP1,and THBS1 Genes in Intracranial Meningioma Patients

Abstract

Background: Cytogenetic, molecular and epigenetic changes are all known to take place in the pathogenesis of meningiomas. In this study, we aimed at investing methylation of MGMT (DNA repair), CDKN2A (cell cycle control), GSTP1 (detoxification), and THBS1 (angiogenesis inhibitor) genes, which are known to be unmethylated in normal tissue, in meningioma samples. Materials and Methods: Methylation specific polymerase chain reaction was used to study promoter regions methylation of genes in 36 patient samples. Results: Methylation in promoter regions of MGMT, CDKN2A, GSTP1, and THBS1 genes were found in 11.1%, 8.3%, 2.8%, and 0% of the cases, respectively. About 19.4% of cases revealed promoter methylation of at least a single gene, whereas only 2.8% of cases revealed methylation of more than one gene. Based on their World Health Organization 2007 grade; 6.3% of grade I cases, 35.3% of grade II cases, and 33.3% of grade III cases showed hypermethylation in the promoter regions of the genes studied. No statistically significant relation was found between promoter zone methylation and factors such as age, sex, histopathology, grade, or recurrence. Conclusions: Further research on promoter zone methylation will help expose the methylation profile and pathogenesis of meningiomas, which will consequently guide to a deeper understanding of the pathogenesis of the disease, thus ensuring a better understanding of the prognosis and considering novel treatment options.

Introduction

Meningiomas originate from arachnoidal cap cells. They constitute ∼20% of all intracranial tumors. The reported incidence is 6/100,000 (Louis et al., 2000). The peak is in the sixth and seventh decades, and women are affected twice as much as men (Lamszus, 2004). Meningiomas can be encountered at all sites where arachnoidal cap cells are found, but convexity and the sphenoid wing are the most frequent anatomical locations (Haddad et al., 2004).

Symptomatology depends on the location and dimensions of the tumor and volume of the peritumoral edema. More than 80% of the meningiomas are slowly growing benign tumors and are regarded as grade I tumors by the World Health Organization (WHO) classification of central nervous system tumors (Louis et al., 2000). Nearly 5% to 15% of meningiomas are WHO grade II tumors.

These tumors are histologically characterized by several features such as increased mitotic activity, increased cellularity, high nucleus-to-cytoplasm ratio, prominent nucleoli, uninterrupted patternless or sheetlike growth, and foci of necrosis. Recurrence occurs in 30%-40% of the cases.

Approximately 1% to 3% of meningiomas are anaplastic, corresponding to WHO grade III. These tumors show histological features of frank malignancy far more than the abnormalities present in atypical meningiomas and are usually fatal, with a median survival of <2 years (Louis et al., 2000; Lamszus, 2004).

In recent years, genetic alterations have been held responsible for the formation and progression of meningiomas, and many studies have been performed in this respect. These studies have revealed that the loss of heterozygosity at the long arm of chromosome 22 (22q) is the most common genetic alteration seen in meningiomas, and is seen in about 40% to 70% of the patients (Dumanski et al., 1987; Seizinger et al., 1987). The tumor suppressor gene associated with Neurofibromatosis Type II (NF2) is located at 22q12.2. The inactivation of merlin, an NF2 gene product, has been shown to be functionally important in meningioma tumorigenesis (Lamszus, 2004). NF2 mutation has been observed in nearly 60% of sporadic meningiomas. Loss of 22q heterozygosity and NF2 mutation often occur simultaneously (Ruttledge et al., 1994; Wellenreuther et al., 1995; Ueki et al., 1999). Deletion of the short arm of chromosome 1 (1p) is the second most common chromosomal abnormality in meningiomas, and its frequency escalates with increasing malignancy grades, suggesting that 1p deletion might be involved in tumor progression (Bello et al., 1994; Weber et al., 1997). The third most common genetic abnormality is loss of the long arm of chromosome 14 (14q), and its relation to meningioma progression has been demonstrated (Lamszus, 2004). Other chromosomal aberrations involved in progression include losses of 6q, 9p, 10q, 18q, and gains of 1q, 9q, 12q, 15q, 20q (Weber et al., 1997; Lamszus, 2004). The DAL1 gene is localized at 18p11.3 and shows a loss of expression in 76% of sporadic meningiomas (Gutmann et al., 2000; Perry et al., 2000; Lamszus, 2004). Inactivation of cell-cycle-related proteins such as p14 (ARF), cyclin dependent kinase inhibitor 2A (CDKN2A), and 2B (CDKN2B); all mapped to 9p21; are frequently found in atypical and malignant meningiomas, indicating that the G1/S phase cell cycle checkpoint is an important aberration in high-grade meningiomas (Bostrom et al., 2001).

Alongside these changes at both chromosome and gene level, epigenetic alterations have also been reported to be important in human tumorigenesis (Brena and Costello, 2007). Epigenetic changes can be defined as heritable molecular changes in the genome without any alteration in the actual DNA sequence. They can affect gene expression or the function of the protein product (Esteller, 2008). The best known epigenetic change is DNA methylation, which is basically a chemical modification involving the addition of methyl (-CH3) groups to the fifth carbon of cytosine of the cytosine-guanine (CpG) dinucleotide. When the promoter region of a gene is methylated, the expression of that gene is repressed (Esteller, 2002).

Methylation changes can be through two mechanisms: loss or gain of methylation. Decrease in methylation in originally hypermethylated genes disturbs genomic stability; thus; for example, activating protooncogenes, which, in turn, may result in cancer development. Another important methylation disturbance responsible for cancer formation is the hypermethylation of promoter regions of tumor suppressor genes. It is a known fact that tumor suppressor genes can be inactivated by either deletion or mutation. Silencing of the promoter regions as a result of hypermethylation is another gene inactivation mechanism shown to be responsible for many types of cancers. Promoter hypermethylation may develop during either tumor cell formation or progression of the disease (Esteller, 2002).

To date, only a limited number of studies related to methylation profiles in cancers have been performed. In this study, we examined the role of epigenetic changes in the formation and progression of three grades of intracranial meningiomas. We aimed at defining promoter region methylation profiles of the MGMT, CDKN2A, GSTP1, and THBS1 genes and investigating the possible effect on the progression of the disease in 36 patients.

Materials and Methods

Patients

Thirty-six adult patients with a pathological diagnosis of meningioma who had been operated on between 1998 and 2008 at the Baskent University Hospitals in Ankara, Konya, and Adana were included in this study. The cases were classified according to the WHO 2000 Grading Scheme for Meningiomas (Louis et al., 2000), and there were 16 grade I, 17 grade II, and 3 grade III cases. Data on sex, age, histological subtype, WHO grade, and recurrence status were obtained and retrospectively recorded. Demographic and clinical properties of the cases are shown in Table 1.

Table 1.

Demographic and Clinical Properties of Patients

Variables	Descriptive statistics, n (%)
Sex
Male	16 (44.4)
Female	20 (55.6)
Age
21-30	1 (2.8)
31-40	4 (11.1)
41-50	5 (13.9)
51-60	16 (44.4)
61-70	5 (13.9)
71-80	3 (8.3)
81-90	2 (5.6)
Tumor Subtype
Meningothelial	5 (13.9)
Transitional	7 (19.4)
Angiomatous	2 (5.6)
Psammomatous	1 (2.8)
Fibrous	1 (2.8)
Atypical	15 (41.6)
Chordoid	1 (2.8)
Clear-cell	1 (2.8)
Anaplastic	3 (8.3)
WHO Grade
I	16 (44.4)
II	17 (47.2)
III	3 (8.4)
Recurrence
Yes	5 (13.9)
No	31 (86.1)

WHO, World Health Organization.

Samples, DNA extraction, and methylation specific polymerase chain reaction

DNA was isolated from 30 μm sections of paraffin-embedded meningioma tissue samples using the NucleoSpin^® tissue isolation kit (Macherey Nagel). DNA samples were subjected to bisulphite modification using the CpGenome DNA modification kit (Methylamp; Epigentek). The modified DNA samples were then amplified in separate reactions using dual primers specific for the unmethylated or the methylated status of the promoter of each gene. The specific primers, product sizes, annealing temperatures, and cycle numbers are shown in Table 2 (Liu et al., 2005). Polymerase chain reaction (PCR) products were separated on 2% agarose gels and visualized by ethidium bromide staining. For every gene studied, DNA isolated from a normal dura mater sample was used for normal tissue control, and DNA isolated from a placental sample and subsequently methylated was used for a methylated tissue control (Fig. 1).

FIG. 1.

Examples of methylation specific polymerase chain reaction products of unmethylated and methylated samples for all four genes on 2% agarose gel, shown alongside with their respective unmethylated dura mater controls, methylated placenta controls and negative controls (water). bp, base pairs; D, dura mate control; M, methylated; NC, negative control; Pl, methylated placenta control; U, unmethylated.

Table 2.

In House Polymerase Chain Reaction Conditions for All Four Genes

	Primers	M-Primers	U-Primers	Product size (bp)	Annealing temperature, (°C)/cycles
CDKN2A	F	5′-TTATTAGAGGGTGGGGCGGATCGC-3′	5′-TTATTAGAGGGTGGGGTGGATTGT-3′	M-150	66/35
	R	5′-GACCCCGAACCGCGACCGTAA-3′	5′-CAACCCCAAACCACAACCATAA-3′	U-151
MGMT	F	5′-TTTCGACGTTCGTAGGTTTTCGC-3′	5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′	M-81	60/35
	R	5′-GCACTCTTCCGAAAACGAAACG-3′	5′-AACTCCACACTCTTCCAAAAACAAAACA-3′	U-93
GSTP1	F	5′-TTCGGGGTGTAGCGGTCGTC-3′	5′-GATGTTTGGGGTGTAGTGGTTGTT-3′	M-89	60/35
	R	5′-GCCCCAATACTAAATCACGACG-3′	5′-CCACCCCAATACTAAATCACAACA-3′	U-95
THBS1	F	5′-TGCGAGCGTTTTTTTAAATGC-3′	5′-GTTTGGTTGTTGTTTATTGGTTG-3′	M-74	59/35
	R	5′-TAAACTCGCAAACCAACTCG-3′	5′-CCTAAACTCACAAACCAACTCA-3′	U-115

F, forward; R, reverse; U, unmethylated; M, methylated.

Statistical analyses

The Spearman's rho correlation statistical analysis method was used to show the relationship between methylation and age, sex, recurrence, tumor grade, and histopathology in SPSS 11.0. If the correlation coefficient was lower than 0.05 (two-tailed), the relationship between the parameters was evaluated as significant. All other calculations were performed using SPSS 11.0.

Results

The age of the patients ranged from 28 to 87 years with a mean age of 55.5±13.04. The female:male ratio was 1.25 to 1. The follow-up period ranged between 2 and 130 with an average of 40 months. Tumor progression was noticed in none of the cases. In 7 of the 36 cases (19.4%), promoter region hypermethylation was observed in at least 1 of the genes studied. In one of these seven cases, methylation was observed in >1 gene. The clinicopathological properties of the cases and methylation status of four genes are shown in Table 3. Promoter hypermethylation was observed in 1 of 16 WHO grade I (6.3%) cases, in 6 of 17 WHO grade II (35.3%) cases, and in one of three cases with WHO grade III (33.3%) tumors. Recurrence occurred in 5 (13.88%) cases. The recurrent cases did not show an increase in their grade during the follow-up period.

Table 3.

The Clinicopathological Properties of Patients and Methylation Status of Four Genes

Case no	Age	Gender	WHO grade	Tumor subtype	Recurrence (months)	Follow-up period (months)	Vital status	MGMT	CDKN2A	GSTP1	THBS1
1	32	Male	I	Meningothelial		19	Alive	M	U	U	U
2	28	Female	I	Meningothelial		22	Alive	U	U	U	U
3	60	Female	I	Meningothelial	230	36	Alive	U	U	U	U
4	59	Male	I	Meningothelial		38	Alive	U	U	U	U
5	66	Male	I	Meningothelial		40	Alive	U	U	U	U
6	50	Male	I	Transitional		38	Alive	U	U	U	U
7	65	Female	I	Transitional	18	42	Alive	U	U	U	U
8	44	Male	I	Transitional	26	72	Alive	U	U	U	U
9	56	Male	I	Transitional		40	Alive	U	U	U	U
10	50	Female	I	Transitional		42	Alive	U	U	U	U
11	42	Male	I	Transitional		62	Alive	U	U	U	U
12	53	Male	I	Transitional		80	Alive	U	U	U	U
13	39	Male	I	Angiomatous		49	Alive	U	U	U	U
14	87	Female	I	Angiomatous		38	Alive	U	U	U	U
15	66	Female	I	Psammomatous		40	Alive	U	U	U	U
16	38	Male	I	Fibrous		90	Alive	U	U	U	U
17	54	Male	II	Atypical		7	Alive	M	U	U	U
18	57	Female	II	Atypical		29	Alive	U	U	U	U
19	42	Male	II	Atypical		41	Alive	U	U	U	U
20	81	Female	II	Atypical		40	Alive	U	U	U	U
21	60	Female	II	Atypical	36	9	Dead	U	M	U	U
22	40	Male	II	Atypical		40	Alive	U	U	U	U
23	61	Female	II	Atypical	94	10	Alive	M	M	U	U
24	58	Female	II	Atypical		56	Alive	U	U	U	U
25	72	Female	II	Atypical		2	Dead	U	U	U	U
26	52	Female	II	Atypical		64	Alive	U	U	U	U
27	58	Female	II	Atypical		71	Alive	U	U	U	U
28	51	Female	II	Atypical		60	Alive	U	U	U	U
29	77	Female	II	Atypical		130	Alive	U	U	U	U
30	55	Female	II	Atypical		18	Alive	U	M	U	U
31	62	Male	II	Atypical		22	Alive	U	U	U	U
32	52	Female	II	Chordoid		88	Alive	U	U	M	U
33	53	Male	II	Clear-cell		44	Alive	U	U	U	U
34	54	Female	III	Anaplastic		64	Alive	U	U	U	U
35	75	Male	III	Anaplastic		90	Alive	M	U	U	U
36	58	Female	III	Anaplastic		32	Alive	U	U	U	U

M, methylated; U, unmethylated.

Promoter region hypermethylation in the CDKN2A gene was demonstrated in one of the grade II cases showing recurrence and in one other patient with grade II tumor in both the CDKN2A and MGMT genes. No promoter hypermethylation was observed in any of the four genes in patients with grade I meningioma showing recurrence. In 4 (11.1%) patients, MGMT was hypermethylated, and all of them were monoallelic methylation. In 3 (8.3%) patients, promoter hypermethylation was observed in the CDKN2A gene, and in all of them, both alleles were methylated (biallelic methylation). Only 1 case (2.8%) showed monoallelic methylation of the GSTP1 gene. Methylation of the THBS1 gene was not seen in any of the cases (Fig. 1).

No statistically significant correlation was derived between factors such as age, sex, tumor histopathology, grade and recurrence, and the methylation of promoter regions of MGMT, CDKN2A, GSTP1, and THBS genes (Table 4).

Table 4.

Nonparametric Correlation Analysis (Spearman's Rho) of Hypermethylation of the Studied Four Genes and Age, Sex of the Patients, Grade, and Histopathology of the Tumor and Recurrence

	MGMT	CDKN2A	GSTP1	THBS1
Age	0.824	0.978	0.775	——
Gender	0.203	0.433	0.379	——
Tumor subtype	0.536	0.331	0.344	——
WHO Grade	0.281	0.219	0.574	——
Recurrence	0.509	0.482	0.694	——

Discussion

The underlying molecular and genetic mechanisms of the epigenetic alterations are not fully understood, but it is known that disturbances involving DNA methylation play a role in the formation of many types of cancer. In this study, promoter region methylation of the four genes related with tumorigenesis was studied.

The MGMT gene is located on the long arm of chromosome 10 (10q26). It plays an important role in DNA repair, and has been the subject of many studies. Esteller et al. (2001) have reported promoter region hypermethylation of MGMT in 39% of colon cancers, in 21% of lung cancers, in 25% of lymphomas, in 34% of brain tumors, and in 32% of head and neck tumors. Liu et al. (2005) reported 6% MGMT promoter hypermethylation in a group of 48 meningioma cases. They did not observe a significant correlation between hypermethylation and tumor type or grade. DeRobles et al. (2008) have reported no MGMT hypermethylation in a group of 36 cases. We encountered MGMT promoter region hypermethylation in 11.1% of cases. One case with grade I, two cases with grade II, and one case with grade III meningioma showed hypermethylation. There was no statistically significant correlation between hypermethylation and sex, age, grade, histologic subtype, and recurrence.

CDKN2A is a tumor suppressor gene located at the p21 region of chromosome 9, and belongs to the INK4 kinase family. CDKN2A and RB1 genes regulate the progression from G1 to S phase in cell-cycle progression. Disturbance of this pathway leads to uncontrolled cell proliferation and tumor development (Ruas and Peters, 1998). Although many tumors display variable degrees of hypermethylation, lymphomas, and colon, gastric, and lung tumors show this characteristic more often than others (Esteller et al., 1998). There are studies that have reported varying degrees of CDKN2A hypermethylation in glioma patients (Yin et al., 2002; Wakabayashi et al., 2009). One study reported CDKN2A hypermethylation in 5 meningioma cases out of 23 (21.7%). Four of the cases had grade II-III, and 1 case had grade I meningioma (Tse et al., 1998). Liu et al. (2005) reported CDKN2A hypermethylation in five cases from a group of 48 meningioma cases. All cases displaying hypermethylation were grade II.

Genetic and epigenetic alterations in the CDKN2A gene may result in disruption of checkpoints regulating cell cycle progression from G1 to S phase. It is believed that this phenomenon plays a role in the pathogenesis of atypical and malignant meningiomas. We have observed CDKN2A gene promoter hypermethylation in 8.3% of the cases, and all patients with hypermethylation were grade II. Our findings did not reveal a statistically significant correlation between CDKN2A gene hypermethylation and sex, age, grade, histopathologic subtype, and recurrence. Although some evidence suggests that CDKN2A gene hypermethylation plays a role in pathogenesis of atypical menigiomas, confirmatory knowledge on the issue from larger chord studies that cover greater number of patients is required.

GSTP1, a member of the glutathione-S-transferase family gene, is located on the long arm of chromosome 11 (11q13). GSTP1 catalyzes the conjugation of carcinogens with glutathione providing detoxification and easy excretion of these compounds. Though it is not capable of DNA repair, it prevents DNA damage through this mechanism (Esteller, 2000).

Esteller et al. (1998) reported GSTP1 gene hypermethylation in 83% of prostate, 20% of renal, and 31% of breast cancers. In the same study, hypermethylation was observed in none of the 18 meningioma patients. In another study, GSTP1 hypermethylation was reported in 71.7% of pituitary adenomas (Yoshino et al., 2007). Liu et al. (2005) have observed no hypermethylation in grade I, 32% in grade II, and 54% in grade III meningiomas in a group of 48 cases. On the basis of these findings, they concluded that GSTP1 promoter region hypermethylation is associated with meningioma grade. In our study, only one case harboring a grade II meningioma showed hypermethylation (2.8%), and no statistically significant association was found between hypermethylation and sex, age, grade, histologic subtype, and recurrence. The small number with hypermethylation in grade II-III meninigomas can be explained by the limited number of cases in these groups.

The THBS1 gene is located on the long arm of gene 15 (15q14). THBS1 inhibits angiogenesis by disturbing the adhesion, progression, and motility of endothelial cells, and inducing their apoptosis (Guo et al., 1997). Gene silencing after THBS1 hypermethylation may cause the abolition of the antiangiogenic effect on tumor cells (Panetti et al., 1997). The loss of THBS1 expression has been shown to be related to progression and recurrence of some human neoplasms (Liu et al., 2005). Loss of expression due to hypermethylation has been reported in glioblastoma multiforme, prostate tumors, and hematopoetic neoplasms (Li et al., 1999).

Meningiomas are rich in vasculature, but there are not enough studies concerning THBS1 hypermethylation loss. Bello et al. (2004) found 30% THBS1 hypermethylation in a group of 98 intracranial meningiomas. They observed 28% hypermethylation in grade I and 37% in grade II-III tumors, and no association was found between hypermethylation and grading. Liu et al. (2005) noted no hypermethylation in grade I meningiomas, but there was hypermethylation in 7 cases out of the 32 cases harboring grade II-III tumors. Six of these cases had atypical meningiomas, and the authors proposed a relation between THBS1 gene hypermethylation and neovascularization of atypical meningiomas.

In the present study, we did not observe THBS1 gene hypermethylation in any of the cases. It may be due to the small number of cases studied. Another reason would be the quality of DNA samples affecting PCR amplification. However, since other PCR experiments using the same DNA samples have resulted in hypermethylation of the other three genes, the latter reason seems less probable.

Studies so far have shown the importance of hypermethylation in gene activation seen in atypical and malignant meningiomas. Transcriptional loss due to promoter region hypermethylation has been believed to be a reversible process, and the treatment of high-grade meningiomas with demethylating agents has been suggested (Liu et al., 2005).

Altogether, our results reflect a picture of the methylation profile of atypical meningiomas regarding genes with important roles in cell cycle control and DNA damage repair. We believe that future epigenetic research containing a greater volume of genes and a larger patient population will help to better understand the role of hypermethylation in the pathogenesis of meningiomas. This may also explain the mechanisms behind the problems of recurrence and high-grade tumor formation. Demethylating agents, either as a primary treatment modality or as an adjuvant to surgery, may play an important role in the management of meningiomas.

Footnotes

Acknowledgments

This study was approved by Baskent University Institutional Review Board (Project no: KA 08-18-2008-AP-195) and supported by Baskent University Research Fund.

Disclosure Statement

No competing financial interests exist for the authors.

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